cancer research

WASHINGTON UNIVERSITY

Division o f Biology and Biomedical Sciences

Program in Molecular Cell Biology

Dissertation Examination Committee:Philip Majerus, Chair

Robert Arch Guojun Bu

Stuart Komfeld Maurine Linder

Linda Pike

THE CHARACTERIZATION OF THE HUMAN INOSITOL(l,3,4,5,6)P5 2-KINASE

By

John Wilson Verbsky

A dissertation presented to the Graduate School o f Arts and Sciences

o f Washington University in partial fulfillment o f the

requirements for the degree o f Doctor o f Philosophy

May 2006

Saint Louis, Missouri

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Acknowledgements

I would like to offer my thanks to Phil Majerus for being so patient with me; his rigor and

reputation as a scientist were proven to me and will remain forever a model to emulate;

his kindness and genuine care for the well-being o f his students is less well known, but

those o f us who have witnessed it shall attest for it.

.. .to my committee for their effort on my behalf. Their advice was not sought enough

considering its helpfulness and direction. I regret not engaging you more frequently.

... to my family for standing behind me during some tough times. I learned from you, in

part, the meaning o f family during this ordeal. Specifically I would like to thank my

twin, and closest friend, James, without whom I would not have made such a journey.

.. .to Alex, Marina, Mo, Cecil, Mochi, and Shao without whose aid this would not have

been possible. You taught me the meaning o f family from those not technically my

family, although the gift o f a car was more fitting a mother to a son than a mentor to a

student. I admire greatly your dedication to science. You will always be considered

close friends by one who doesn’t keep many.

.. .to Cecil for keeping the candy drawer stocked, and the beer flowing.

.. .to Yang Sun for his impatience.

ii


And finally, to the people who always make me smile...

.minha bela esposa Carla; eu te amo mais que a lua, os astros, e a propria vida;

and my boys, Andrew and Christopher, whom I will always try to make proud. Ilove and miss you every day.

iii


Table of ContentsAcknowledgements..............................................................................................................................iiTable o f Contents................................................................................................................................ ivList o f Figures......................................................................................................................................viAbbreviations......................................................................................................................................viiAbstract o f the Dissertation...........................................................................................................viiiChapter 1 : INTRODUCTION...........................................................................................................1

Introduction....................................................................................................................................... 2Synthesis o f InsP6 ............................................................................................................................3Physiological roles o f InsP6........................................................................................................... 8

Early studies o f InsP6.................................................................................................................. 8Nuclear roles for InsP6..............................................................................................................10InsP6 in exocytosis.....................................................................................................................13Receptor mediated endocytosis.............................................................................................. 15InsP6 in channel regulation......................................................................................................17

Objectives........................................................................................................................................ 21References....................................................................................................................................... 22

Chapter 2 : THE SYNTHESIS OF INOSITOL HEXAKISPHOSPHATE..........................26Abstract............................................................................................................................................27Background.....................................................................................................................................27Materials and Methods................................................................................................................. 28Results.............................................................................................................................................. 30

Identification and Isolation o f a Gene Encoding the Putative Human InsP5 2-Kinase....................................................................................................................................................... 30Expression o f Human InsP5 2-kinase Complements Yeast Mutants Deficient inEndogenous I p k l......................................................................................................................30Expression o f Human InsP5 2-Kinase in Sf21 Cells and Kinetic Studies...................30Northern Analysis o f InsPs 2-Kinase................................................................................... 31Alignments o f the Sequences o f Putative InsPs 2-Kinases............................................. 31

Discussion....................................................................................................................................... 31References....................................................................................................................................... 32

Chapter 3 : THE PATHWAY FOR THE PRODUCTION OF INOSITOLHEXAKISPHOSPHATE IN HUMAN CELLS.........................................................................33

Abstract............................................................................................................................................34Background.....................................................................................................................................34Materials and Methods................................................................................................................. 35Results.............................................................................................................................................. 36

Overexpression o f the Inositol 5/6-Kinase Results in Elevated Levels o f InsP4, InsPs,and InsP6 .....................................................................................................................................36Silencing o f Ins(l,3 ,4 )P3 5/6-Kinase Decreases InsP5 and InsP6 Levels...................... 36Cells Expressing the 5-Kinase Show No Increase in InsP5 or InsP6 Levels................. 38Gene Silencing o f the 5-Kinase Results in Decreased InsPs and InsP6 L evels 39

iv


Cells Overexpressing the 2-Kinase Produce InsP6 by Depleting the Available InsPs....................................................................................................................................................... 41Cells Transfected with a siRNA to the 2-Kinase Block Production o f InsP6 andAccumulate InsP5......................................................................................................................41

Discussion....................................................................................................................................... 41References....................................................................................................................................... 43

CHAPTER 4 : DISRUPTION OF THE MOUSE INOSITOL 1,3,4,5,6PENTAKISPHOSPHATE 2-KINASE GENE, ASSOCIATED LETHALITY, ANDTISSUE DISTRIBUTION OF 2-KINASE EXPRESSION..................................................... 44

Abstract............................................................................................................................................ 45Background..................................................................................................................................... 45Materials and Methods................................................................................................................. 45Results:.............................................................................................................................................46

ES line XA232 from a gene trap screen at BayGenomics successfully targets themouse InsP5 2-kinase................................................................................................................46The insertion o f the pGTOpfs construct in the XA232 ES line is embryonic lethal. 47 MEFs generated from mice heterozygous for the trapping construct show decreased2-kinase activity.........................................................................................................................47Tissue dependent expression o f the 2-kinase in adults and embryos............................48

Discussion:......................................................................................................................................49References:...................................................................................................................................... 50

CHAPTER 5 : INCREASED LEVELS OF INOSITOL HEXAKISPHOSPHATE (InsP6) RESULT IN AN INCREASED AMOUNT OF RIP AND PROTECTION OF HEK293CELLS FROM TNFa AND FAS INDUCED APOPTOSIS................................................... 51

Abstract............................................................................................................................................52Background.....................................................................................................................................52Materials and Methods................................................................................................................. 53Results:.............................................................................................................................................53

HEK 293 cells expressing a stably transfected RNAi to the 2-kinase result in alteredInsP6 profiles.............................................................................................................................. 532-kinase expressing cells are resistant to TNFa mediated apoptosis, whereas 2-kinase RNAi lines are more susceptible to apoptosis....................................................... 54The 2-kinase RNAi construct can overcome the protection from apoptosis o f 5/6-kinase expression.......................................................................................................................54Increased InsP6 protects against FAS mediated apoptosis, while decreased InsP6levels renders cells more susceptible to FAS mediated apoptosis.................................54Alterations in InsP6 levels do not affect receptor internalization, caspase 8 activity,or TNF receptor number.......................................................................................................... 542-kinase over-expressing cells and 2-kinase RNAi lines showed altered levels o f theanti-apoptotic protein RIP....................................................................................................... 55

Discussion:...................................................................................................................................... 56References:...................................................................................................................................... 57

Chapter 6 : CONCLUSIONS AND FUTURE DIRECTIONS................................................58References:......................................................................................................................................65

v


List of FiguresFigure 1-1. The synthesis pathway o f InsP6 in humans, yeast, D. melanogaster, and A.

thaliana.......................................................................................................................................... 4Figure 2-1 Alignment o f the putative InsPs 2-kinases............................................................... 28Figure 2-2 Expression o f human InsPs 2-kinase rescues InsP6 production in a S.

cerevisiae ipkl null strain........................................................................................................29Figure 2-3 Complementation o f the synthetic lethal phenotype o f the g le l-2 ip k l-4

double mutant by expression o f the putative human InsP5 2-kinase............................. 29Figure 2-4 Kinetics studies o f human InsP5 2-kinase............................................................... 29Figure 2-5 Northern blot analysis o f human InsPs 2-kinase mRNA levels......................... 30Figure 2-6 The alignment o f the putative and the cloned InsP6 2-kinases........................... 31Figure 3-1 Proposed pathways for production o f InsP6 in human cells (A) or yeast,

Drosophila melanogaster, and Arabidopsis thaliana (B)................................................ 35Figure 3-2 HPLC profiles o f [3H]inositol-labeled HEK-293 cells expressing Ins(l,3 ,4 )P3

5/6-kinase.................................................................................................................................... 37”3

Figure 3-3 HPLC profiles o f [ H]inositol-labeled HeLa cells expressing the 5/6-kinaseRNAi construct.......................................................................................................................... 38

Figure 3-4 Overexpression o f human 5-kinase...........................................................................39Figure 3-5 Gene silencing o f the human 5-kinase..................................................................... 40Figure 3-6 HPLC profiles o f [3H]inositol-labeled HEK-293 cells expressing the

Ins(l,3 ,4 ,5 ,6 )P5 2-kinase..........................................................................................................41Figure 3-7 HPLC profiles o f [3H] inositol-labeled HEK-293 transfected with an siRNA to

InsPs 2-kinase.............................................................................................................................42Figure 3-8 The human inositol phosphate pathway showing the in vivo and in vitro

activities reported for 5/6-kinase, 5-kinase, and 2-kinase................................................42Figure 4-1 The mouse 2-kinase locus on chromosome 13...................................................... 46

•7

Figure 4-2 [ H] Inositol labeling o f soluble inositol phosphates o f WT or heterozygousMEFs............................................................................................................................................ 47

Figure 4-3 2-kinase expression in adult and embryonic mice................................................ 48Figure 4-4 Pathways for synthesis o f InsP6 in different organisms. (A) Homo sapiens. (B)

Saccharomyces cerevisiae, Drosophila melanogaster, and Arabidopsis thaliana.... 49 Figure 5-1 HPLC profiles o f [3H]inositol-labeled HEK293 cells expressing a RNAi

construct to the 2 -kinase.......................................................................................................... 54Figure 5-2 APOPercentage staining o f cells expressing the 2-kinase or the 2-kinase

RNAi............................................................................................................................................ 55Figure 5-3 PARP Western blots o f TNF-treated 2-kinase (2-K) lines.................................. 55Figure 5-4 2-Kinase RNAi construct expression overcomes the protective effect o f 5/6

kinase expression in HEK293 cells.......................................................................................55Figure 5-5 Expression o f the 2-kinase affords protection from Fasmediated apoptosis,

whereas depletion o f InsP6 results in an increased susceptibility to Fas mediatedapoptosis......................................................................................................................................55

Figure 5-6 Transferrin uptake assays in 2-kinase-overexpressing ce ll.................................56Figure 5-7 Cell lines expressing the 2-kinase contain relatively higher levels o f RIP,

whereas cells deficient in InsP6 contain relatively lower levels o f RIP........................56

vi


Abbreviations

2-kinase, inositol 1,3,4,5,6-pentakisphosphate 2-kinase

5-kinase, inositol 1,3,4,6-tetrakisphosphate 5-kinase

5/6-kinase, inositol 1,3,4-trisphosphate 5/6-kinase

EST, expressed sequence tag

HEK, human embryonic kidney

IPMK, inositol polyphosphate multikinase

InsP3, inositol 1,4,5-trisphosphate

Ins(l,3 ,4 )P3, inositol 1,3,4-trisphosphate

Ins(l,3 ,4 ,6 )P4, inositol 1,3,4,6-tetrakisphosphate

Ins(l,3 ,4 ,5 )P4, Inositol 1,3,4,5-tetrakisphosphate

InsPs, inositol 1,3,4,5,6-pentakisphosphate

InsP6, inositol 1,2,3,4,5,6-hexakisphosphate

HPLC, high performance liquid chromatography

mAh, monoclonal antibody

MEF, mouse embryonic fibroblast

PARP, poly(ADP-ribose) polymerase

PP-InsP4, disphosphoinositol tetrakisphosphate

RIP, receptor-interacting protein

RNAi, RNA interference


Abstract of the Dissertation

THE CHARACTERIZATION OF THE HUMAN INOSITOL(l,3,4,5,6)P5 2-KINASE

By

John Wilson Verbsky

Doctor o f Philosophy in Biology and Biomedical Sciences (Molecular Cell Biology)

Washington Unversity in St. Louis, 2006

Professor Philip Majerus, Chairperson

Inositol hexakisphosphate, InsPs or phytic acid, is the most abundant inositol

phosphate in the world, and yet its function in the biology o f animals is largely contested.

The cloning in 1999 o f the first gene responsible for the production o f InsP6 from InsPs in

yeast, the InsPs 2-kinase known as IPK1 in yeast, finally allowed a truly in vivo study that

described a concrete function for InsP6 in the export o f mRNA from the nucleus. At the

initiation o f the work presented in this thesis, no other 2 -kinase homolog had been

discovered.

This thesis describes the characterization o f the first mammalian InsPs 2-kinase.

The first section will describe: the cloning o f the first mammalian InsPs 2-kinase using

conserved motifs from the yeast sequence to uncover potential genes; the expression and

biochemical characterization o f the protein; and the confirmation o f its role as a 2 -kinase

in vivo in expression studies in yeast mutant for the 2 -kinase.

The second part o f the thesis w ill address the major synthesis pathway o f InsP6 in

mammalian cells using over-expression and RNAi treatment o f the genes involved in the

viii


synthesis o f the higher inositol phosphates. This work is necessary to confirm the

function o f the 2 -kinase with respect to apoptosis.

The third part o f this thesis will describe the generation o f a mutant mouse line

that is deficient for the 2-kinase. In addition to showing that loss o f the 2-kinase results

in embryonic lethality, this work will investigate the pattern o f in vivo expression o f the

2 -kinase in the brains, hearts, and testicles o f mouse embryos and adult mice.

The final part o f this thesis will investigate a role for the higher inositol

phosphates in the regulation o f apoptosis. This effect was seen in cell lines that express

the 5/6-kinase, a gene that synthesizes a potential early substrate for the generation o f

InsPs and InsP6. It will describe a possible mechanism for the protection. This work will

also address the role o f InsP6 on endocytosis, a physiological function widely attributed

to InsP6.

ix


Chapter 1 : INTRODUCTION

Sed nil dulcius est, bene quam munita tenere Edita doctrina sapientum templa serena,

Lucretius, D e rerum natura, ii, lines 7-8


Introduction.Inositol hexakisphosphate, (InsPe), or phytic acid, was the first inositol phosphate

discovered over 80 years ago by Postemak[l], when he proved that the principle storage

form o f phosphate in the seeds o f plants was identical to synthetic InsP6. As such, InsP6

is the most abundant inositol phosphate in the world[2]. Inositol phosphates come in two

forms: phosphatidyl inositols, in which inositol containing as many as three phosphates is

bound to the lipid phosphatidic acid and therefore insoluble; and soluble inositol

phosphates, including InsP6, which only contain phosphate groups linked to the carbon

backbone o f the inositol ring. There are many inositol phosphates due to the possibility

o f phosphorylation o f one or more o f the inositol carbons, and many isomers o f inositols

carrying the same number o f phosphates.

Inositol phosphates gained prominence many years after their discovery when

Streb et al. found that Ins(l,4 ,5 )P3 (InsP3) was a calcium mobilizing second

messenger[3]. Shortly thereafter it was found that InsP3 is metabolized into other inositol

phosphate isomers[4]. Within a few years, InsP6, which was thought to be mostly

confined to plants, was found to be a component o f mammalian cells[5]. Since that time,

there have been many attempts to attribute physiological functions to InsP6 in vitro, many

fraught with difficulty because o f the problem o f working with a molecule that is so

negatively charged. The charge may have non-physiologic consequences; InsP6 can

chelate cations, and therefore alter in vitro reaction conditions, and the negative charge

may interact electrostatically with proteins in a non-physiologic manner[6 ]. In vivo

studies will avoid a lot o f the problems associated with experimentation with InsP6.

2


Synthesis of InsP6The common precursor o f all soluble inositol phosphates, including InsP6, in

mammalian cells is InsP3, which is produced by the cleavage o f phosphatidyl inositol

(4 ,5)P2 (PIP2) by phospholipase C, yielding InsP3 and diacylglycerol. InsP3 is then

metabolized to a number o f more highly phosphorylated inositol species through the

actions o f several phosphatases and kinases (fig. 2). InsP3 can be phosphorylated by a 3-

kinase, giving rise to Ins(l,3 ,4 ,5)P4, and then dephosphorylated by inositol a

polyphosphate 5-phosphatase which can act on this InsP4 isomer, resulting in

Ins(l,3 ,4 )P3, and on InsP3 itself, resulting in Ins(l,4 )P2. Menniti et al. [Menniti, 1990

#50] saw that in addition to the expected increase o f the Ins(l,4 ,5 )P3 isomer, the

Ins(l,3,4)P3 isomer increased in response to PLC activation with bombesin. In this

model, InsP6 is derived from the Ins(l,3,4)P3 isomer. Ins(l,3 ,4 )P3 is the substrate for

Ins(l,3 ,4 )P3 5/6-kinase, which phosphorylates either the D5 or D6 position o f the inositol

ring[7]; the major phosphorylation event occurs at the D 6 position. The resulting

Ins(l,3 ,4 ,6 )P4 is in turn the preferred substrate for Ins(l,3,4,6) 5-kinase[8], resulting in

Ins(l,3 ,4 ,5 ,6 )P5 (InsPs). This is the major inositol pentakisphosphate isomer in cells,

lacking a phosphate group only at the D2 position o f inositol. InsP6 is produced by

phosphorylation at the D2 position by an InsPs 2-kinase.

3


Saccharomyces cerevisiae, Drosophila melanogaster, Arabadopsis thaliana

IPK2

lns(1,4,5)P3

Homo sapiens

lns(1,4,5,6)P4

lns(1,4,5)P3

lns(1,3,4)P3

HC

> HO

lns(1,3,4,5)P4

5-Ptase

>lnsP3 5/6-K

lns(1,3,4,6)P4

IPK2

lns(1,3,4,5,6)P5

lnsP4 5-K

IPK1

lns(1,2,3,4,5,6)P6

lnsP5 2-K

lns(1,3,4,5,6)P5 lns(1,2,3,4,5,6)Pe

Fig. 1. The synthesis pathway of lnsP6 in humans, yeast, D. melanogaster, and A. thaliana.

Figure 1-1. The synthesis pathway of InsP6 in humans, yeast, D. melanogaster, and A thaliana.

Although the activity o f this 2-kinase was partially purified by Phillipy et al. from

soy beans[9], the identity o f a 2-kinase gene was unknown until 1998, when York et

al. [10] discovered the gene during a synthetic lethal screen with a yeast mutant involved

in mRNA export. This screen identified three genes involved in inositol phosphate

biosynthesis, a phospholipase C homolog (PLC1), a kinase that phosphorylates InsP3 to

InsPs (IPK2), and a 2-kinase (IPK1) (fig. 1). Since InsPs and InsP6 were completely

absent in the phospholipase C mutant, this was in vivo confirmation that InsPs and InsP6

were products o f the InsP3 released by PIP2 hydrolysis. Although other synthesis

pathways o f InsP6 occur in nature, for instance the ability o f Dictostelium to synthesize

InsP6 directly from inositol[l 1], the likely pathway for InsP6 in mammals remains the one

through InsP3.

4


Yeast IPK1 mutants have no InsP6 when the cells are labeled with [3H]-inositol,

but instead accumulate InsP5 and a pyrophosphate form o f InsP5, PP-InsP4, confirming its

in vivo activity as an InsPs 2-kinase. The genes responsible o f the production o f InsPg

among three fungi, S. cerevisiae, S. Pombe, and C. albicans share only a slight homology

amongst themselves (-20% identity)[12]. This low degree o f sequence conservation

even among fungal species hampered attempts to identify the 2 -kinase gene in mammals

by using homology searches.

The pathway for the production o f InsPs, the substrate for 2-kinase, is somewhat

controversial (fig. 1). In yeast, InsP3 is converted directly to Ins(l,4 ,5 ,6 )P4 and to InsP5

by Ipk2 [10, 13]. This pathway differs from that proposed by the work o f Menniti in that

no isomerization o f Ins(l,4 ,5)P3 to kis(l,3,4)P3 is required; therefore the intermediate

InsP4 isomer is not Ins(l,3 ,4 ,6 )P4, but Ins(l,4 ,5 ,6 )P4 [13]. Deletion o f IPK2 causes an

increase in Ins(l,4 ,5 )P3, proving that there is no other pathway to go from InsP3 to InsP5

in yeast. In addition, yeast does not possess a 5/6-kinase, nor has an Ins(l,3 ,4 )P3 isomer

been seen in metabolically labeled yeast extracts.

The Drosophila and Arabadopsis homologs o f Ipk2 have also been cloned, and

they can complement the yeast IPK2 gene in mutant lines[14, 15], The Drosophila and

Arabadopsis homologs similarly produce InsPs from Ins(l,4 ,5 )P3 through the

Ins(l,4 ,5 ,6 )P4, as does yeast. Interestingly, the authors identified a 5-kinase activity o f

the Drosophila and Arabadopsis homologs o f Ipk2 on Ins(l,3 ,4 ,6 )P4. This activity is

necessary for the proposed pathway in mammalian cells. Since they argue that the

alternate pathway works through Ins(l,4 ,5 ,6 )P4, which is already phosphorylated at the

D5 position, they did not find this activity relevant to the pathway. Yet it could possibly

5


raise a question as to their conclusions that Arabadopsis and Drosophila use the same

synthesis pathway as yeast, especially when one considers that Arabadopsis contains

three copies o f the 5/6-kinase gene. This conclusion is also called into question by work

in Zea mays. A mutant has been identified that produces low levels o f phytic acid, and

this mutant has been shown to encode a 5/6-kinase homolog[16]. This suggests that the

synthesis o f InsP6 in Zea mays requires 5/6-kinase, which the work in Arabadopsis would

deny. Whether Arabadopsis uses the same pathway to InsPs as Zea mays remains to be

seen. Plants may have developed more than one synthesis pathway due to the need to

make large quantities o f phytic acid. Or, it is possible that phytic acid synthesis may

differ depending on the plant structure in which it is produced. The study o f 5/6-kinase

in Zea mays shows that expression o f 5/6-kinase is high in the embryo, the organ where

phytic acid accumulates in seeds.

The possibility o f the direct phosphorylation o f InsP3 to InsP5 in these metazoans

has called into question the validity o f the mammalian pathway proposed above,

especially since Fujii et al. [17] analyzed the role o f the rat IPK2 homolog and found that

it too could produce InsPs from InsP3 when over-expressed in Rat-1 cells. This result is

troubling, because previous in vitro work suggested that the intermediate InsP4 produced

by the rat Ipk2 protein was Ins(l,3 ,4 ,5 )P4, not Ins(l,4 ,5 ,6 )P4 as seen in yeast, Drosophila,

and Arabadopsis. This work did not address the role o f the 5/6-kinase in determining the

synthesis pathway o f InsP5. Whether their results are simply the effect o f the over

expression, or whether it suggests differences in activities o f the phosphatases and

kinases that metabolize Ins(l,3,4,5)P4 in different cell lines, must be addressed. The

6


requisite pathway for the production o f InsPs, and ultimately, InsP6 should be determined

in mammalian cells.

The significance o f the source o f InsPs will be seen below in studies on InsP6. If

the substrate o f the 2-kinase lies downstream o f the activity o f 5/6-kinase and 5-kinase in

mammals, the physiologic effects seen by manipulation o f these genes may actually be

due to alterations o f the levels o f downstream products, e.g. InsP6. Although the pathway

to InsPs differs between mammals and yeast, the enzyme responsible for the conversion

o f InsPs to InsP6 should be similar, because both mammals and yeast use the same InsPs

isomer to make InsP6. InsP6 in turn is the substrate for InsP6 kinases, three isoforms o f

which have been described in mammals. These proteins phosphorylate InsP6 [18] to a

single pyrophosphate form, diphosphoinositol pentakisphosphate (PP-InsP(5)/InsP(7)),

and a di-pyrophosphate form, bis(diphospho)inositol tetrakisphosphate (bis-PP-

InsP(4)/InsP(8)). One must also consider the possibility that an effect seen with

manipulation o f 2 -kinase may be due to its effect on a downstream metabolite.

7


Physiological roles of InsP6.

E arly studies o f InsP 6.Phytic acid has long been studied in nutrition. Phytic acid is a major phosphate

source in plants, specifically in the developing seeds. From one to a few per cent o f the

total mass o f seeds is phytic acid, and phytic acid represents 65% to 85% o f the total

phosphate mass in seeds[19]. Upon germination, phytases release phosphate from phytic

acid, which is used for the developing plant’s metabolism; interestingly, germinating soy

beans possess significant 2 -kinase activity, suggesting that the phosphate stores o f phytic

acid themselves are insufficient[2 ], or that there are functions o f the 2 -kinase protein

itself. Ingestion o f large amount o f phytic acid have been associated with both negative

and potentially positive sequelae. Although soluble at acidic pH, phytic acid is a

polyanion, and as such it has a potent ability to chelate cations. For cultures that depend

upon plant sources for food, this presents a problem with respect to nutrition. Phytic acid

can chelate iron and zinc, forming salts which are largely excreted, and can result in iron

or zinc deficiency[20]. Furthermore, it presents a problem with respect to animal waste;

most non-ruminants excrete phytic acid, which is a major source o f phosphate pollution.

This is a troubling aspect o f the diet o f farm animals. Animal feed is supplemented with

either phytases or phosphate, because phytic acid itself is not a source o f phosphate.

There are attempts to limit this problem by breading plant with low levels o f phytic

acid[ 19].

The high fat, low fiber diet o f western, industrialized nations has long been

considered to promote cancer as compared to that o f nations which have a diet rich in

8


plants. Since plants have an abundance o f phytic acid, this compound has been a

candidate for the agent that prevents cancers. Studies have been reported that generally

confer an anti-cancer effect on InsP6 (see [21] and references therein). Most o f the

studies used cancer models in which rats are fed with a diet supplemented with InsP6 and

report an decreased incidence o f cancer; or cancer cell lines are treated with InsP6 and an

inhibition o f growth or a stimulation o f differentiation o f the tumor lines is reported.

These studies lack some credibility because o f the problem o f absorption o f InsP6; it is

highly charged, and has not been shown to cross membranes. The only binding partners

described for InsP6 are intracellular proteins (see below), and could not contribute to its

transport across membranes. One does not know whether the effects seen in the in vitro

studies are due to pH change, electrostatic interaction, or ion chelation, and as such are

inconclusive.

The one cancer model that does not completely suffer these shortcomings is colon

cancer. Protection from colon cancer could be attributed to the chelating properties o f

InsP6 on iron. InsP6 chelates iron and inhibits its ability to generate hydroxyl

radicals[22]. This could inhibit cellular oxidative damage in the colon. To support this,

InsP6 has been reported to inhibit oxidation o f DNA by H2O2, and H2O2 plus Cu(II)[23].

Since the lumen o f the colon does contain abundant InsP6, the issue o f absorption is

reduced, although not absent, because the anti-oxidant effect would presumably be

confined to the lumen o f the colon. Alternatively the anti-oxidant effect may simply

result from the chelation and excretion o f iron, thereby lowering total iron levels.

Nonetheless, higher cellular levels o f InsP6 may act as antioxidants; the question remains

i f the ingestion o f phytic acid can actually raise cellular InsP6 levels.

9


N uclear ro les f o r InsPg.Until the mid 1990’s, InsP6 suffered from the definition as a storage form o f

phosphate in plants. The discovery o f the yeast genes involved in the synthesis o f IsnPs

and InsP6 rejuvenated studies o f the more highly phosphorylated inositols. Furthermore,

the fact that they were discovered in a genetic screen, and could be studied in vivo finally

removed the difficulties o f working with InsP6 in vitro.

Using a temperature sensitive mutant o f g le l, defective for the essential mRNA

export factor G lel, York et al. conducted a screen that looked for genes that were

synthetically lethal with GLEl. Synthetic lethal screens identify pairs o f genes that are

not lethal by themselves, but are lethal in combination with each other. Thus they

frequently suggest functional relationships between proteins. This screen specifically

identified the three gene products that together are responsible for converting

PtdIns(4 ,5 )P2 to InsP6 [10, 13]. This included the previously characterized phospholipase

C, PLC1[24], and two inositol polyphosphate kinases, IPK1, the yeast InsP5 2-kinase, and

IPK2, the gene to which the mammalian InsP4 5-kinase is homologous. Besides the

genetic linkage between mutants defective in InsP6 production and the g le l mRNA

export mutant, strains lacking the IPK1 gene alone showed an accumulation o f mRNA in

their nuclei[10]. This directly implicates the enzyme that produces InsP6 in mRNA

export. This work was confirmed in mammalian cells expressing the S. dublin protein

SopB, a virulence factor that acts as an indiscriminate inositol phosphate phosphatase and

can deplete InsP6 from cells in culture [25]; these cells also show nuclear accumulation o f

mRNA consistent with the phenotype seen in yeast.

10


Another nuclear role for InsP6 was discovered when Hanakahi et al. were

attempting to isolate the agent responsible for the stimulation o f non-homologous end

joining (NHEJ) o f double stand DNA breaks in vitro [26]. NHEJ requires a complex o f

proteins including DNA-PKcS (catalytic subunit), the Ku70/80 heterodimer, XRCC4, and

DNA ligase IV. The authors used an in vitro assay containing this protein complex and

labeled DNA, to which they added chromatographic fractions, to isolate the factor that

stimulates joining o f the labeled DNA. Interestingly, this factor turned out to be InsPg.

Inositol hexakissulphate (InsSe) did not, suggesting that the effect is specific to InsP6.

Later work determined that InsP6 was binding to the Ku70/80 heterodimer [27] and not

DNA-PKcs, an interesting fact considering that DNA-PKcS contains a m otif homologous

to phosphatidyl inositol 3-kinase, which one may postulate could participate in inositol

binding.

In addition to its role in NHEJ, the Ku 70/80 heterodimers may also play a role in

telomere capping and the maintenance o f telomeres. Ku has been found to bind telomere

ends in both yeast and mammalian cells[28-30], where its presence has been postulated to

stabilize the telomere ends from aberrant end joining with other chromosomes or aberrant

telomerase dependent telomere lengthening. York et al. [31] report shortened telomers in

yeast mutant for the 2-kinase, but attribute this effect to an increase in the levels o f PP-

IP4, the pyrophosphate resulting from the phosphorylation o f InsPs, which is shown to be

a negative inhibitor o f telomere length. Nonetheless, inhibition o f the activity o f 2-

kinase, which results in an increase in PP-IP4, could be a means by which cells regulate

telomere ends. The contradictory roles o f Ku o f stimulating end joining in NHEJ while

11


preventing joining at the telomeres, could be due to other factors, possibly the presence o f

InsP6 itself.

To the growing list o f nuclear functions o f InsP6 can also be added chromatin

remodeling. Shen et al. [32] recently reported that InsP6 could inhibit the activity o f two

nucleosome remodeling complexes, NURF and INO80. This in vitro inhibition was not

seen with either inositol hexakissulphate (InsSe), cation chelation with EDTA, or with

InsPs, and is likely significant, although the effect did require high levels o f InsP6. InsP6

could also inhibit the ATPase activity o f these complexes. These two complexes, along

with the SWI/SNF ATPase containing remodeling complex, are known to be involved in

the expression o f the yeast INOl gene, encoding the inositol-1-phosphate synthase.

These authors were therefore looking to see whether the metabolites which ultimately

result from the activity o f the synthase are likely to be involved in its regulation. Though

InsP6 did not inhibit the remodeling by the SWI/SNF complex, InsP4 and InsP5

stimulated remodeling. This result was consistent with the finding that yeast Ipk2

mutants that cannot produce InsP4 or InsPs were unable to recruit the remodeling

complexes INO80 and SWI/SNF to the PH 05 promoter[33]. Chromatin remodeling

could be affected by 2-kinase activity, converting InsPs, which is stimulatory, to InsP6

which is inhibitory. It would be informative to look at chromatin remodeling in yeast

with excess InsP6.

12


InsP^ in exocytosis.InsP6 has also been implicated in exocytosis. Exocytosis is the process o f vesicle

fusion to the plasma membrane. The proteins involved in this process have been well

described and include SNAREs, synaptotagmin, and the small G protein, Rab3A[34].

SNAREs help dock the vesicles near the membrane. Synaptotagmin is a calcium binding

protein that also binds to SNAREs; it is considered the sensor o f calcium that triggers

vesicle fusion. In the presence o f calcium, synaptotagmin partially inserts into the

plasma membrane, providing the force necessary to fuse the vesicle membrane to the

plasma membrane. Rab3 A provides the directionality o f exocytosis and is implicated in

control o f Ca2+ mediated vesicle fusion at the synapse[35]. Rab3A is bound to the

vesicles in its GTP bound form, and after vesicular fusion, it hydrolyzes GTP to GDP.

The GDP bound form is extracted from the plasma membrane in a calcium dependent

process, and returned to another vesicle, where it exchanges GDP for GTP.

The highly phosphorylated inositol phosphates have been implicated in

exocytosis. InsP6 along with InsP4 and InsPs, have been reported to bind the C2b domain

o f synaptotagmin[36, 37]. In the squid giant axon presynapse, injection o f these inositol

phosphates blocks synaptic transmission. Interestingly, its C2b domain is also the

domain to which the lipid inositol phosphate, PIP2, binds. The relevance o f the report o f

InsP6 binding again should be questioned, since the PIP2 may represent the physiological

relevant binding partner. One the other hand, InsP6 may compete with PIP2 for binding

to this domain.

InsP6 has also been reported to stimulate insulin release, and thus exocytosis, in

permeabilized insulinoma cells [38]. This effect did not alter free calcium levels, which

13


the authors measured to rule out the possibility o f the chelating effect o f InsP6 on calcium

levels. The authors show that InsP6 stimulates the activity o f protein kinase C in vitro.

Protein kinase C is a calcium and phospholipid dependent protein kinase that is thought

to potentiate exocytosis[39]. InsP6 did not stimulate insulin release in the presence o f

PKC inhibitors, supporting the in vitro data. Later work showed a general stimulation o f

exocytosis measured as an increase o f capacitance by patch clamp techniques. This

stimulation o f exocytosis was blocked when the protein kinase C e isoform was depleted

by RNAi[40], but not other protein kinase C isoforms. This effect then seems to be

modulated by the effect o f InsP6 on protein kinase C. Again, a concern regarding the

validity o f this work remains; early work on the activity o f protein kinase C found that it

was stimulated by PIP2 and PIP3, and that though InsP6 mimicked this result, InsS6 did

also[41]. It remains to be seen whether the effect on protein kinase C is not due to the

simple charge effect o f the molecule.

A second inositol polyphosphate kinase was implicated in exocytosis when Luo et

al. [42] isolated a protein they termed GRAB (guanine nucleotide exchange factor for

Rab3A) in a yeast two hybrid screen with the InsP6 kinase l(InsP6 K l). GRAB is an

exchange factor for Rab3 A, stimulating the exchange o f GDP for GTP on Rab3 A, and

playing a role in the regulation o f its activity. Since GRAB also binds InsP6K l, this

interaction may regulate exocytosis. Since its substrate is InsP6, its activity may

coordinate phosphorylation o f InsP6 with exocytic events, although the significance in the

binding o f InsP6 K l to GRAB is unknown at present.

14


R ecep tor m ed ia ted endocytosis.InsP6 has also been implicated in receptor mediated endocytosis, although the in

vitro work is controversial. InsP6 has been shown to bind the clathrin accessory proteins

AP2 and AP180 [43-46] and to inhibit clathrin cage assembly in vitro[47, 48]. The

accessory proteins recognize the intracellular domain o f receptors, while also associating

with clathrin itself. Thus, the accessory protein recruit receptors into clathrin coated pits.

This would presumably inhibit endocytosis in cells with high levels o f InsP6. (3-arrestin,

which recruits G protein coupled receptors into coated pits for endocytosis and down

regulation, similar to the action o f accessory proteins on other receptors, has also been

shown to bind InsP6[49]. In these studies [3H]-InsP6 is bound to P-arrestin or AP2 and

competed o ff with phosphorylated lipid inositols. This would suggest that the lipid

binding may be the physiologically relevant binding, even though the K<j o f InsP6 is

nearly an order o f magnitude lower than the next best binding partner[46]. Nonetheless,

InsP6 injection has been used recently in Xenopus oocytes to block the ability o f P-

arrestin to internalize and desensitize receptors [50].

On the other hand, others have found InsP6 to stimulate endocytosis in vitro, as

measured by a decrease in capacitance in patch clamp experiments, possibly by the

stimulation o f protein kinase C and an inhibition o f synaptojanin [51]. Synaptojanin is a

phosphatidyl inositide phosphatase that dephosphorylates PIP2 to PIP. In extracts

prepared in the presence o f InsP6, an increase in the level o f PIP2 was reported, consistent

with an inhibition in the activity o f synaptojanin, although no direct evidence for this

inhibition was presented. Dynamin, a molecule involved in scission at the neck o f the

15


budding vesicle is recruited by binding to PIP2, and as such an increase in PIP2 would be

consistent with an increase in endocytosis[52].

The high affinity binding sites for the lipid inositol phosphates and InsP6 have

been mapped to regions containing basic stretches o f amino acids in both AP-2 and P-

arrestin. Mutagenizing these amino acids to acidic or neutral amino acids decreases the

binding o f both lipid and soluble inositols. Furthermore, the mutant constructs were

unable to be recruited into clathrin coated pits in vivo, even though the mutagenized AP-2

and p-arrestin show no decreased ability o f binding clathrin in vitro, suggesting that the

lipid inositol binding was crucial for recruitment o f AP-2 and P-arrestin to the membrane.

InsP6 was also shown to disrupt the interaction between arrestin and clathrin, and arrestin

and rhodopsin in vitro [46], whereas a lipid inositol, PtdIns(3,4,5)P3 stimulates the

interaction; this latter result seems to indicate some specificity o f the interaction, which

considering the charge effect o f these molecules, is important. Surprisingly, the

inhibition o f these interactions is not affected by mutagenesis o f the high affinity binding

site, suggesting the presence o f a second, low affinity binding site. The authors did find

evidence for such a site when expressing fragments o f arrestin and looking for InsP6

binding. The presence o f a second affinity binding site may allow for a degree o f

regulation by InsP6.

16


InsPa in channel regulation.A number o f studies have reported the effect o f InsP6 on channel regulation. In

insulin secreting cells, L-type voltage gated calcium channels are inhibited by the activity

o f serine-threonine protein phosphates (PPases). Larsson et al. [53] showed an inhibition

o f the serine/threonine protein phosphatases 1, 2A, and 3 by InsPs and InsP6. Inhibition

o f the phosphatases leads to an increased inward cellular calcium current, which they

were able to measure by patch clamp experiments. The increased inward current was

also seen when InsS6 was included in the patch clamp pipette, and therefore the current

induced by InsP6 may not be physiologically relevant. Their work also looked at InsP6

levels in insulin secreting cells, and they were able to detect an increase in InsP6 levels

after treatment o f these cells with glucose. Since insulin secreting cells respond to the

treatment with glucose by the exocytosis o f insulin, a process requiring the L-type

calcium channels, they argue that an increase implicates InsP6 in stimulation-secretion

coupling in these cells through their inhibition o f protein phosphatases.

The same group reported the same effects o f InsPg on L-type calcium channels in

the hippocampus[54]. When they evoked convulsive seizures in rat brains by electrical

stimulation, the saw increases in InsP6 mass in a number o f regions o f the brain. The

most responsive region was the hippocampus. Voltage gated calcium channels are

abundant in the hippocampus; so they looked to see i f there was a role o f InsP6 in these

channels. They did find that InsP6 induced a greater calcium current through L-type

voltage gated calcium channels in patch clamp experiments on pyramidal cell cultures

from the hippocampus, just as they found with insulin secreting cells. Furthermore, the

same concentration o f InsP5 did not stimulate the increase calcium current. Interestingly,

17


though, they attributed the increased calcium current to an increase in adenylate cyclase

activity, which they measured in hippocampus cell membrane extracts. They argue then

that InsP6 inhibits the serine/threonine protein phosphates in the hippocampus, while also

stimulating the activity o f protein kinase A by a stimulation o f adenylate cyclase and the

resulting increase in cAMP. The resulting phosphorylation o f the L-type calcium

channels increases the channel open probability and enhances the availability o f the L-

typeCa2+ channel[54], resulting in an increased inward calcium current.

Finally, in plants, abscisic acid is produced in drought conditions and induces

changes in the ion channel activity in the plasmalemma and tonoplasts o f guard cells.

The resulting loss o f potassium, associated anions, and water from the cell leads to the

reduction in turgor and closing o f the stomatal pore. Abscisic acid induces a five-fold

increase o f InsP6 in guard cells[55]. Furthermore, using patch clamping o f guard cells,

InsP6 was shown to inhibit the inward potassium channel. Since neither neo-lnsVe nor

scyllo-InsP6 inhibited this current, two stereoisomers o f wyo-InsPe, it is probably not an

artifact. Thus, InsP6 may be the causative agent by which abscisic acid regulates ion loss

and turgor in the guard cells.

18


H ighly p h osph oryla ted inositols in apoptosis.

Recently, Wilson et al. have described other functions for the Ins(l,3,4)P3 5/6-

kinase. In addition to its role as an inositol phosphate kinase, it can also act as a protein

kinase, and it can phosphorylate the proteins c-Jun, IkB, and p53[56]. Since two o f these

proteins have been implicated in apoptosis, it was possible that expression o f the 5/6-

kinase might be involved in the regulation o f apoptosis. Fas and TNFa are both

members o f the TNF receptor superfamily[57-59], and have received a lot o f attention for

their role in apoptosis. TNFa and Fas ligand can both signal a cell to undergo apoptosis

through the recruitment o f FADD to the receptor; this recruits procaspase 8 which

undergoes autocatalysis producing active caspase 8. Active caspase 8 cleaves the

effector caspase 3, which then cleaves in vivo targets (PARP, etc.), resulting in apoptosis.

The signaling through the TNF receptor differs from that o f the FAS receptor in that the

TNF receptor recruits FADD through the adaptor TRADD, whereas the FAS receptor

recruits it directly. The adaptor TRADD, in addition to recruitment o f FADD, can also

recruit other molecules, including TRAF2 and RIP, although the Fas receptor recruits RIP

to itself without the TRADD. RIP and flip, in turn, recruit and activate the IKK complex,

which results in the ubiquitination and degradation IkB , allowing NFkB to translocate to

the nucleus and mediate transcription. NFkB activation results in the transcription o f

proteins, e.g. FLIP and cIAP, that generally protect against apoptosis. As a result, cell

lines, including HEK293, require the inhibition o f the transcription or translation o f

proteins to sensitize them to the apoptotic effect o f TNFa treatment.

In cell lines containing a tetracycline inducible 5/6-kinase construct, TNFa

induced apoptosis is inhibited relative to vector lines as monitored by caspase 8, caspase

19


3, and PARP cleavage. Pretreatment o f cells with tetracycline did not protect against

FAS ligand induced apoptosis in their hands. Sun et al. [60] could not attribute this

protection from apoptosis to decreased stability o f IkB or increased NFkB activity, which

could have anti-apoptotic effects. Since the apoptotic assays showed differing

sensitivities to TNFa in the presence o f cycloheximide, this would also argue against a

role for NFkB transcription in this protection, because, presumably, protein translation

should be inhibited.

The lack o f an effect on IkB or N F kB suggested that the protective effect o f 5/6-

kinase expression may not be due to its function as a protein kinase. Since the 5/6-

kinase is responsible for the production o f Ins( 1,3,4,6)P4, which is thought to be the

precursor for InsP5 and InsP6, one could attribute the protective role o f over-expression o f

5/6-kinase to an increased amount o f these products. Morrison et al. have found that an

InsP6 kinase, which makes InsP7 from InsP6, can stimulate IFN|3 induced apoptosis in

ovarian carcinoma cells[61]. More work by this group implicated the TNF related

apoptosis inducing ligand (TRAIL) and its receptor (DR5) in the initiation o f apoptosis

by INF|3[62]. TRAIL induced apoptosis is similar to TNFa and Fas induced apoptosis in

a number o f ways, including FADD recruitment and caspase activation[63]. Since the

effect ofInsP6K2 expression may influence levels o f upstream inositol phosphates, an

influence on apoptosis by both the 5/6-kinase and the InsP6K2 may be more than

coincidence. In addition to IFN0 stimulation, the InsP6K2 has been reported to

stimulated apoptosis by a number o f sources o f cellular stress, including H2O2,

staurospaurine, etoposide, and hypoxia[64].

20


Objectives.The many functions attributed to InsP6 remain in question because o f the

difficulty in working with InsPe in vitro. Conclusive evidence will require in vivo work

similar to the work done in yeast. Since many o f the studies reported above were carried

out in mammalian cells, the first step toward conclusive in vivo data requires the cloning

and characterization o f a mammalian 2-kinase. This thesis aims to clone the human 2-

kinase using homologies to the yeast 2-kinase. The cloned gene will be used to produce

protein and to confirm its 2-kinase activity. Human cell lines w ill be produced that either

over-express or deplete 2-kinase. The cell lines will be used to address the mammalian

synthesis pathway o f InsP6 and its role in endocytosis and apoptosis. A mouse 2-kinase

knockout will be produced using an embryonic stem cell line that has a gene trapping

construct inserted into the 2-kinase gene. This mouse will be characterized to address the

necessity o f InsP6 for life.

21


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63. Thorbum, A., Death receptor-induced cell killing. Cell Signal, 2004. 16(2): p. 139-44.

64. Nagata, E., et al., Inositol hexakisphosphate kinase-2, a physiologic mediator o f cell death. J Biol Chem, 2005. 280(2): p. 1634-40.

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Chapter 2 : THE SYNTHESIS OF INOSITOL HEXAKISPHOSPHATE

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T h e J o u r n a l o f B io l o g ic a l C h e m is t r yO 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 277, No. 35, Issue of August 30, pp. 31857-31862, 2002 Printed in l/.S-A.

The Synthesis o f Inositol H exakisphosphateCHARACTERIZATION OF HUMAN INOSITOL 1,3,4,5,6-PENTAKISPHOSPHATE 2-KINASE*

Received for publication, June 8, 2002 Published, JBC Papers in Press, June 25, 2002, DOI 10.1074/jbc.M205682200

J o h n W. V erb sk y t, M onita P . W ilsont, M arina V. K isse leva$ , P h ilip W. M ajerust§, a n d S u sa n R. WenteHIIFrom the $Department o f Internal Medicine and the 11Department o f Cell Biology and Physiology,Washington University School o f Medicine, St. Louis, Missouri 63110

The enzym e(s) responsib le for th e production o f inosito l hexakisphosphate (InsP6) in vertebrate ce lls are unknow n. In fungal cells, a 2-ldnase designated Ip k l is responsib le for syn th esis o f InsP6 by phosphorylation of inosito l 1,3,4,5,6-pentakisphosphate (InsPs). B ased on lim ited conserved sequence m otifs am ong fiv e Ip k l prote in s from different fungal sp ecies, w e h ave id entified a hum an genom ic DNA sequence on chrom osom e 9 that encodes hum an in osito l 1,3,4,5,6-pentakisphosphate 2-kinase (InsPs 2-kinase). R ecom binant hum an enzym e w as produced in Sf21 cells, purified , and show n to catalyze the syn th esis o f InsP6 or phytic acid in v itro . The recom binant protein converted 31 nm ol o f InsPs to InsPefaun/mg o f protein (Vmax). The M ichaelis-M enten constant for InsP6 w as 0.4 /am and for ATP w as 21 /am . Saccharom yce8 cerevisiae lack ing IPK 1 do n ot produce InsP0 and show leth a lity in com bination w ith a g le l m utant a llele. H ere w e show that expression of the hum an InsPs 2-kinase in a yeast ip k l null strain restored the synthesis o f InsP6 and rescu ed the g le l-2 i p k l - 4 leth a l phenotype. N orthern analysis on hum an tissu es show ed expression o f the hum an InsP6 2-kinase mRNA predom inantly in brain, heart, p lacenta, and testis. The iso lation o f th e gene responsib le for InsP6 syn th esis in m amm alian ce lls w ill allow for further stud ies o f the InsPe signaling functions.

Cells amplify and regulate signals through the generation of a variety of second messengers. The inositol polyphosphate family of second messengers has grown in complexity with the discovery of new functions for the soluble, more highly phosphorylated inositols. The common precursor of all soluble inositol phosphates in mammalian cells is Ins(l,4,5)P3, which is produced when phospholipase C cleaves phosphatidylinositol 4,5-bisphosphate yielding InsP3 and diacylglycerol. InsP3 is then metabolized to a num ber of more highly phosphorylated inositol species through the actions of several phosphatases and kinases; the cellular functions of these inositol polyphosphates are beginning to be elucidated (1-4). An understanding

* This work was supported by a Kirsch investigator award from the Steven and Michele Kirsch Foundation (to S. R. W.), grants from the National Institutes of Health (HL16634, HL3289), and National Institutes of Health Training Grant HL07088 (to P. W. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

II Present address: Dept, of Cell and Development Biology, Vanderbilt University Medical Center, 3130A Medical Research Bldg. Ill, Nashville, TN 37232.

§To whom correspondence should be addressed: Dept, of Internal Medicine, Box 8125, 660 S. Euclid Ave., St. Louis, MO 63110. Tel.: 314-362-8801; Fax: 314-362-8826; E-mail: [email protected].

This paper is available on line at http://www.jbc.org

of the enzymes responsible for the production of soluble inositol polyphosphates will be critical to establishing their roles in cellular physiology.

The major inositol pentakisphosphate isomer in eukaryotic cells, inositol 1,3,4,5,6-pentakisphosphate (InsPg),1 is converted to inositol hexakisphosphate (InsP6) by phosphorylation a t the D2 position of the inositol ring. A role for the product of the 2-kinase, InsP6 or phytic acid, has been implicated in many cellular processes. InsP6 has been shown to bind the clathrin assembly proteins AP2 and AP3 (5, 6) and to inhibit clathrin cage assembly in vitro (7, 8). InsP6 inhibits serine and threonine protein phosphatases, which are thought to regulate L- type Ca2+ channels in pancreatic islet cells (9). Nonhomologous DNA end joining of double strand breaks is stim ulated by InsP6 (10) through its binding to the Ku70/80 subunits of DNA-PK (11, 12). Most recently, InsP6 has been suggested to stim ulate endocytosis, possibly by the activation of protein kinase C and inhibition of synaptojanin (13).

The first role for InsP6 in vivo was revealed by studies in the budding yeast Saccharomyces cerevisiae (14), in which the production of InsP6 was shown to be required for efficient messenger RNA (mRNA) export. This is based on the results of a genetic screen for mutations th a t were lethal in combination w ith a temperature-sensitive g lel m utant defective for the essential mRNA export factor, G lel (14). The synthetic lethal screen specifically identified the three gene products th a t together are responsible for converting phosphatidylinositol 4,5- bisphosphate to InsP6 (14, 15). This included the previously characterized P lcl (16) and two inositol polyphosphate kinases, Ipk l and Ipk2 (14, 15). Besides the genetic linkage between m utants defective in InsP6 production and the g lel mRNA export m utant, strains lacking the IPK1 gene alone show a marked accumulation of mRNA in their nuclei (14). This directly implicates the enzyme th a t produces InsP6 in mRNA export.

Our ongoing studies have focused on testing whether the Ip k l protein and InsP6 function are conserved across species. Recent studies have identified IPK1 genes from two other fungi, Schizosaccharomyces pombe and Candida albicans (17). Although functionally conserved, the sequence identity is limited to a few small regions with high homology. However, there is, overall, less than 24% identity in all pairwise combinations across the fungal InsP5 2-kinase domains. This lack of signifi-

1 The abbreviations used are: InsPB, inositol 1,3,4,5,6-pentakisphosphate; InsP6, inositol hexakisphosphate; Ins(l,3,4,5)P4, inositol 1,3,4,5- tetrakisphosphate; Ins(l,3,4,6)P4, inositol 1,3,4,6-tetrakisphosphate; Ins(l,3,4)Pg, inositol 1,3,4-trisphosphate; InsP5 2-kinase, inositol 1,3,4,5,6-pentakisphosphate 2-kinase; 5-FOA, 5 fluoroorotic acid; HPLC, high performance liquid chromatography; DTT, dithiothreitol; EST, expressed sequence tag.

31857

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mailto:[email protected]

http://www.jbc.org

31858 Human InsPs 2-Kinase Ipk l

IP5 2-kinase homology

B.C .o lb ic o n s IPK1 S.pombe IPK1-C S .c e r e v i s i a e IPK1 IPS 2 -k in o se

C. a lb ic a n s IPK1 5 . pombe IPK1-C S . c e r e v is io e IPK1 IP 5 2 -k in a s e

C. a lb ic a n s IPK1 S . pombe IPK1-C S . c e r e v is io e IPK1 IPS 2 -k in o se

C. a lb ic a n s IPK1 S . pombe IPK1-C S . c e r e v is ia e IPK1 IPS 2 -k in a s e

C. a lb ic a n s 1PK1 S . pombe IPK1-C 5 . c e r e v is ia e IPK1 IPS 2 -k in o se

C. a lb ic o n s 1PK1 S . pombe IPK1-C S . c e r e v is ia e IPK1 IPS 2 -k in a s e

C. a lb ic o n s IPK1 S . pombe IPK1-C S . c e r e v is ia e IPK1 IPS 2 -k in a s e

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Fig. 1. Alignment o f the putative InsPs 2-kinases. Yeast InsP5 2-kinases (denoted by their yeast gene name, IPK1) and the human InsP5 2-kinases were assembled using the Clustal program. The human, S. cerevisiae, and C. albicans sequences are full-length, whereas the S. pombe Ipkl sequence is restricted to the C-terminal kinase region. The N-terminal first 374 amino acid residues of S. pombe Ipkl comprise a domain that is unrelated to the other Ipkl proteins and show significant amount of homology to an unrelated protein (see Ives et al. (17)). Black boxes represent identity between at least two amino acids. Boxed residues represent similarity between amino acids. Bars designate Box A and Box B as discussed by Ives et al. (17) and the newly defined Box C and Box D (see "Results”). The asterisk denotes the conserved cysteine residue required for activity of the S. cerevisiae Ipkl.

cant homology initially impeded the discovery of a nonfungal InsP5 2-kinase. In particular, the enzymes for de novo synthesis of InsP6 in mammalian cells are unknown. In this report, we have used a strategy of searching for proteins in data bases th a t share small, nearly identical amino acid sequences found in the fungal kinase domains. We have identified and characterized a protein from hum an cells th a t represents the first nonfungal InsP5 2-kinase.

MATERIALS AND METHODSStrains and Media—Escherichia coli strain DH5a was used as the

bacterial host for all plasmids. Bacterial strains were cultured in LB medium and transformed by standard methods. Yeast strains were grown either in 1% yeast extract and 2% peptone or in synthetic minimal medium plus appropriate amino acids supplemented with 2% glucose. Yeast transformations were completed by the lithium acetate method (18). 5-Fluoroorotic acid (5-FOA) was obtained from United States Biologicals and used at a concentration of 0.5 mg/ml. The S. cerevisiae strains used in this study include: SWY2105 (MATa ade2 ade3 ura3 his3 leu2 trpl canl ipkl:\KANr) (kindly provided by S. Johnson) and SWY1793 (MATa ade2 ade3 ura3 his3 leu2 trpl canl glel-2 ipkl-4 pSW611 (GLE11URA31ADE3)) (14).

Cloning of Human lnsPB 2-Kinase—The gene encoding human InsPs 2-kinase was isolated by nested PCR amplification using a Marathon spleen cDNA library (CLONTECH) as template. First-round PCR primers were chosen 90 bp upstream of the initiator methionine and 25 bp downstream from the stop codon (upstream primer: 5'-AGCTCCGTC- CCCGAGTCCTAGC-3'; downstream primer 5'-AAAGACACTGCAGG- GAAAGAGTTAGACC-3'). This product was then used as a template for second-round PCR using a sense primer encoding a BamHI site followed by the sequence starting from amino acid number 2 (sense, 5'-CGCGGATCCGAAGAGGGGAAGTTGGACGAGAATGAATGG-3';

antisense, 5'-AAGCTTGGGGACCTTGTGGAGAACTAATGTGCAATC- TTCGC-3'). PCR was performed with Taq polymerase (Fisher Scientific) using standard protocols. The PCR product was inserted into a TOPO-TA cloning vector (Invitrogen) using the manufacturer’s instructions.

Analysis of Human InsPs 2-Kinase Expression in Yeast Mutant Strains—The sequence encoding the human InsP5 2-kinase was inserted into a yeast expression vector by replacing the NcoUSnaBl fragment of the plasmid pSW747, which contains most of the GLE1 gene (17) with a cDNA fragment amplified from the TOPO-TA cDNA InsP6 2-kinase clone as template (sense primer, 5'-CATGCCATGGGG- AAGAGGGGAAGTTGGACGAG-3'; antisense primer, S’-CCGGAATT- CGGGAAAGAGTTAGACCTTGTGGAG-3'). The resulting construct places the human InsP5 2-kinase gene behind the GLE1 promoter and the resulting protein is a fusion to the first eight amino acids of Glel. Yeast strains were transformed using standard protocols (18) and grown on synthetic minimal media lacking leucine (19). The ipkl null strains (SWY2105) containing either an empty LEU2/CEN plasmid (pRS315) (20) or the LEU2ICEN plasmid containing human InsP5 2-kinase were labeled with [3H]inositol (30 pCi/ml for 36 h). Soluble inositol phosphates were isolated from the yeast cells as described (17). Equal amounts of radioactivity from the two strains were loaded onto a Partisphere SAX (4.5 x 126 mm) strong anion exchange HPLC column along with a [32P]InsP6 standard (see below) and separated at 1 ml/min with a 0-1.7 m gradient of ammonium phosphate (pH 3.5) over a period of 20 min followed by 30 min at 1.7 M ammonium phosphate. To test complementation of the glel-2 ipkl-4 synthetic lethality, the yeast strain SWY1793 was transformed with the appropriate LEU2 plasmids. The resulting strains were streaked onto 5-FOA plates and grown for 4 days at 23 °C.

Purification of Human lnsPB 2-Kinase from Sf21 Cells—Full-length human InsP6 2-kinase was subcloned into the pBacPAK9 vector (CLONTECH) using the restriction sites BcoRI and Noil. The resulting

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Human InsP5 2-Kinase Ipk l 31859

A 500

400

300

200

100

0 10 20 30 40

empty vector

40 min

B 500

400

PP-IP.300

200

100

10 20 30 40 min

human 1P52 kinaseno vector

Fig. 3. Complementation o f the synthetic lethal phenotype of the glel-2 ip k l-4 double mutant by expression o f the putative human InsPs 2-kinase. The S. cerevisiae glel-2 ipkl-4 double mutant strain harboring a GLEHURA3/CEN plasmid (SWY1793) was transformed with an empty LEU2/CEN plasmid or a LEU2/CEN plasmid containing the putative human InsP5 2-kinase gene. Colonies from the untransformed SWY1793 strain and the two transformed strains were tested for growth on 5-FOA medium at 23 °C for 4 days.

nmol IP6 2 0 min mg

1 /V

1/S2001 0 0

nM ATP

Fig. 2. Expression o f human InsPB 2-kinase rescues InsPfl production in a S. cerevisiae ip k l null strain. The ipkl:\KANr mutant strain (SWY2105) expressing an empty LEU2ICEN plasmid (A) or a LEU2/CEN plasmid containing the gene encoding human InsP6 2-kinase (B) were grown in media containing [3H]inositol. The soluble inositol phosphates were separated by HPLC as described under “Materials and Methods.” 32P-Labeled InsPB and InsP6 were run as internal standards {dashed lines). The labels indicate the identity of the various inositol phosphates.

His- and FLAG-tagged fusion protein was expressed in Sf21 cells using the BacPAK™ baculovirus expression system (CLONTECH). Cells were infected for 3 days, pelleted, and lysed by sonication (3 x 10 s) in 20 ml of lysis buffer (20 mM HEPES, pH 7.6,140 mM NaCl, 10% glycerol, 0.1% Nonidet P-40, o.5 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 2 pM pepstatin, 40 pM iodoacetamide, 20 pM bestatin, and 40 pM leupeptin). The lysate was allowed to bind to M2 anti-FLAG-agarose beads (Sigma) for 1 h, washed with Tris-buffered saline containing 0.5 mM DTT, and eluted with FLAG peptide (0.1 mg/ml, Sigma). The resulting eluate was run on an SDS-polyacrylamide gel and stained with Coomassie Blue, revealing a single band of Mr 50,000 (not shown).

Production of Radiolabeled Inositol Species—[3H]InsP5 was produced starting from [3H]Ins(l,3,4,5)P4 (PerkinElmer Life Sciences). Ins(l,3,4,5)P4 was converted to Ins(l,3,4)P3 by treatment with recombinant OCRL inositol polyphosphate 5-phosphatase purified from Sf9 cells (21). Ins (1,3,4)P3 was converted to Ins(l,3,4,6)P4 using recombinant GST-Ins(l,3,4)P3 5/6-kinase purified from E. coli (22). The OCRL 5-phosphatase in the reaction converted any Ins(l,3,4,5)P4 product of 5/6-kinase back to Ins(l,3,4)P3, resulting in the production of Ins(l,3,4,6)P4 as the only product with nearly 100% conversion of the

nmol IP6 2 0 min mg

.081 /V

.04

1/S

vM IP5Fig. 4. Kinetics studies of human lnsP8 2-kinase. Human InsP5

2-kinase purified from Sf21 cells was assayed as described under “Material and Methods.” Conversion from InsP0 to InsP6 was monitored by HPLC. The results represent three independent experiments (mean ± standard deviation). A , activity assays performed with constant amounts of InsP5 (10 pM) and varying amounts of ATP. B, activity assays performed at constant ATP concentration (500 pM) and varying amounts of InsP6. Insets are the Lineweaver-Burk plots.

substrate. Reaction mixtures contained 2.5 pg of recombinant OCRL, 50 mM HEPES, pH 7.5, 3 mM MgCl 2, 50 pM ATP, 2.5 x 10 6 cpm of [®H]Ins(l,3,4,5)P4t 10 mM phosphocreatine, 800 units of phosphocreatine kinase, 1 mM DTT, and 7.5 pg of GST-5/6-kinase in 1 mj. Reactions

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31860 Human InsPB 2-Kinase Ipk l

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Fig. 5. Northern blot analysis o f human InsPs 2-kinase mRNA levels. The cDNA corresponding to the full-length human InsP6 2-kinase gene was radiolabeled and used to probe a multiple tissue Northern blot (CLONTECH). The resulting band was ~3 kb.

proceeded for 1 to 2 h until all substrate was converted to product. [3H]Ins(l,3,4,6)P4 was converted to [3H]Ins(l,3,4,5,6)P6 by adding 1.5 Ug of recombinant Ins(l,3,4,6)P4 5-kinase and incubating for about 2 h until all substrate was converted to product.2 The identity of [3H]Ins(l,3,4,5,6)PB was confirmed by its co-elution on a Partisphere SAX HPLC column with a [32P]Ins(l,3,4,5,6)PB standard produced from Ins(3,4,5,6)P6 by a chick red blood cell extract (23). This [32P]InsP6 was also used as the substrate to make a [32P]lnsP6 standard using partially purified InsP5 2-kinase from soy bean extracts (24).

Kinetic Analysis of InsP6 2-Kinase Activity—The optimal conditions for assay of FLAG purified human lnsPs 2-kinase were determined to be 50 mM HEPES (pH 7.0), 100 mM KC1,1 mM DTT, 10 mM MgCl2, and 0.1-0.5 pg of human InsP6 2-kinase in 100 pi at 37 °C for 30-60 min. Assays were performed using [3H]InsP5 mixed with unlabeled lnsP6 at a constant specific activity for each assay. The Km for InsP6 was determined at 0.5 mM ATP while varying the InsP6 concentration from 0.25 to 4 pM; the Km for ATP was determined at 10 pM InsP5 while varying the ATP from 5 to 200 pM. Reactions were performed using 0.18 pg of enzyme for 1 h and were stopped with an equal volume of 60 mM ammonium phosphate (pH 3.5) and 2 mM InsP6 to aid in recovery from a Whatman Partisphere SAX strong anion exchange column (4.6 x 125 mm). InsP5 was separated from InsP« at 1 ml/min with a 10-min gradient from 0 to 1.7 m ammonium phosphate (pH 3.5) followed by 30 min at 1.7 m. Kinetic data are the result of three independent experiments. Because the assay regularly converted more than 20% of lnsP6 to InsP6 its concentration was calculated by taking the log average of the starting and ending InsP5 concentrations.

Northern Analysis—The full-length cDNA encoding human InsP6 2-kinase was gel-purified using Qiaex II gel extraction kit and labeled with [a-32P]dCTP using a random hexamer labeling kit (Rediprime II, Amersham Biosciences). The probe was hybridized to a multiple tissue Northern blot following the manufacturer’s instructions (CLONTECH).

RESULTSIdentification and Isolation o f a Gene Encoding the Putative

H um an InsP6 2-Kinase—Based on the Ip k l protein sequence from S . cerevisiae, genes encoding InsP6 2-kinases in S. pombe and C. albicans were identified previously (17). F urther use of these sequences and data base searching algorithms allowed us to identify partial clones th a t likely encode two additional fungal InsP5 2-kinases, one each from Kluyveromyces lactis and Saccharomyces servazzi. The alignment of all the fungal sequences allowed the identification of a number of regions of short though significant homology throughout the sequence (designated Boxes A-D, Figs. 1 and 6) even though the overall level of conservation is limited. U sing the BLAST program for short, nearly exact m atches (NCBI), we found th a t amino acids D(L/V)DLK(P/S)X(E/M) of Box D from the fungal Ipk ls m atched a predicted gene and a num ber of hum an ESTs th a t all mapped to chromosome nine. A consensus from an alignm ent of all the ESTs was compared w ith the fungal Ip k l sequences using the C lustal method (25). The resulting align-

2 S. Chang, A. Miller, Y. Feng, S. Wente, and P. Majerus, manuscript submitted.

m ent is shown in Fig. 1. Although the overall identity was quite low (less th an 20%), the hum an sequence did share significant identity w ith all of the fungal Ip k l cDNA sequences. In particu lar, it was sim ilar to all th ree of the four boxes of homology defined by the fungal Ip k l sequences. The hum an sequence also included the conserved cysteine in Box C, the point m utation of which renders the enzyme inactive in yeast (17). We therefore postulated th a t th is gene m ight be a candidate hum an orthologue of the fungal Ipk l. The full- length cDNA, isolated as described under “M aterials and Methods,” was used for fu rther studies confirming identification of a hum an InsP6 2-kinase.

The Expression o f H um an In$Ps 2-Kinase Complements Yeast M utants Deficient in Endogenous Ip k l—To test for w hether the gene encoding the potential hum an InsPs 2-kinase is functional, the protein was expressed in S. cerevisiae cells. A L E U 2/C E N yeast expression plasmid was constructed with the full-length hum an cDNA under the control of the S. cerevisiae GLE1 promoter. The plasmid expressing the hum an InsP6 2-kinase was then transformed into two m utant S. cerevisiae yeast strains: SWY2105, a null of the yeast IPK1 gene (ipkl::KANr), and SWY1789, the g le l-2 ip k l-4 double m utant synthetic lethal strain. As controls, strains were independently transformed with an empty LE U 2/C E N plasmid. To analyze the production of inositol polyphosphates, the ip k l null strains were grown in the presence of [3H]inositol, the inositol phosphates were isolated from total cell lysates, and samples were separated by HPLC. As previously reported, the ip k l null strain produced no InsP6 and accumulated InsP5 and an undefined PP-InsP4 metabolite ((14); Fig. 2A). In contrast, samples from the cells w ith the putative hum an InsP6 2-kinase plasmid resulted in an elution profile with reduced InsP5 and PP-InsP4 peaks, as well as a new peak th a t ran immediately after the PP-InsP4 peak and co-eluted with the 32P InsP6 standard (Fig. 2B). Thus, the potential hum an InsP5 2-kinase can produce InsP6 in a S. cerevisiae strain lacking the yeast Ipkl.

To test directly w hether the hum an InsP5 2-kinase functionally complements the yeast IPK1 gene, we tested for whether the plasmid expressing the hum an InsP5 2-kinase could rescue the growth defect of a gle l-2 ip k l-4 double m utant. The g le l-2 ip k l-4 double m utant is lethal unless a plasmid harboring either wild type GLE1 or IPK1 is present (14). Using the double m utant strains m aintained by a GLE1 / URA3 / CEN plasmid (SWY1793) and transformed with either the hum an InsP6 2-kinase, LEU2 / CEN, or empty LEU2 / CEN plasmid, growth was tested on media containing 5-FOA. 5-FOA is metabolized into a toxic product by the Ura3 protein, and only strains th a t lack or can lose a plasmid containing the URA3 gene will grow on 5-FOA (26). Thus, growth will happen only if the hum an InsP5 2-kinase can rescue the lethal phenotype and allow cells to grow without the GLE1 / URA3 / CEN plasmid. The strain containing the empty LEU 2/C EN plasmid did not form colonies, reflective of the g le l-2 ip k l-4 lethal phenotype (Fig. 3). However, the strain expressing the potential hum an InsP5 2-kinase gene did grow on 5-FOA, suggesting th a t the hum an protein is functionally conserved. Based on its ability to both produce InsP6 in yeast and rescue the g le l-2 ip k l-4 synthetic lethality, we conclude th a t we have identified the hum an InsP6 2-kinase.

Expression o f H um an InsP5 2-Kinase in Sf21 Cells and Kinetic Studies—Repeated attem pts to express the hum an InsP6 2-kinase in bacterial expression systems yielded mostly insoluble protein; therefore, a baculovirus expression system was employed. The gene encoding the hum an InsP6 2-kinase was inserted into a baculovirus expression vector containing N- term inal polyhistidine and FLAG peptide epitope tags. The expression plasmid was used to transfect Sf21 cells. Three days

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Human InsP5 2-Kinase Ipk l 31861

BOX A BOX C BOX BSC ----------- G R G T ^ ^ ^ ^ P |N Q L L M

h s l» ffiQ H 3 v -A T G X W K Q IS K ^ ^ ^ K sG N K Q R L ^ H qVQdm ^ ^ ■ o G W L ■ ® 3 M Q L I«-H N G K IK R L G H ^^^B f SGTPSRc e ^ ^ ■ O C F F ■ m IL Q IM SC G Q SH FSE M Y D ^^^B f SGNYCR ^ Ba g ^ ^ ■ qgwl ■ yB l h q x im l - q k k s ia k is k ^ ^ H y sg k pv r ^ BZD ■ ■ ■ C G F L B O TM H Q H I*F-SQ G EISK TSEB B B B f SGSKER lcB B a tq

Fig. 6. The alignment of the putative and the cloned InsPe 2-kinases. Sequences were isolated from BLAST searches of EST and genomic data bases and aligned with the fungal and mammalian sequences using the Clustal method. The regions listed correspond to the boxes of homology as discussed in the text and in the legend for Fig. 1. The sequences are as follows: sp, S. pombe, sc, S. cerevisiae; ca, C. albicans; kl, K lactis; ss, S. servassi; hs, Homo sapiens; dm, Drosophila melanogaster, ce, Caenorhabditis elegans; ag, Anopheles gambiae; zm, Zea mays. The D. melanogaster and C. elegans sequences are predicted from genomic sequences, and therefore the boxes near the C terminus could not be predicted with confidence. Those residues that are almost perfectly conserved are shaded.

post-infection, the cells were harvested and lysed, and protein to be the binding partners for InsP6 in the DNA-PK complex,was purified by affinity absorption with M2 anti-FLAG-agarose the yeast Ku proteins do not bind InsP6 under the same con-beads and elution w ith FLAG peptide. The Michaelis-Menten ditions (11). This suggests th a t the role of InsP6 in nonhomolo-kinetic param eters were determined by following the conver- gous end joining DNA repair may be specific to mammaliansion of [3H]InsP5 to InsP6 a t multiple concentrations of InsP6 cells. Whereas previous studies of the role of InsP6 in higherand ATP (Fig. 4). These assays directly confirmed the in vitro eukaryotes have been conducted by in vitro experiments or byactivity of the protein as an InsPs 2-kinase. The apparent Km correlating a specific function w ith an inositol phosphate promeasurements for InsPg and ATP were determined to be 0.43 file, studies directed a t the enzymatic source of InsP6 have beenand 21 pM, respectively, by double reciprocal plots of 1/V versus limited by the lack of a InsP5 2-kinase gene or protein. Here we1/S (Fig. 4, insets). The apparent Vmax was confirmed by both have identified the first nonfungal InsPs 2-kinase.sets of kinetic data to be ~31 nmol of InsP6 formed/min/mg of One strategy th a t we have used previously to analyze the protein. roles of inositol polyphosphates in mammalian cells has been to

Northern Analysis o f InsP6 2-Kinase—The full-length InsP6 deplete cells of inositol metabolites by heterologous expression2-kinase gene was labeled and used to probe a human, multi- 0f an inositol phosphate phosphatase from Salmonella dublintissue Northern blot, showing the ubiquitous expression across SopB (27). When SopB is overexpressed in a tetracycline-de-all tissues w ith high levels of transcript in heart, brain, testis, pendent system in human cells, there are rapid perturbationsand placenta (Fig. 5). jn cellular levels of multiple soluble inositol phosphates. In

Alignments o f the Sequences o f Putative InsP5 2-Kinases— particular, total cellular InsP5 and InsP6 levels are depleted.Using the sequence of hum an InsP5 2-kinase, we searched both Coincidentally, polyadenylated RNA accumulates in the nu-genomic and EST data bases to compile alignments of our deus, and protein synthesis is markedly inhibited, thus sug-cloned and the putative InsP6 2-kinases. Our searches have gesting a conserved role for the InsP5 2-kinase of yeast andyielded a number of putative InsP5 2-kinases across many mammaiian ^ in mRNA export. However, this study couldnonfungal species (Fig. 6). All of the putative InsP5 2-kinases not the relative importance of different inositolcontained most of the boxes of homology defined by the yeast phosphates in the mRNA export mechanism, because severalalignments (Fig. 1). Box A is almost perfectly conserved among inositol p ^ p ^ p h ^ m perturbed. Isolation of the humanall InsP6 2-kinases with the consensus sequence EXKPK Box IngP 2.16naae ^ nQW aIk)W gene deletion studieg inC was initially defined by the sequencei CRXC found m all the which Qnly Insp production is depleted. This is a goal of ourfungal and hum an sequences. The addition of the nonfungal future studiesInsPs 2-kinases expanded the repon of homology of Box C. ^ ^ r t we haye confirmed the conservationexposing the sequence (F/Y)GPLDL, which was found almost . . T o i • c , .. „ ,TT ,_ ® . . «, ’ , „ . . between InsP5 2-kinase of yeast and mammalian cells. We showperfectly represented in all sequences except for the fungi ,, , , T ti m • * ̂ j r- • r„ . Tj- i j o . f, , that human InsP6 2-kinase can complement the deficiencies ofC. albicans, K. lactis, and o. servazzi. Of the other two boxes of T „ , _x. : 0 . .. A ̂ ,, . vv xv , .. i i .i „ InsPfi production m a o . cerevisiae ipk l null m utant stram andhomology, Box B seems less well conserved: when the nonfun- ,, ̂ A A _ ,, , , . - , , . * ,. ’ . , r the synthetic lethality of the g lel-2 ip k l-4 m utant stram. Suchgal homologues are included in the alignment, the residues of , J, , . „ ,, t n o i * ,, . ... . . . , ,.n. broad cross-species complementation is unusual, espeaally whenthe hum an InsPR 2-kinase th a t line up w ith this box shift ̂ , i X*.(compare Fig. 1 with Fig. 6). Box D, which was used to identify * e 0VeraU pr0tein ^ u e n c e homology is so low. This s^ g e s ts

, t T-, o i • ii j , . , that enzymatic activity and InsP6 production alone is sufficientthe hum an InsPfi 2-kinase, is well conserved between human, _ . , . T , , . . _ . , , ,fungal sequences (except S. pombe), com, and Anopheles- be- for the role of Ipk l m mRNA . f P 01̂ . It remains possible thatcause the amino acid sequences from C. elegans and Drosophila P™ tfn-protem interaction motifs and targeting sequences couldare both predicted from genomic sequences, it is possible th a t also be conserved and remains to be discovered,they are lacking the homology found in Box D because of errors 11 ,s notable th a t the apparent Vm„ of hum an InsPs 2-kmasein gene prediction. 18 qvute low a t 30 1111101 of InsPe formed/min/mg of protein.

However the Vmax values for the fungal proteins are also quite DISCUSSION low (17) as is the value for the mammalian Ins(l,3,4)P3 5/6-

Although a role for InsP6 in mRNA export has been clearly kinase (60 nmol of InsP4 formed/min/mg of protein) (22). Theevinced through studies in budding yeast (14), the other pro- la tte r is the first enzyme in the pathway of synthesis of InsP6posed cellular functions of InsP6 require the study of higher in mammalian cells. W hether these low levels of activity resulteukaryotes. For instance, studies of the role of InsP6 on L-type from some as yet unidentified cofactor or post-translationalCa2+ channels were performed on insulin-secreting B cells (9). modification th a t is missing from the in vitro assays remains to In addition, although the mammalian Ku proteins were shown be determined.

31


31862 Human InsPs 2-Kinase Ipk l

The hum an InsP5 2-kinase mRNA is found in most tissues, although to varying degrees. The abundance in the brain is consistent w ith its proposed function in endocytosis and its possible effect on the synaptic protein synaptojanin (13). The mRNA level matches th a t of the InsP3 5/6 kinase (22), both showing robust expression in heart and brain. This is not surprising because they both act in the pathway th a t produces InsP6. The apparent paucity of the InsP6 2-kinase in some tissues is surprising, considering the range of functions proposed for InsP6. Gene deletion experiments will allow us to assess the role of InsP6 in vivo.

The discovery of the putative, nonfungal InsP5 2-kinases will also aid in our understanding of the function of InsP6. The alignment of the fungal, human, and the putative InsP5 2-kinases from com, Drosophila, Anopheles, and C. elegans highlights the most conserved residues among the InsP5 2-kinases; these will provide good targets for mutagenesis to discover which residues are necessary for enzyme activity, its regulation, or possibly its interaction with other proteins. Interestingly, the cysteines in Box C are perfectly conserved among all of the sequences except for com. ESTs of other plants (potato, rose, wheat) also lack the conserved cysteines, even though Box C itself is maintained. It will be interesting to inquire whether there is a connection between this residue substitution and the peculiarities of the plant enzymes. The discovery of a putative p lant InsP6 2-kinase is also interesting because of the desire for a low phytate seed to m itigate the problems associated with high InsP6 levels in seeds, i.e. pollution and m alnutrition (28). The identification of the hum an Ins5P 2-kinase sets the stage for breakthroughs in analyzing the role of InsP6 production in animal development and disease.

Acknowledgments—We thank all of the members of the Majerus and Wente laboratories for reagents and helpful discussions, in particular Sylvia Johnson for the SWY2105 strain and Aimee Miller for aid in isolating soluble inositol phosphates from yeast.

REFERENCES1. Majerus, P. W., Kisseleva, M. V., and Norris, F. A. (1999) J. Biol. Chem. 274,

10669-106722. York, J. D., Guo, S., Odom, A R., Spiegelberg, B. D., and Stolz, L. E. (2001)

Adv. Enzyme Regul. 41, 57-713. Shears, S. B. (2001) Cell. Signal. 1 3 ,151-1584. Irvine, R. F., and Schell, M. J . (2001) Nat. Rev. Mol. Cell. Biol. 2 ,327-3385. Theibert, A. B., Estevez, V. A , Ferris, C. D., Danoff, S. K , Barrow, R. K.,

Prestwich, G. D., and Snyder, S. H. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 3165-3169

6. Chadwick, C. C., Timerman, A. P., Saito, A., Mayrleitner, M., Schindler, H.,and Fleischer, S. (1992) J. Biol. Chem. 267, 3473-3481

7. Norris, F. A., Ungewickell, E., and Majerus, P. W. (1995) J. Biol. Chem. 270,214-217

8. Ye, W., Ali, N., Bembenek, M. E., Shears, S. B., and Lafer, E. M. (1995) J. Biol.Chem. 270,1564-1568

9. Larsson, O., Barker, C. J ., Sjbholm, A., Carlqvist, H., Michell, R. H., Bertorello,A., Nilsson, T., Honkanen, R. E., Mayr, G. W., Zwiller, J., and Berggren, P. O. (1997) Science 278,471-474

10. Hanakahi, L. A., Bartlet-Jones, M., Chappell, C., Pappin, D., and West, S. C.(2000) Cell 102, 721-729

11. Hanakahi, L. A , and West, S. C. (2002) EMBO J. 21, 2038-204412. Ma, Y., and Lieber, M. R. (2002) J. Biol. Chem. 277, 10756-1075913. Hoy, M., Efanov, A. M., Bertorello, A. M., Zaitsev, S. V., Olsen, H. L., Bokvist,

K., Leibiger, B., Leibiger, I. B., Zwiller, J ., Berggren, P. O., and Gromada, J. (2002) Proc. Natl. Acad. Sci. U. S. A 9 0 ,6773-6777

14. York, J. D., Odom, A. R., Murphy, R., Ives, E. B., and Wente, S. R. (1999)Science 285, 96-100

15. Odom, A. R., Stahlberg, A., Wente, S. R., and York, J . D. (2000) Science 287,2026-2029

16. Flick, J., and Thomer, J . (1998) Genetics 148, 33-4717. Ives, E. B., Nichols, J., Wente, S. R., and York, J . D. (2000) J. Biol. Chem. 275,

36575-3658318. Ito, H., Fukuda, Y., Murata, K , and Kimura, A. (1983) J. Bacteriol. 153,

163-16819. Sherman, F., Fink, G. R., and Hicks, J. B. (1986) Methods in Yeast Genetics,

Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY20. Sikorski, R. S., and Heiter, P. (1989) Genetics 1 2 2 ,19-2721. Zhang, X., Jefferson, A. B., Auethavekiat, V., and Mqjerus, P. W. (1995) Proc.

Natl. Acad. Sci. U. S. A. 92,4853-485622. Wilson, M. P., and Msgerus, P. W. (1996) J. Biol. Chem. 271, 11904-1191023. Stephens, L. R., Berrie, C. P., and Irvine, R. F. (1990) Biochem. J. 269, 65-7224. Phillippy, B. Q., Ullah, A. H., and Ehrlich, K. C. (1994) J . Biol. Chem. 269,

28393-2839925. Higgins, D. G., and Sharp, P. M. (1989) Comput. Appl. Biosci. 5,151-15326. Sikorski, R. S., and Boeke, J . D. (1991) Method Enzymol. 194, 302-31827. Feng, Y., Wente, S. R., and Majerus, P. W. (2001) Proc. Natl. Acad. Sci. V. S. A

98,875-87928. Raboy, V. (2002) J. Nutr. 132 ,503S-505S

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Chapter 3 : THE PATHWAY FOR THE PRODUCTION OF INOSITOL HEXAKISPHOSPHATE IN HUMAN CELLS

33


T h e J o u r n a l o f B io l o g ic a l C h e m is t r y

© 2005 by The American Society for Biochemistry and Molecular Biology, Inc.Vol. 280, No. 3, Issue of January 21, pp. 1911-1920, 2005

Printed in U.SA.

The Pathw ay for the Production o f Inositol H exakisphosphate in Human Cells*

Received for publication, October 12, 2004, and in revised form, November 4, 2004 Published, JBC Papers in Press, November 5, 2004, DOI 10.1074/jbc.M411528200

J o h n W. V erbsky$§, S hao-C hun C hang$§, M on ita P . W ilson^, Y asuh iro M ochizukiH, an d P h ilip W. Majerus$||From the $Department o f Internal Medicine, Division o f Hematology, Washington University School o f Medicine,St. Louis, Missouri 63110 and the 11Laboratory o f Molecular Cell Biology, School o f Life Science, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan

The yeast and D rosoph ila pathw ays lead ing to the production o f inosito l hexakisphosphate (InsPe) have been elucidated recently. The in vivo pathw ay in h u m ans has been assum ed to be sim ilar. H ere w e show that overexpression o f Ins(l,3 ,4)P3 5/6-kinase in hum an cell lin es resu lts in an in crease o f inosito l tetrakisphosphate (InsP4) isom ers, inosito l pentak isphosphate (InsP5) and InsP6, w hereas its d ep letion by RNA in terference decreases th e am ounts o f these in osito l phosphates. Expression o f Ins(l,3,4,6)P4 5-kinase does n ot in crease the am ount o f InsPs and InsPe, although its d ep letion does block InsP6 and InsP6 production , show ing that it is necessary for production o f InsP5 and ln sP e. E xpression o f Ins(l,3>4,5,6)Ps 2-kinase in creases th e am ount o f InsPe by d ep leting th e InsPe in the cell, and d ep letion o f 2-ki- n ase decreases th e am ount o f InsP6 and causes an in crease in InsP8. T hese resu lts are con sisten t w ith a path w ay that p roduces InsP6 through th e sequentia l action o f ln s(l,3 ,4)P 3 5/6-kinase, Ins(l,3 ,4 ,6)P4 5-kinase, and lns(l,3,4,5,6)P5 2-kinase to convert Ins(l,3 ,4)P 3 to ln sP 6. Furtherm ore, the ev id en ce im plicates 5/6-kinase as the rate-lim iting enzym e in th is pathw ay.

Ins(l,2,3,4,5,6)P6 (InsPg)1 has been implicated in many cellular processes. It is required for mRNA export from the nucleus in yeast (1) and hum an cells (2). InsP6 binds to the clathrin assembly proteins AP2 and AP180 (3, 4) and inhibits clathrin cage assembly in vitro (5, 6). InsP6 inhibits serine and threonine protein phosphatases, which are thought to regulate L type Ca2+ channels in pancreatic islet cells (7). Nonhomolo- gous DNA end joining of double strand breaks is stim ulated by

* This work was supported by National Institutes of Health GrantsHL 55272, H L16634, and H107088 and by American Heart AssociationGrant AHA-0475014N. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ These authors contributed equally to this work.I To whom correspondence should be addressed: Division of Hematol

ogy, Box 8125, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110. Tel.: 314-362-8839; Fax: 314-362-8826; E-mail: [email protected].

1 The abbreviations used are: InsPfl, inositol hexakisphosphate; HEK, human embryonic kidney; HPLC, high performance liquid chromatography; Ins(l,4)P2, inositol 1,4-bisphosphate; Ins(l,3,4)P3, inositol 1,3,4- trisphosphate; Ins(l,4,5)Pa, inositol 1,4,5-trisphosphate; Ins(l,3,4,5)P4, inositol 1,3,4,5-tetrakisphosphate; Ins(l,3,4,6)P4, inositol 1,3,4,6-tetrakisphosphate; InsP5, inositol 1,3,4,5,6-pentakisphosphate; 2-kinase, inositol 1,3,4,5,6-pentakisphosphate 2-kinase; 5/6-kinase, inositol 1,3,4- trisphosphate 5/6-kinase; 5-kinase, inositol 1,3,4,6-tetrakisphosphate 5-kinase; IPMK, inositol polyphosphate multikinase; RNAi, RNA interference; siRNA, small interfering RNA.


InsP6 through its binding to the Ku70/80 subunits of DNA-PK (8, 9). Most recently, InsP6 has been suggested to stim ulate endocytosis, possibly by the activation of protein kinase C and inhibition of synaptojanin (10). The many roles for InsP6 necessitates an understanding of the pathway leading to its production.

InsP6 is synthesized ultimately from Ins(l,4,5)P3 (Fig. 1). The action of phospholipase C on the lipid phosphatidylinositol (4,5)-bisphosphate yields Ins(l,4,5)P3 and diacylglycerol. Ins(l,4,5)P3 can then be phosphorylated by an Ins(l,4,5)P33-kinase to Ins(l,3,4,5)P4 or dephosphorylated by an inositol polyphosphate 5-phosphatase to Ins(l,4)P2 (11); Ins(l,3,4,5)P4 can be also be dephosphorylated by 5-phosphatases, yielding Ins(l,3,4)P3. When looking a t the formation of the soluble inositol phosphates upon phospholipase C activation in ra t pan- creatoma cells, Menniti et al. (12) saw th a t in addition to the expected increase of the Ins(l,4,5)P3 isomer, Ins(l,3,4)P3 is also increased, as well as Ins(l,3,4,6)P4. They therefore argued th a t in vivo Ins(l,4,5)P3 is phosphorylated to Ins(l,3,4,5)P4 by an Ins(l,4,5)P3 3-kinase, dephosphorylated to Ins(l,3,4)P3 by a5-phosphatase, and phosphorylated to Ins(l,3,4,6)P4 (Fig. 1A). The increase of Ins(l,3,4)P3 was also seen by Wong et al. (13)in WRK-1 cells stim ulated with vasopressin. Wilson and Majerus (14) isolated a cDNA encoding the hum an kinase which catalyzes the conversion of Ins(l,3,4)P3 to Ins(l,3,4,6)P4 and named it Ins(l,3,4)P3 5/6-kinase because of its ability to phosphorylate both the D-5 and D-6 positions of the inositol ring. The product of 5-kinase activity of 5/6-kinase on Ins(l,3,4)P3 is the same as th a t of the Ins(l,4,5)P3 3-kinase, Ins(l,3,4,5)P4, and as such would lead back to Ins(l,3,4)P3 after the action of a 5-phosphatase. The above data would suggest th a t the hum an pathway for the production of InsP6 works through the isomerization of Ins(l,4,5)P3 to Ins(l,3,4)P3, and the sequential phosphorylation of Ins(l,3,4)P3 to Ins(l,3,4,6)P4 by the 5/6 kinase, of Ins(l,3,4,6)P4 to Ins(l,3,4,5,6)P5 by a 5-kinase, and of InsP6 to InsP6 by a 2-kinase (Fig. 1A).

An alternate pathway to the one discussed above was proposed when the yeast pathway was discovered through genetic screens (Fig. IB). In yeast, Ins(l,4,5)P3 is converted directly to InsP6 by the sequential action of two proteins: Ipk2, which produces InsP5 in a two-step phosphorylation of Ins(l,4,5)P3, first to Ins(l,4,5,6)P4 and then to InsP5; and Ipk l, which produces InsP6 (1,15). This pathway differs from th a t proposed by Menniti et al. (12) in th a t no isomerization of Ins(l,4,5)P3 to Ins(l,3,4)P3 is required and in th a t the intermediate InsP4 isomer is not Ins(l,3,4,6)P4 but Ins(l,4,5,6)P4 (15). Deletion of IPK2 causes an increase in Ins(l,4,5)P3, whereas loss of IPK1 causes a loss of InsP6 and an accumulation of InsP6, proving th a t there is no other pathway to go from InsP3 to InsP6 in yeast. In addition, yeast cells do not possess a 5/6-kinase, nor

1911

34



http://www.jbc.org

1912 Pathway o f InsP6 Synthesis

A. Homo sapiens

"5-Ptase

lnsP4 5-K (hIPMK)

lnsP5 2-K

lnsd,3,4,5,6)P5 lns(1,2,3,4.5,6)P6

B. Saccharomyces cerevisiae, Drosophila melanogaster, Arabidopsis thaliana

lns(1l4,5)P3

1PK2 IPK2 IPK1

lns(1,4,5,6)P4 108(1.3.4,5,6)Ps lnsa2,3.4.5.6)P6Fig. 1. Proposed pathways for production of InsPe in human cells (A) o r yeast, Drosophila melanogaster, and Arabidopsis

thaliana (B).

has an Ins(l,3,4)P3 isomer been seen in metabolically labeled yeast extracts. The Drosophila and Arabidopsis homologs of IPK2 have been cloned and can complement the yeast Ipk2 deletion m utant (16, 17). The Drosophila and Arabidopsis homologs first produce Ins(l,4,5,6)P4 from Ins(l,4,5)P3, and then InsP5 from th is InsP4 isomer, as does yeast. Interestingly, when the authors were determining the activities of the Drosophila and Arabidopsis homologs of Ipk2, they found a significant 5-kinase activity on Ins(l,3,4,6)P4. This activity is necessary for the pathway proposed in hum an cells. Because the yeast pathway works through Ins(l,4,5,6)P4, which is already phosphorylated a t the D-5 position, they suggest th a t this activity is not relevant for the production of InsP6 in these organisms.

A cDNA encoding the ra t homolog of the yeast Ipk2 was isolated using a conserved sequence found in inositol phosphate kinases (18). These authors found th a t ra t Ipk2 could catalyze the conversion of Ins(l,4)P2 to Ins(l,4,5)P3, Ins(l,4,5)P3 to Ins(l,3,4,5)P4, Ins(l,3,4,5)P4 to Ins(l,3,4,5,6)PB, and Ins(l,3,4,5,6)P5 to the pyrophosphate PP-InsP4. The extensive kinase activity led them to name the enzyme inositol polyphosphate m ultikinase (IPMK) (19). The 3- and 6-kinase activities m atch those of yeast Ipk2, although the ra t homolog produces Ins(l,3,4,5)P4 first, whereas Ipk2 in yeast produces Ins(l,4,5,6)P4 first. Nonetheless it was inferred th a t the pathway to InsP6 in mammals may not proceed through the Ins(l,3,4)P3 isomer. Yet this work did not include any in vivo description of the activities, no other InsP4 isomers were tested, the reactions were performed for long times with large quantities of enzyme, and no kinetic data were presented.

A cDNA encoding the hum an homolog of Ipk2 was described in two studies th a t came to different conclusions regarding its action (20, 21). Nalaskowski et al. (20) showed th a t the hum an homolog could produce Ins(l,3,4,5)P4 from Ins(l,4,5)P3, and Ins(l,3,4,5,6)P5 from this InsP4 in vitro. Its ability to produce InsPg from InsP3 led them to conclude th a t this was the hum an homolog to the ra t IPMK and yeast Ipk2, although the InsP4 isomer, Ins(l,3,4,5)P4, again differs from th a t in yeast, Arabidopsis, and Drosophila. Chang et al. (21) also isolated the hum an homolog of Ipk2, which they called Ins(l,3,4,6)P4 5-ki-

nase because of its novel substrate specificity. This protein is much more active as a 5-kinase on Ins(l,3,4,6)P4 than as a6-kinase on Ins(l,3,4,5)P4; its catalytic processivity (k ^ ) is 43 times greater for the former isomer. This suggests th a t it phosphorylates Ins(l,3,4,6)P4 to InsP5 in vivo, not Ins(l,3,4,5)P4 to InsPg as suggested by the work of Saiardi et al. (19) and Nalaskowski et al. (20), neither of which tested the Ins(l,3,4,6)P4 isomer. Second, although Chang ef al. (21) found that the human homolog could act as a 3-kinase on Ins(l,4,5)P3 in vitro (its was only 1.6 times less than for Ins(l,3,4,6)P4), it did not complement an Ipk2 deletion m utant in yeast. Therefore, in this in vivo experiment, the human 5-kinase does not convert Ins(l,4,5)P3 to InsP6. This was not a surprising result considering that the human and rat proteins did not make Ins(l,4,5,6)P4 as does yeast, but instead produced Ins(l,3,4,5)P4 from Ins(l,4,5)P3. Chang et al. (21) concluded that the human homolog of yeast Ipk2 and rat IPMK is primarily an Ins(l,3,4,6)P4 5-kinase; we will refer to this protein as such These data suggested that the pathway in human cells does work through Ins(l,3,4)Pa in vivo, not Ins(l,4,5)P3, by the sequential actions of 5/6-kinase to make Ins(l,3,4,6)P4, 5-kinase to make InsPg, and 2-kinase to make InsP6. Here we present in vivo data that confirm this pathway in human cells.

MATERIALS AND METHODS

All chemicals were reagent grade or better. Restriction endonucleases, DNA-modifying enzymes, and general reagents were from Amer- sham Biochemicals, Roche Applied Science, Fisher, Invitrogen, New England Biolabs, Promega Corp., Sigma, and Stratagene unless stated otherwise. PCR was performed using the Pfu DNA polymerase per the protocol from Stratagene. Oligonucleotide synthesis and DNA sequencing were performed by the Protein and Nucleic Acid Chemistry Laboratory, Washington University, St. Louis, MO. Acrylamide solution, Bio-Safe Coomassie Blue stain, and the Bradford protein assay kit used for protein work were purchased from Bio-Rad. A SuperSignal West Pico kit used for detection of Western transfer blots was from Pierce. Radiolabeled inositol phosphates [3H]Ins(l,4,5)P3, [3H]Ins(l,3,4,5), and pHJInsPg were purchased from PerkinElmer Life Sciences and Amer- sham Biosciences.

Strains, Plasmids, and Growth Conditions—Methods for Escherichia coli growth and selection were described previously (22, 23). E. coli strain XL-lBlue (Stratagene) was used as the bacterial host for all plasmids unless stated otherwise. Bacterial strains were cultured in LB (1% tryptone, 0.5% yeast extract, 1% NaCl, pH 7.4) medium supple-

35


Pathway o f InsP6 Synthesis 1913merited with 100 #ig/ml ampicillin where appropriate and were transformed by standard methods (22, 23). All bacterial strains were grown at 37 “C.

Western Blot Analysis—Specified tissue culture cells were treated with trypsin, washed with phosphate-buffered saline, and resuspended in 25 mM HEPES, pH 7.5, 3 mM MgCl2, 150 mM NaCl, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM ATP, and proteinase inhibitors (Complete Mini EDTA-free, Roche Applied Science). Cells were lysed by two freeze-thaw cycles in an ethanol-dry ice bath, and particulate debris was removed by centrifugation at 10,000 x g in an Eppendorf centrifuge at 4 °C. The protein concentration of the clarified lysate was determined using the Bradford assay (Bio-Rad) as per the manufacturer’s protocol. Samples (5 ng) were loaded onto a 10% gel for SDS-PAGE and subsequently electroblotted onto polyvinylidene difluoride membranes (Immo- bilon-P, Millipore). For detection of 5-kinase overexpression, anti-Myc antibody (Cell Signaling) was used, and for a loading control, anti-API antibody was used. The appropriate horseradish peroxidase-conjugated secondary antibody and the SuperSignal West Pico Chemiluminescent Substrate (Pierce) were used to visualize the appropriate band.

5-Kinase Enzyme Activity Assay—For each sample, the clarified cellular lysate was prepared as described above. Enzymatic activity was determined as described previously (21) and summarized below. Enzyme was added to 50 mM HEPES, pH 7.2, 100 mM KC1, 100 /ig/ml bovine serum albumin, 8 mM MgCl2, 5 mM ATP, and 1,000-2,000 cpm of [aH]Ins(l,3,4,6) to a total reaction volume of 50 p.1 at 37 °C for the desired times. The reaction was stopped by the addition of 1 ml of H20, and the sample was loaded onto a 500-pl Dowex column (AG 1-X8 formate, mesh 200-400, Bio-Rad) equilibrated in water. The column was washed eight times with 1 ml of 1M ammonium formate and 0.1 m formic acid to elute the substrate. The product was eluted with 2 ml of2 M ammonium form ate and 0.1 M formic acid and counted in a liquid scintillation counter.

Synthesis of Radiolabeled Inositol Phosphates—The synthesis of the substrate [3H]Ins(l,3,4,6) was described previously (21). 32P*Labeled standards for HPLC analysis were synthesized based on previously published methods for (32P]Ins(l,4,5)P3 (24), [32P]Ins(l,3,4,5) (25), and [32P]Ins(l,3,4,5,6)P5 (26). [32P]Ins(l,2,3,4,5,6)P6 was made with the following modifications to the protocol of Stephens et al. (27); 32Pj-labeled mung beans were homogenized in water, filtered through Whatman No.3 paper, and loaded on a column of Dowex chloride resin (AG 1-X4, 100-200 mesh, Bio-Rad). The unincorporated phosphate was removed by extensive washing with 0.25 N HC1, and the [32P]InsP6 was eluted with 1.5 N HC1. InsP6 was lyophilized multiple times to remove acid. All standards were confirmed by their elution times relative to commercially purchased 3H-labeled inositol phosphates on an Adsorbosphere SAX HPLC column (Alltech) with a linear gradient of 0-100% 1 M ammonium phosphate, pH 3.5, at 1 ml/min over 120 min.

Construction of Stably Transfected Cell Lines Overexpressing Inositol Phosphate Kinases— HEK-293 cells expressing 5/6-kinase were constructed as reported previously (28). The 2-kinase-expressing cells and 5-kinase-expressing cells were constructed using the tetracycline-indu- dble vector pcDNA4/TO (Invitrogen). The 2-kinase gene used previously for expression in Sf9 cells (29) was subcloned into the pcDNA4/TO vector using the BamHI and Notl restriction sites; the resulting construct has 2-kinase preceded by a FLAG tag. The human 5-kinase cDNA was subcloned from the plasmid host pTrcHisA (Invitrogen) (21) by PCR with 5’-CATGCCATGGCAACAGAGCCACCATCCCCCCTC-3' and 5 '-CGGGGTACCAATTGTCTAAAATACTTCGAAGTAC-3 ’ primers. The resulting PCR product has 5-prime Ncol and 3-prime Kpnl restriction sites and is inserted into the Ncol/Kpnl sites of the plasmid pTrcHis2B (Invitrogen) in-frame with a 3-prime Myc tag. A second PCR with 5'-CGGGGTACCTCCGTTATGGCAACAGAGCCACCATCCCC- C-3' and 5'-CGCGGATCCTCAATTCAGATCCTCTTCTGAGATGAG-3' primers using the pTrcHis2B construct of the human 5-kinase generated a product with 5-prime Kpnl and 3-prime BamHI restriction sites flanking the human 5-kinase cDNA in-frame with a 3-prime Myc tag; this construct was ligated into the mammalian expression plasmid pcDNA4/TO. All PCR products were verified by DNA sequencing.

Stable cell lines were constructed by transfecting these constructs into HEK-293 TRex cells (Invitrogen) using Lipofectamine 2000 (Invitrogen), plating serial dilutions of the transfected cells, and selecting them in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum (Tet system approved, Clontech), 2 mM glutamine, 100 units/ml penicillin G, 10 pg/ml streptomycin, 0.25 pg/ml amphotericin B, 5 x̂g/ml blasticidin (Invitrogen), and 0.4 mg/ml Zeocin (Invitrogen) until single colonies were obtained. Stable cell lines were maintained in medium containing Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum (Tet system approved, Clontech), 2 mM glutamine, 5 pg/ml blas

ticidin, and 0.3 mg/ml Zeocin. These gene products were induced with 0.2 jig/ml tetracycline unless otherwise noted. Expression of the human 5-kinase was verified by Western blot analysis using monoclonal antibodies recognizing the Myc epitope (Cell Signaling) and by enzyme activity assays, whereas expression of 2-kinase was verified by Western blot analysis using monoclonal antibodies recognizing the FLAG epitope (Sigma) of 2-kinase.

Gene Silencing of the Inositol Phosphate Kinases—The preannealed siRNA oligonucleotides used for gene silencing of the inositol phosphate kinases were synthesized by Dharmacon RNA Technologies. The oligonucleotides used for the human 5-kinase were sense 5'-GGAUGGAG- UCUCCAGAUUAdTdT-3' and antisense 5'-AAAUCUGGAGACUCCA- UCCdTdT-3', for the luciferase control were sense 5'-CUUACGCUGA- GUACUUCGAdTdT-3' and antisense 5'-UCGAAGUACUCAGCGUAA- GdTdT-3', and for 2-kinase were sense 5'-GAAGACCUCGGAAGAGA- UUdTdT-3' and antisense 5'-UAUCUCUUCCGAGGUCUUCdTdT-3'. Tissue culture cells were grown to 85-95% confluence in 12-well plates with or without tetracycline in standard media and transfected with 1.6 pg of siRNA oligonucleotide using Lipofectamine 2000 per the manufacturer’s protocol. After 6 h, the cells were treated with trypsin, transferred to 6-well plates, and then grown overnight in 5% C02 at 37 °C. A second transfection with 4 pg of siRNA oligonucleotide was performed using Lipofectamine 2000, and after a 6-h incubation, the cells were transferred to 10-cm plates containing media with or without 10 pCi/ml pH]inositol and with or without 0.2 pg/ml tetracycline. After 3 days, cells were harvested for Western blot analysis and HPLC of soluble inositol phosphates. Densitometry of the bands from the Western blot analysis was performed using the Kodak one-dimensional 3.5 software with the Kodak Image Station 440CF. Construction of the 5/6-kinase RNAi stable cell line was described previously (30).

Analysis of Soluble Inositol Phosphates—Cells were grown in complete media containing 10 pCi/ml [3H]inositol for at least 72 h with or without tetracycline induction. Cells were lysed in methanol and 0.5 N HC1 (2:1) and extracted with chloroform. The aqueous phase was separated, dried, and resuspended in water. 32P-Labeled standards were added to each sample. The samples were applied to a 4.6 x 250-mm Adsorbosphere SAX HPLC column and eluted with a linear gradient of0-1.0 M ammonium phosphate, pH 3.5, over 120 min at a flow rate of 1 ml/min, using a modification of the method of Hughes et al. (31). For 2-kinase overexpression or RNAi lines a Whatman Partisphere SAX strong anion exchange column (4.6 X 125 mm) was used running a 30-min gradient from 0 to 1.7 M ammonium phosphate, pH 3.5, followed by a 30-min step of 1.7 m ammonium phosphate, pH 3.5. Radioactivity was measured using the inline detector 0-RAM (IN/US System Inc.), and the identity of the individual inositol phosphates was assigned on the basis of coelution with known standards.

RESULTS

Overexpression o f the Inositol 516-Kinase Results in Elevated Levels o f InsP4, InsPs, and InsP6—Stable HEK-293 cells expressing a tetracycline-inducible 5/6-kinase were labeled with [3H]inositol for 3 -4 days, their soluble inositol phosphates extracted, and equal counts of soluble inositol phosphates were separated on an Adsorbosphere SAX HPLC column. Two sets of labeling experiments were performed, one of log phase growing cells (Fig. 2, A and B ) and another of confluent cells (Fig. 2, C and D). 5/6-Kinase-expressing lines were either uninduced (Fig. 2, A and C) or induced with 0.2 p.g/ml tetracycline (Fig. 2, B and D). Induced cells had elevated levels of Ins(l,3,4,6)P4 (7-fold and 2-fold increase in log phase and confluently growing cells, respectively), and they also showed elevated levels of InsP5 (3-fold and 5-fold, respectively) and InsP6 (1.7-fold and 2-fold, respectively). This suggests th a t the product of the 5/6- kinase reaction, Ins(l,3,4,6)P4, is phosphorylated to InsP6, which is then phosphorylated to InsP6. A large amount of In8(3,4,5,6)P4 is seen in labeling of confluently growing 5/6- kinase-overexpressing cell lines (Fig. 2, C and D), which is not seen consistently in labeling of log phase growing cells (Fig. 2, A and B). Ins(3,4,5,6)P4 is thought to arise from the action of a1-phosphatase on InsP5 (32). I t therefore mirrors the rise in InsP6.

Silencing o f Ins(l,3,4)P3 516-Kinase Decreases InsP5 and InsP6 Levels—Overexpression of 5/6-kinase showed th a t its

36


1914

A

■5c2&

oX

400

300

200

100

0

Pathway of InsP6 Synthesis

HEK-293 5/6-Kinase (- Tetracycline)

u»ns(1,3,4,5,6)P5

k J L

lns(3,4,5,e)p4/lns<1,4,5,6)P4

lnsd,3,4,5)P4lns(1,3,4,6)P4

ln»(1,4,5)Pj 1

20 40 60Time (min)

SO

lns(1,2,3,4,5.6)P6 .

i100 120

B 400 rVS§ 300 -V

* 200 -c

8 100 _X |

CO 10 w

HEK-293 5/6-Kinase (+ Tetracycline)

!n5(1,3,4,5,6)Ps

I

lns(3,4,5,6)P4/ln3(1,4,5,6)P4 |ns(1.3,4,5)P4 lf»S(1,3,4,8)P4

ins(1,4,5)Ps

,4 xv. JL. l_ ^

l l

Jr»s(1,2,3t4,5,6}P6-

L20 40 60

Time (min)80 100 120

HEK-293 5/6-Kinase (- Tetracycline)

z

&COE§OX

oX

400

lns(1,2,3,4,5.6)Pe .300 lns(3,4,5,6)p4/lns(1,4,5.6)P4lns(1,3,4,5)P4

lns(1,3,4,8)P, lns(1,4.5)P3 —i

200

100

400 LueC

0 20 60 80Time (min)

HEK-293 5/6-Kinase (+ Tetracycline)400

lns(1.2,3,4,5,6)Ps .300lns(3,4,5,8)PVIns(1.4.5.6)P4

lns(1,3,4,5)P4 lns(1,3.4,6)P4

lns(1,4,5)Pj - t

200

100

0 20 40 60 80Time (min)

Fig. 2. HPLC profiles of [3H]inositol-labeled HEK-293 cells expressing Ins(l,3,4)Ps 5/6-kinase. A, log phase cells grown without tetracycline. B, log phase cells grown with tetracycline. C, confluent cells grown without tetracycline. D, confluent cells grown with tetracycline. All cells were labeled with [3H] inositol for 3 days, and their soluble inositol phosphates were extracted and separated by Adsorbosphere SAX HPLC. The identity of the labeled peaks was confirmed by internal standards (not shown).

activity was sufficient to produce Ins(l,3,4,6)P4, InsP5) and InsP6. We then asked w hether it was necessary for production of the higher inositol phosphates, using HeLa cells stably

transfected w ith the 5/6-kinase RNAi construct (30). When labeled with [3H]inositol, these cells show a decrease in Ins(l,3,4,6)P4 (to 12.7% of control levels), in InsP5 (to 13%) and

37


Pathway o f InsP6 Synthesis 1915

A.HeLa pSuper

9)■5S£

o01

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800

lns(3,4.S,6)P4/ln*(1,4,5,6)P4 -----ln*(1,3,4.5)P4 ----lns(1(3,4,6)P4 —j

lns(1,4,5)P3 - ^ I

600

400

200

60 80 1000 20 40Time (min)

B. HeLa 5/6-Kinase RNAi800

&3C 6005<DCl 400tnc

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0

rns(3,4,5,6)P4/int(1,4,5,6)P4 (n*(1.3,4.5)P4 in«(1l3,4te)P4

lns(1,3,4}P3

a A . , , 11

lns<1,3,4,5,6)P5

20 40 60Time (min)

JL

lns(1.2,3.4,5.6)Pe

/

80 100

C. 400

300

200

100

5535 40 45

HeLa pSuper HeLa 5/6-Kinase RNAi

Fig. 3. HPLC profiles of [^jinositol-labeled HeLa cells expressing the 5/6-kinase RNAi construct. A, cells lines stably transfected with the pSuper vector alone. B, cell lines stably transfected with the pSuper vector containing an RNAi target sequence to the 5/6-kinase gene. C, expanded portions of chromatograms in A and B corresponding to the area of the InsP3 isomers were lined up according to their internal Ins(l,4,5)P3 standard (not shown). Lines were labeled with [3H]inositol for 3 days, and their soluble inositol phosphates were extracted and separated by Adsorbosphere SAX HPLC.

in InsP6 (to 71%). Ins(3,4,5,6)P4 is also decreased (to 20%), consistent w ith a loss of its source, InsP5. When the portions of the chromatograms corresponding to the InsP3 isomers are lined up by the internal standards and expanded, one clearly sees an increase in Ins(l,3,4)P3 in the 5/6-kinase RNAi cell line compared with the vector cell line (Fig. 3C). Therefore, 5/6- kinase is necessary for production of Ins(l,3,4,6)P4 and also for InsP5 and InsP6.

Cells Expressing the 5-Kinase Show No Increase in InsPs or InsP6 Levels—HEK-293 cells were stably transfected with the cDNA encoding 5-kinase under the regulation of a tetracycline- inducible system (T-REx, Invitrogen) as described under “Materials and Methods.” In the presence of tetracycline, a 10-fold increase in the 5-kinase enzymatic activity was observed, whereas without tetracycline, the activity is sim ilar to th a t of

the control HEK-293 cells stably transfected with vector DNA w ith or w ithout tetracycline (Fig. 4A). Induction of 5-kinase was confirmed by the presence of Myc-tagged protein only in the presence of tetracycline in the stable cell line, whereas no recombinant 5-kinase protein was observed in vector cells (Fig. 4B) or in the uninduced stable cell line.

To determine the effect of overexpression of 5-kinase on soluble inositol phosphates in vivo, 5-kinase stable HEK-293 cells were labeled with 10 p.Ci/ml [3H]inositol for 3 days in the presence or absence of tetracycline. Soluble inositol phosphates were extracted and resolved by Adsorbosphere SAX HPLC. Interestingly, unlike the 5/6-kinase results, overexpression of 5-kinase does not increase the level of InsP6, the product of the 5-kinase enzyme reaction, or InsP6 (Fig. 4D) compared with uninduced cells (Fig. 4C). The soluble inositol phosphate pro-

38


1916

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g> 5000

1 4000 EQ . 3000

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Tetracycline (0.2 pg/ml)

Pathway of InsPg Synthesis

B.

---------- 1 i-----------Vector 5-kinase

kDa50

38

Vector 5-kinaseI I I Tetracycline

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HEK-293 5-kinase (- Tetracycline)1500

1000 tn6(3.4,5,6)P</in6(1,4,5,6)P4 --------ins(1(3,4l5)P4 --------lns(1,3,4l6)P4 j500

800

0 20 40 60 100Time (min)

D. HEK-293 5-kinase (^Tetracycline)

c2&8c3OoX

1200

800 ln9(3,4,5t6)P4/lns(1,4,5,6)P4 lns(1,3,4,5)P4

lns(1,3,4,6)P4400

■ L - , — —

40 v- / > . 60Time (min)0 20 80 100

Fig. 4. Overexpression of human 5-kinase. A, activity assays for phosphorylation of Ins(l,3,4,6)P4 to InsP5 using cell extracts from induced (0.2 pg/ml tetracycline) or uninduced vector or 5-kinase overexpression lines. Each data set represents three independent assays; the error bars are the S.D. for the data set. B, Western blot analysis of 5 pg of protein from cell lysates in A. The recombinant 5-kinase was visualized by anti-Myc primary antibody. C, uninduced 5-kinase stable cells labeled with pH]inositol for 3 days. D, induced 5-kinase stable cells labeled with pH]inositol for 3 days. Soluble inositol phosphates were extracted from cells and separated on an Adsorbosphere SAX HPLC column, and the HPLC chromatograph is shown. The reference locations of isomers of InsP4, InsP6, and lnsP6 were determined by the addition of known 32P-labeIed standards. The radioactivity (cpm) of each sample was normalized to the total cell number.

files are identical to those of the control vector cells w ith or without tetracycline (data not shown), and the lipid inositol phosphate profiles are not altered by the overexpression of 5-kinase (data not shown). These experiments were performed multiple times w ith the same results. Hence, increased 5-kinase activity does not a lter the level of InsPB and InsP6 in HEK-293 cells, suggesting th a t production of InsP5 is limited by the availability of the substrate of 5-kinase, Ins(l,3,4,6)P4,

Gene Silencing o f the 5-Kinase Results in Decreased InsP6 and InsP6 Levels—Because the overexpression experiments did not produce a change in higher inositol phosphates, we performed gene silencing experiments using synthetic siRNA oligonucleotides to confirm th a t the 5-kinase protein was necessary for synthesis of InsP5 and InsP6 in vivo. HEK-293 cells were transfected w ith no siRNA oligonucleotide, siRNA oligo

nucleotide directed against the luciferase gene, or siRNA oligonucleotide directed against the 5-kinase gene. To determine the magnitude of gene silencing, we used the overexpressing 5-kinase HEK-293 stable cell line because we do not have antibodies th a t can consistently detect endogenous levels of 5-kinase. When 5-kinase stable cells induced with tetracycline were transfected w ith a siRNA oligonucleotide directed against the 5-kinase gene, the overexpressed 5-kinase protein is decreased significantly on W estern blot analysis compared with either no siRNA oligonucleotide or siRNA oligonucleotide directed against the luciferase gene (Fig. 5A). To determine the magnitude of gene silencing, we compared the relative optical density of the 5-kinase band on the W estern blot w ith th a t of the loading control API; we observed a —90% decrease in the 5-kinase protein (Fig. 5B). Naive HEK-293 cells were used for

39


Pathway o f InsP6 Synthesis 1917

A. B.

kDa48.837.1

82.2

siRNA Otigo

anti-MycAb (5-kinase)

anti-AP1 Ab

2 0.8 TO1 5 06w SIS 0.4q ce cg Sf 0.2w m8 ~ o

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c. HEK-293

ScIsa2c33X

3000

2000Ins (3,4,6,fl)fVlns(1,4,5,6)P4

lna(1,3,4.5)P4 lns(1,3,4,6)P41000

20 40 60 80 1000 Time (min)

D. HEK-293 siRNA Luciferase

28.

oX

3000

2000Ins (3.4.5,6)P4/lns(1,4,5,6)P4

lns(1,3.4,5)P4 In s(1,3.4.6)P41000

40 60 800 20 100Time (min)

Sc18

oX

3000

2000

1000

HEK-293 SiRNA 5-kinase

\ S

lns(1,3.4,5,6)P5 •

Ins (3,4.5,6)P4/lns<1,4,5,6)p4 lns(1>3,4,S)P4 lns(1,3.4,6)P4

lns(1,2,3,4,5,8)Pe

1 ■A-20 40 _ . . . 60Time (min) 80 100

Fig. 5. Gene silencing of th e hum an 5-kinase. A, Western biot analysis of 5 pg of lysate from induced 5-kinase cells transfected with no siRNA oligonucleotide, the luciferase siRNA oligonucleotide, or 5-kinase siRNA oligonucleotide as described under “Materials and Methods.” The 5-kinase was visualized with anti-Myc antibody, and the loading control was determined by anti-API antibody. B, relative densitometry of bands in A determined by Kodak Image Station 440CF. The optical density ratio of 5-kinase to API was plotted on the y axis. C-E, HPLC profiles of naive HEK-293 cells transfected with no siRNA oligonucleotide (C), the luciferase siRNA oligonucleotide (D), or 5-kinase siRNA oligonucleotide (E), and labeled with (3H]inositol as described under “Materials and Methods.” Soluble inositol phosphates were extracted and separated on an Adsorbosphere SAX HPLC column, and the HPLC chromatograph is shown. The reference locations of isomers of InsP4, InsP6, and InsP6 were determined by the addition of known 32P-labeled standards. The radioactivity (cpm) of each sample was normalized to protein.

labeling experiments so th a t we could determine the effect of silencing the endogenous 5-kinase. These cells were tran sfected with siRNA oligonucleotides, labeled with 10 /xCi/ml [3H]inositol, the soluble inositol phosphates were extracted,

and equal counts were resolved on an Adsorbosphere SAX HPLC column. In cells transfected with 5-kinase siRNA oligonucleotide, the level of InsP6 was decreased to 29.4% and 33.1% compared w ith the level observed in cells mock transfected or

40


1918 Pathway of InsPe Synthesis

g HEK-293 + 2-kinase + tetracydine (0.1 wj/ml)

0 5 10 15 20 25 30

A 1000 HEK"293 + vector ♦tetracycline (0.1 ng/ml)

800

600

400

200

150 5 10 20 25 30

Time (min) Time (min)F ig . 6. HPLC profiles o f [®H]inositol-labeled HEK-293 cells expressing the Ins(l,3,4,5,6)Pa 2-kinase. TRex vector cells (A) or 2-kinase-

expressing cells (B) were grown in the presence of 0.1 jig/mi tetracycline and [3H]inositol for 3 days, and their soluble inositol phosphates were extracted and separated on Partisphere SAX HPLC. The identity of the labeled inositol phosphates was confirmed by 32P-labeled internal standards.

transfected with the luciferase siRNA oligonucleotide, respectively, and the level of InsP6 was decreased to 26.8% and 28.4%, respectively (Fig. 5, C -E ). Similar results were observed in 5-kinase stable cells w ith or without overexpression of the 5-kinase protein and in naive HeLa cells (data not shown). These findings are consistent with 5-kinase functioning in the pathway for the synthesis of InsP5. Interestingly, HEK-293 cells transfected w ith the 5-kinase siRNA oligonucleotide did not accumulate Ins(l,3,4,6)P4, the preferred substrate (21); however, in HeLa cells transfected with the 5-kinase siRNA oligonucleotide, the Ins(l,3,4,6)P4 level increased by 70% (data not shown). Additionally, the peak representing the isomer Ins(3,4,5,6)P4 decreased in both HEK-293 and HeLa cells transfected with 5-kinase siRNA oligonucleotide, which is expected because this isomer is derived from InsP6.

Cells Overexpressing the 2-Kinase Produce lnsP6 by Depleting the Available InsPs—To assess further the in vivo pathways to InsP6, stable HEK-293 cell lines were constructed with a tetracycline-inducible 2-kinase gene and labeled with [3H]- inositol for 4 days in the presence of 0.1 pg/ml tetracycline. The soluble inositol phosphates were extracted, and equal counts were resolved on a Partisphere HPLC column as described under “Materials and Methods.” Compared with the cells stably transfected with an empty vector (Fig. 6A), the soluble inositol phosphate profile of the cells expressing 2-kinase showed a decrease of InsP6 (to 20% of vector lines) and concomitan t increase of InsP6 (1.5-fold) (Fig. 6B). This result was consistent from run to run and from multiple clones. In all cases, expression of 2-kinase decreased the amount of InsP6. Interestingly, InsP6 does not seem to be replenished. The results from the 5-kinase-expressing lines would suggest that this is because of a lack of substrate for 5-kinase. These results confirm th a t 2-kinase is sufficient for production of InsP6 in vivo.

Cells Transfected with a siRNA to the 2-Kinase Block Production o f InsP6 and Accumulate InsPs—2-Kinase is necessary for InsP6 production, as shown by silencing 2-kinase in naive

HEK-293 cells using siRNA oligonucleotides (Fig. IB). As a control we used oligonucleotides to the luciferase gene (Fig. 7A). When these cell lines were labeled w ith [3H]inositol and their soluble inositol phosphates purified and separated by HPLC, there was a decrease in InsP6 relative to the controls by about 50%, confirming th a t 2-kinase is necessary for production of InsP6 in vivo. In addition, these lines had a 4-fold increase of InsP5. Stable cell lines expressing 2-kinase RNAi and labeled w ith [3H]inositol showed a decrease of InsP6 to 30% of controls (data not shown). The increase in InsP5 and relatively smaller decrease of InsP6 suggest th a t cells conserve InsP6. During our attem pts to produce stable knock-outs of 2-kinase using a RNAi expression construct, we were only able to produce a few stable cell lines, none of which had a complete depletion of InsP6 (data not shown); we had a sim ilar experience trying to produce stable RNAi cell lines of 5/6-kinase. Feng et al. (2) showed th a t depleting the higher inositol phosphates by expressing the Salmonella protein, SopB, a phosphatase th a t breaks down inositol phosphates, caused the cells to ball up and stop dividing. Thus it may be difficult to get a more complete silencing of 2-kinase because the effects are toxic to cells.

DISCUSSION

Here we provide in vivo evidence th a t the hum an pathway to the higher inositol phosphates proceeds through the Ins(l,3,4)P3 isomer via the action of 5/6-kinase to produce Ins(l,3,4,6)P4, 5-kinase to produce InsP6, and 2-kinase to produce InsP6. Expression of 5/6-kinase in hum an cells results in an increase of all of the isomers downstream of Ins(l,3,4)P3 in the pathway, Ins(l,3,4,6)P4, InsP5, InsP6, and Ins(3,4,5,6)P4 from InsP5. Silencing 5/6-kinase results in an increase of Ins(l,3,4)Ps and a decrease of all inositol phosphates downstream of Ins(l,3,4)P3. When we express 5-kinase in human cells, we do not detect an increase in InsP5. Silencing 5-kinase by siRNA expression results in a decrease in InsPs and InsP6. Expression of 2-kinase results in an increase in InsP6 and

41


HEK-293 + Luciferase siRNA

800

lns(1,3,4,5,6)Ps -----

1 6 0 0

0 400

200

Time (min)

Pathway o f InsP6 Synthesis

B . HEK-293 + 2-kinase siRNA

1919

20 30 40Time (min)

Fig. 7. HPLC profiles o f [sH]inositol-labeled HEK-293 transfected with an siRNA to lnsP 8 2-kinase. A, cells transfected with luciferase siRNA. B, cells transfected with siRNA to the 2-kinase gene. Both lines were grown in the presence of [3H]inositol for 3 days, and their soluble inositol phosphates were extracted and separated on Partisphere SAX HPLC columns. The identity of the inositol phosphate isomers was determined by a2P-labeled internal standards (not shown).

concomitant loss of InsP5, whereas depleting 2-kinase results in a decrease in lnsP6 and an accumulation of InsP5. These results confirm th a t the pathway proposed for the production of InsP6 in ra t cells by Menniti et al. (12) is the pathway used for the production of InsP6 in hum an cells.

Because overexpression of 5/6-kinase results in an increase in Ins(l,3,4,6)P4, InsP5, and InsP6, whereas overexpression of 5-kinase alone does not resu lt in an increase of InsP5, the production of InsP5 m ust be limited by the availability of substrate, Ins(l,3,4,6)P4. This is supported by expression of 2-kinase, during which InsP6 is produced a t the expense of all of the available InsP6, which is not replenished. Therefore, our data suggest th a t the rate-limiting step in this pathway is the production of Ins(l,3,4,6)P4 by 5/6-kinase. Overexpression of Ins(l,3,4)P3 5/6-kinase showed th a t this protein is sufficient to cause increases in both InsP6 and InsP6. Silencing th is gene resulted in an increase in its substrate, Ins(l,3,4)P3, and a decrease in its product, Ins(l,3,4,6)P3, as expected, and also a decrease in InsP5 and InsP6, showing th a t 5/6-kinase is also necessary for their production in vivo. The results from the RNAi experiments confirm th a t the activities described for 5-kinase and 2-kinase in vitro are likewise necessary for production of InsP6 in vivo.

In yeast, Arabidopsis, and Drosophila, the pathway can operate directly through Ins(l,4,5)P3 without isomerization to Ins(l,3,4)P3, but the evidence argues against such a pathway in hum an cells. F irst, Seeds et al. (16) showed th a t expression of the Drosophila Ipk2 gene from the actin promoter in Drosophila resulted in a 5-fold increase in InsP6 and InsP6, but the expression of 5-kinase, the hum an homolog of Ipk2, does not a lter the levels of InsP5 and InsP6 in hum an cells. Thus, in Drosophila Ipk2 is sufficient for production of InsP6 from Ins(l,4,5)P3, whereas in hum an cell lines 5-kinase is not. Also, silencing of 5/6-kinase should not affect the higher inositol phosphates if its activity were uninvolved in the pathway, but we show th a t silencing 5/6-kinase blocks higher inositol phosphate production. Furthermore, the expression of Drosophila Ipk2 in an ipk2-null yeast strain restores InsPg production, whereas expression of the hum an 5-kinase in yeast does

lns(1,3,4)P3

X vV 5~Ph

'6-kinase

5-phosphatase

5-kinaselns(l,3,4,6)F^ lns(1,3,4,5)FJ ? .....» lns(1,3,4,5,6)P5

- 3-kinase— 5-kinase —

ins(1,3,4,5,6)Ri lns(1,4,5)P3

-2-kinase

ln s f |

F ig . 8. The human inositol phosphate pathway showing the in vivo and in v itro activities reported for 5/6-kinase, 5-kinase, and2-kinase. We did not see 5-kinase phosphorylation of Ins(l,3,4,5)P4 to InsP6 in our overexpression experiments, although it does occur in vitro and is thus questioned.

not (21). Therefore, the hum an 5-kinase cannot convert Ins(l,4,5)P3 to InsP5. These data show th a t these organisms use different pathways.

An examination of the activities of the Ipk2 homologs on Ins(l,4,5)P3 illustrates the differences between the pathways. Yeast, Arabidopsis, and Drosophila produce Ins(l,4,5,6)P4 from Ins(l,4,5)P3, which is then converted to InsP5, whereas the ra t IPMK and the hum an 5-kinase produce Ins(l,3,4,5)P4 in vitro (Fig. 8). The work of Chang et al. (21) showed th a t the human 5-kinase could phosphorylate the D-3 position of Ins(l,4,5,6)P4 when expressed in a m utant yeast stra in th a t produces this InsP4 isomer, but it could not produce InsP6 from Ins(l,4,5)P3 itself in a yeast ipk2 null strain (21). Therefore, the 6-kinase activity described for 5-kinase in vitro is absent in this in vivo experiment, either on Ins(l,3,4,5)P4 or on Ins(l,4,5)P3. Thus, although yeast convert Ins(l,4,5)P3 to Ins(l,4,5,6)P4, the human homolog cannot. Ins(l,4,5,6)P4 is not a substrate for 5-phosphatases, whereas Ins(l,3,4,5)P4 is a substrate. Subse

42


1920 Pathway of InsP6 Synthesis

quent dephosphorylation of Ins(l,3,4,5)P4 would result in Ins(l,3,4)P3. Therefore, the product of any activity th a t 5-kinase would have on Ins(l,4,5)P3 could be converted to Ins(l,3,4)P3 by a 5-phosphatase. This is analogous to the ability of 5/6-kinase to produce Ins(l,3,4,5)P4 from Ins(l,3,4)P3; it too will be degraded by 5-phosphatases back to Ins(l,3,4)P3, which can then be made into Ins(l,3,4,6)P4. This Ins(l,3,4,6)P4 isomer is the committed isomer for higher inositol phosphate synthesis in hum an cells, as is Ins(l,4,5,6)P4 in yeast. The only activities thus described on these two InsP4 isomers are kinase reactions leading to InsP5. Nonetheless, we do not see changes in the inositol phosphate profiles of labeled cells overexpressing 5-kinase, e.g. an increase in Ins(l,3,4,5)P4, Ins(l,3,4)P3, or the downstream products of its phosphorylation, Ins(l,3,4,6)P4, InsPg, or InsP6. Thus we believe th a t the role of 5-kinase in the production of InsP6 is to convert Ins(l,3,4,6)P4 to InsP5.

The formation of Ins(3,4,5,6)P4 seen in these experiments is also relevant to the control of this pathway. Ins(3,4,5,6)P4 is a potent inhibitor of 5/6-kinase (31). When the supply of InsP5 is sufficient for InsP6 production and the metabolic needs of the cell, Ins(3,4,5,6)P4 builds up and inhibits 5/6-kinase. This would presumably shut down the synthesis of InsP6 and, therefore, InsP6. Another observation seen in these experiments is the ability of the cell to preserve InsP6 a t the expense of InsP5. This is seen in the RNAi experiments of 5/6-kinase, and it was seen during numerous 5-kinase siRNA transfections, during which InsPg levels are less than InsP6. Given the num ber of functions attributed to InsP6, this is not a surprising result. When we attem pted to silence 2-kinase and 5/6-kinase using stable transfections of the RNAi constructs, we had difficulty generating 2-kinase and 5/6-kinase RNAi lines, whereas the control RNAi lines were abundant. None of the lines we did generate had complete depletion, suggesting th a t the loss of InsP6 is toxic to cells.

We have determined the in vivo pathway for the production of InsP6 in hum an cells. Although the pathway we have determined differs from the yeast, Drosophila, and Arabidopsis pathways, it does follow the pathway proposed for ra t cells by Menniti et al. (12). Humans may have evolved a pathway that requires 5/6-kinase to produce InsP6, possibly for the greater control of the pathway; in support of this, we believe th a t 5/6-kinase is the rate-limiting enzyme in the hum an pathway. It is interesting th a t the activity of 5-kinase on Ins(l,3,4,6)P4, which is necessary in the hum an pathway, has been described for both the Drosophila and Arabidopsis Ipk2 proteins and thus seems to be conserved. W hat has changed is the predominance of 5/6-kinase in the hum an pathway. Some questions do remain. Is there a 5/6-kinase in Drosophila? Why does Arabidopsis have three copies of 5/6-kinase if they are unnecessary for production of InsPg?

Acknowledgment—We thank Cecil Buchanan for the production of[32P]Ins(l,4,5)Pa.

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43


Chapter 4 : DISRUPTION OF THE MOUSE INOSITOL 1,3,4,5,6 PENTAKISPHOSPHATE 2-KINASE GENE, ASSOCIATED LETHALITY, AND TISSUE DISTRIBUTION OF 2-KINASE EXPRESSION.

44


m m

Disruption of the mouse inositol 1,3,4,5,6- pentakisphosphate 2-kinase gene, associated lethality, and tissue distribution of 2-kinase expressionJo h n V erbsky*, Kory L av inet , a n d Philip W . M ajerus**

•D epartm en t o f Internal Medicine, Division o f Hem atology, and d e p a r tm e n t o f M olecular Biology and Pharmacology, W ashington University School of Medicine, St. Louis, MO 63110

Contributed by Philip W. Majerus, May 3, 2005

Many functions have been suggested for inositol 1,2,3,4,5,6- hexakisphosphate (InsPe), including mRNA export nonhomologous end-joining, endocytosis, and ion channel regulation. However, it remains to be demonstrated that InsPg is necessary for in vivo survival. We previously isolated a cONA encoding the mammalian inositol 1,3,4,5,6-pentakisphosphate (InsPs) 2-kinase (2- kinase), the enzyme that converts InsPs to lnsP6. We used the sequence to search the BayGenomics databases and identify an ES cell line (XA232) that has a gene trap construct embedded in the 2-kinase gene. We obtained a mouse from this line, produced heterozygotes, and confirmed that the heterozygotes contain the trapping construct and have diminished 2-kinase activity. Breeding the XA232 heterozygotes produced no homozygous offspring; thus, loss of 2-kinase is lethal in mice. Dissections of embryonic day-8.5 uteri yielded no homozygous embryos; thus, the mice die before day 8.5 postcoitum. The gene trap construct contains a p-galactosidase/neomycin reporter gene, allowing us to stain heterozygotes for p-galactosidase to determine tissue-specific expression of 2-kinase protein. 2-kinase is expressed in the hippocampus, the cortex, the Purkinje layer of the cerebellum in the brain, in cardiomyocytes, and in the testes of adult mice. At day 9.5 postcoitum, 2-kinase was expressed in the notochord, the ventricular layer of the neural tube, and the myotome of the somites. Intense staining was also seen in the yolk sac, suggesting that InsPg is necessary for yolk sac development or function. Furthermore, failure of yolk sac development or function is consistent with the early lethality of 2-kinase embryos.

em bryonic lethal | g ene dele tion | inositol signals

M any functions of inositol 1,2,3,4,5,6-hexakisphosphate (InsPe) have been discovered since the time when it was

thought to be merely a storage form of phosphate in plants (1). The first inositol 1,3,4,5,6-pentakisphosphate (InsPs) 2-kinase (2-kinase) gene, encoding the enzyme that produces InsPs, was isolated in a screen for yeast defective in mRNA export from the nucleus (2); this function was also associated with InsPs in mammalian cells (3). We cloned the 2-kinase gene from humans shortly thereafter (4). InsPs has been implicated in a number of physiological functions by in vitro work, including nonhomologous end-joining of double-strand DNA breaks (5), endocytosis (6, 7), and ion channel regulation (8, 9).

The many functions with which InsPs has been associated suggest that the 2-kinase may be necessary for life. This suggestion is implied in other work we have done with tissue culture lines. Using a stably transfected RNAi construct to the 2-kinase gene, we were able to deplete InsPs in HEK293 lines (unpublished data). We were only able to obtain a few clones, and even in these clones, some InsPs was retained, suggesting that a complete loss of InsPs is lethal. O ther evidence for this lethality came from tetracycline-inducible expression of the inositol phosphate phosphatase SopB (3) in HEK293 cells, which breaks down InsPs. These cells stopped dividing and detached from the tissue culture plate.

We now describe a disruption of the 2-kinase gene in mice to further assess its importance. Two strategies are used to generate gene deletions in mice (10): (/) knockout protocols, which remove part of a gene by homologous recombination, rendering the gene nonfunctional, and (h) gene trapping protocols, which randomly insert a trapping construct downstream of an exon, resulting in a fusion of the gene product to the reporter protein encoded for in the construct. The trapping construct contains a gene encoding a /3-galactosidase/neomycin-resistance fusion protein that will be driven by the endogenous promoter of the trapped gene. Randomly generated knockout lines can be produced by introducing the trapping construct into ES cells and selecting for resistance to neomycin. The goal is to produce knockouts of all mouse genes (11) expressed in ES cells. The presence of /3-galactosidase allows the assessment of the expression level of the protein in mouse tissues; protein levels are thus correlated to the intensity of the /3-galactosidase expression. A number of consortiums are currently producing ES cell lines that contain trapped exons, and they are depositing the sequences in searchable databases.

Here, we describe the mouse derived from the ES cell line XA232 from BayGenomics (which can be accessed at h ttp :// baygenomics.ucsf.edu), which has trapped exon 1 of the 2-kinase gene. We show that mouse embryonic fibroblasts (MEFs) heterozygous for the construct show decreased 2-kinase activity and that no mice homozygous for the construct are found from matings of heterozygotes. Therefore, loss of the 2-kinase is lethal. We also used /3-galactosidase staining to describe areas of prominent expression in both adult and embryonic mouse tissues.

Materials and MethodsReagents. All chemicals were reagent grade or better. Restriction endonucleases, DNA modifying enzymes, and general reagents were from Amersham Pharmacia, Fisher, Invitrogen, New England Biolabs, Sigma, and Stratagene unless stated otherwise. PCR was performed by using TaqDNA polymerase from Invitrogen. Oligonucleotide synthesis and DNA sequencing were performed by the Protein and Nucleic Acid Chemistry Laboratory, Washington University. DMEM was from Fisher, and inositol-free DMEM was produced by the Tissue Culture Support Center, Washington University.

Mice. A male chimeric mouse generated from the ES cell line XA232 was obtained from BayGenomics. The ES cell line XA232 was generated by using a gene trap protocol with the

Abbreviations: MEF, mouse embryonic fibroblast; InsPs, inositol 1,3,4,5,6-pentakisphosphate; InsPs, inositol 1,2,3,4,5,6-hexakisphosphate; 2-kinase, InsPs 2-kinase; En, embryonic day n; PP-lnsPs, disphosphoinositol tetrakisphosphate.

*To whom correspondence should be addressed at: Department of Internal Medicine, Washington University School of Medicine, P.O. Box 8125, 660 South Euclid Avenue, St. Louis, MO 63110. E-mail: phil#im.wustl.edu.

O 2005 by The National Academy of Sciences of the USA

8448-8453 j PNAS | Ju n e 14 ,2005 j vol. 102 j no. 24 w w w .pnas.org /cg i/do i/10 .1073/pnas.0503656102

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http://www.pnas.org/cgi/doi/10.1073/pnas.0503656102

Ao 10 Kilobases from start codon

Ex2 ^ \ E x 3 ^ - \ E x 4

(inti for8359) —-4=(int2rev7

Ex 1 B-galactosidasa neo Ex 2 Ex 3

(int1for8359)* ♦ (rev2long)

B

Exon 1 B-galactosidase neo

B-galactosidase neo vector sequence

Q Mouse# 165 166int1fbr8359/int2rev ~ r z z t J 3 H I 3 B l intifor8359/rev2tong (+/-) (+/+)

Fig. 1. The m ouse 2-kinase locus on chrom osom e 13. (A) The genom ic sequence on chrom osom e 13 corresponding to th e 2-kinase locus w as ob tained from th e National Center for Biotechnology inform ation, and th e exons w ere determ ined by com parison to th e cDNA sequence. Num bering for th e region s tarts a t codon 1 o f th e 2-kinase gene. The prim ers used for screening, th e relative insertion site o f th e pGTOpfs construct, and th e resulting fusion pro te in a re indicated. (B) A m ap of th e trapp ing construct, pGTOpfs. En2, engrailed 2 intron; neo, neomycin-resistance gene. (O PCR genotyping on tw o offspring from m atings o f heterozygotes; th e prim er pairs used to g e n e ra te each PCR product are indicated.

trapping construct pGTOpfs containing the intron from the engrailed 2 gene upstream of the gene encoding the /3-galactosidase/neom ycin-resistance fusion p ro te in (see h t tp : / / baygenomics.ucsf.edu). C57 BL6 mice are from The Jackson Laboratory.

PCR-Based Genotyping of Mice. Genotype of mice was determined by using two parallel PCRs, both using the same forward primer from intron 1, intlfor8359 (5'-AGAAGCCTGAGGAGCAT- GTTCGAT-3'), but different reverse primers; one primes in the second intron of the 2-kinase gene, int2rev, (5'-CATTTC- CCTATCCTGGGCAGCA-3'), whereas the second primes within the /3-galactosidase gene of the trapping construct, rev21ong (5'-GACGACAGTATCGGCCTCAGGAAGATCG- CACTC-3'). PCR was carried out by using standard techniques. PCR conditions were 30 cycles at 95°C for 1 min, 58°C for 1 min, and 72°C for 2 min. DNA was prepared from 0.5 cm of clipped tail from 15-day-old mice and purified by the Puregene Genomic DNA Purification Kit (Gentra Systems) per the manufacturer’s protocols. One microliter of final DNA solution was used per 50 p.1 of PCR. Reaction products were electrophoresed on 1% agarose gels and stained with ethidium bromide.

Generation and Maintenance of MEFs. Embryonic day (E) 12embryos were used to generate MEFs. Embryos were dissected from the uterus of pregnant mice, separated from their yolk sac, and then homogenized with a razor blade in a plOO tissue culture plate. Homogenized embryos were aspirated 10 times in 10 ml of DMEM supplemented with 10% FBS/100 units/ml penicillin/ 100 fig/ml streptomycin; MEF lines were maintained in the same media. MEFs were split 1:2 by using a solution of PBS plus 0.5 mM EDTA to release cells from tissue culture plates. Genotyping was performed on the MEFs as described for mouse tails.

FHJInositol Labeling of Heterozygote and WT MEFs. MEFs were metabolically labeled with [3H]inositol (ARC, St. Louis) for 4 days. MEF lines heterozygous for the trapping construct or M EF lines from W T litterm ates were plated at =“30% con-

Verbsky e f a/.

fluency in a solution of 80% inositol-free DM EM /20% DM EM supplemented with 10% FBS and 3 /xCi/ml (1 Ci = 37 GBq) [3H]inositol for 2 days, split 1:2 in the same conditions, and allowed to grow for 2 more days. The cells from two plOO tissue culture plates were lysed in m ethanol/0.5 M HC1 (2:1) and extracted with chloroform. The aqueous phase was separated, dried, and resuspended in water. The soluble inositol phosphates were separated on a W hatman Partisphere SAX strong anion exchange HPLC column (4.6 x 125 mm) running a 20-min gradient from 0 to 1.7 M ammonium phosphate (pH 3.5), followed by a 30-min step of 1.7 M ammonium phosphate (pH 3.5). Radioactivity was measured by using an inline detector, /3-RAM (IN /U S Systems, Tampa, FL), and the identity of the individual inositol phosphates was assigned on the basis of elution times of 32PO,)-labeled internal standards.

/3-Galactosidase Staining of Mouse Tissues and Mouse Embryos./3-Galactosidase staining was performed as described in ref. 12. Briefly, tissues were dissected in PBS and fixed in LacZ fixative (0.2% glutaraldehyde/0.1 M MgCl2/5 mM EGTA in PBS) overnight at 4°C. Tissues were then washed in LacZ wash buffer (2 mM MgCl2/0.01% sodium deoxycholate/1% Nonidet P-40 in PBS). After washing, tissues were sectioned on a cryotome at 12-p.m thickness. Sections were then stained in X-Gal staining solution (1 mg/ml X-Gal/5 mM potassium ferrocyanide/5 mM potassium ferricyanide in LacZ wash buffer) until appropriate color development. After color development, tissue sections were washed, counterstained with hematoxylin and eosin, and mounted. Whole-mount staining was performed as above except that tissues were stained immediately after fixation and washing.

ResultsES Cell Line XA232 from a Gene Trap Screen at BayGenomics Successfully Targets the Mouse 2-Kinase. A search of the BayGenomics database (http://baygenomics.ucsf.edu) to find a potential source for a 2-kinase knockout mouse yielded one entry, XA232, which contained the sequence of the first exon of the 2-kinase gene. Sequences in this database are generated by sequencing of

PNAS | Ju n e 14 ,2005 | vol. 102 | no. 24 | 8449

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http://baygenomics.ucsf.edu

Table 1. Genotypes of heterozygous matingsS tag e + / + + / -

AdultE12.5E8.5

6647

1295

15

5'-RACE products from mRNA of ES cell lines that contain insertions of the trapping construct pGTOpfs. We obtained a chimeric male mouse generated from the XA232 ES cell line. We crossed this mouse to C57 BL6 females to generate heterozygous mice containing one copy of the potentially trapped 2-kinase gene.

The entry of the BayGenomics database indicated that the trapping construct had inserted into the first intron of the 2-kinase gene. The genomic sequence of the mouse 2-kinase gene was obtained from the National Center for Biotechnology Information. Fig. 1 depicts a map of the 2-kinase locus on mouse chromosome 13 that was generated by comparing the sequence we obtained to the cDNA sequence of the mouse 2-kinase gene. The first intron of the 2-kinase gene is 9.8 kb. We initially used a neomycin PCR to determine genotype until we developed a PCR screen for the trapping construct. We found the location of the trapping construct in the intron by screening the entire first intron of the 2-kinase gene by PCR on neomycin-positive mice, using forward primers spaced 500-1,000 bp apart and reverse primers from various locations within the trapping construct. One PCR primer pair generated a consistently positive reaction. This 1.2-kb reaction product was cloned and sequenced. The trapping construct was confirmed to have inserted ^ 9 kb into intron 1 of the 2-kinase gene. The same forward primer was paired with a reverse primer located just downstream of the second exon of the 2-kinase gene (Fig. 1) to generate a 1.5-kb product. These two reactions made up the screening method used to genotype 2-kinase mice (Fig. 1C). Of the first 100 mice, every neomycin-positive mouse was also positive for the intlfor8359/rev21ong PCR product, confirming that the XA232

line has only one insertion of the pGTOpfs construct. Furthermore, we confirmed by RT-PCR on brain mRNA that the insertion results in a fusion between the /3-galactosidase gene and exon 1 of the 2-kinase gene (data not shown), thus confirming the 5 '-RACE results of BayGenomics.

The Insertion of the pGTOpfs Construct in the XA232 ES Cell Line Is Embryonic Lethal. The confirmed heterozygote mice were mated to generate homozygous mice. Tail clippings from 195 offspring from the heterozygous matings were screened by PCR, with none showing the 1.2-kb product alone; therefore, none was found to be homozygous for the construct (Table 1). The ratio of heterozygous mice to W T mice was 2:1, exactly as expected for a homozygous lethal mutant. When we preformed dissections of the uteri of pregnant mice on day 12 postcoitum to generate MEFs, we noticed that approximately one in four placentae did not contain embryos but did contain blood and debris, which were likely resorbed embryos. Furthermore, no homozygous mutant mice were found by PCR screening of the M EF lines produced. We looked earlier in embryogenesis and performed dissections at day 8.5 and 9.5. Even by day 8.5, all of the uteri from pregnant heterozygous mice contained resorbed embryos. The empty placentae did contain debris, suggesting that the embryos died shortly before day 8.5, but we were unable to isolate them from the surrounding placental tissue for genotyping. Genotyping of the yolk sac from day-8.5 embryos was conducted (Table 1). No embryos were found homozygous for the trapping construct, and the ratio of heterozygotes to WT embryos was again 2:1. Thus, homozygosity for the trapping construct inserted in the 2-kinase gene is lethal.

MEFs Generated from Mice Heterozygous for the Trapping Construct Show Decreased 2-Kinase Activity. We attem pted to confirm that the insertion of the trapping construct had disrupted the 2-kinase gene by measuring 2-kinase activity in crude extracts of mouse brains, but we were unable to do so. Therefore, we made MEFs from day-12 embryos to label the cells with [3H]inositol and com pare the soluble inositol phosphate pro-

B _ MEF line #3 (+•/+) w 2000 ......

^ MEF line#3 #8

>ntffor83S9ArCfriv Q M | | H U

*nio'8359frt!v2tong(p.3*l> { + /+ ) (+ /-)

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-Ins(1,3,4.5,6)p5

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PP-lnsP,iPt i

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Fig. 2. [3H]lnositol labeling o f soluble inositol phosphates o f WT or heterozygous MEFs. (A) Genotyping o f th e tw o MEF lines g en e ra ted from E12 embryos from o ne litter used fo r [3H]inositol labeling. G enotype is indicated under gel. (B) MEF lines w ere labeled fo r 4 days in th e presence o f [3H]inositol, th eir soluble inositol phosphates w ere extracted, and equal counts w ere run on a Partisphere HPLC column. Identities o f th e inositol phosphates a re indicated. (Left) MEFs from WT embryos. (R ight) MEFs from heterozygous litterm ates.

8450 | w w w .pnas.org /cg i/do i/10 .1073/pnas.0503656102 Verbsky e t al.

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Es

files (Fig. 2). Heterozygous and W T MEFs were labeled with [3H]inositol for 4 days, the soluble inositol phosphates were extracted, and equal inositol phosphate counts were loaded on a Partisphere HPLC column. Compared with W T MEFs, heterozygotes have a labeling profile consistent with decreased 2-kinase activity. InsPs and disphosphoinositol tetrakisphosphate (PP-InsP,t) levels are markedly elevated in heterozygous MEFs (InsPs levels are 4-fold higher, and PP-InsP4 levels are 2-fold higher), whereas InsP6 levels are almost the same (1,220 cpm for heterozygous mice vs. 1,400 cpm for W T). We conclude that insertion of the trapping construct in the XA232 line disrupts the activity o f one copy of the 2-kinase gene, causing a delay in the conversion of InsPs to InsP6 and therefore an accumulation of the substrate of 2-kinase, InsP5.

Tissue-Dependent Expression of the 2-Kinase in Adults and Embryos.Gene trapping with /3-galactosidase enables rapid identification of cell types and tissues that express the trapped gene. Insertion of /3-galactosidase into intron 1 of the 2-kinase gene produces an exon l-/3-galactosidase fusion protein product. This product is expressed under control of the endogenous

2-kinase prom oter, and, thus, /3-galactosidase staining of heterozygous mice (carrying one copy of the trapped gene) offers a sensitive and accurate method to characterize the expression pattern of 2-kinase.

Northern blot analysis of adult RNAs has shown that the 2-kinase transcript is most prominently expressed in brain, heart, and testes (4). To determine which cell types express 2-kinase, heterozygous adult tissues were stained for /3-galactosidase activity (Fig. 3 A -D ). Staining of sections through the brain revealed specific expression throughout the hippocampus (CA1, CA2, CA3, and dentate gyrus) (Fig. 314), inner layers of the cerebral cortex (Fig. 3B), and Purkinje cells of the cerebellum (Fig. 3C). Sections of the heart (Fig. 3D) demonstrated punctate staining in cardiomyocytes but not in interstitial cells, blood vessels, or valves. Testis staining was diffuse throughout (not shown).

Because homozygous animals presumably die during embryonic development, we examined sites of 2-kinase expression during embryogenesis to gain insight into probable causes of death (Fig. 3 E-K ). Staining of E9.0 heterozygous animals uncovered expression of 2-kinase in multiple tissues. Whole-

* J ' w HB / / FB

Fig. 3. 2-kinase expression in a du lt and embryonic mice. 0-galactosidase staining o f heterozygous adu lt and embryonic tissues was perform ed to characterize expression p a tte rns o f 2-kinase. (A-D) In ad u lt tissues, prom inen t 0-galactosidase activity w as d e tec ted in th e brain (A -O and heart (D). Cryosections o f adult brains uncovered specific staining th ro u g h o u t th e hippocam pus (A), inner layers o f th e cerebral cortex (B), and Purkinje cells of th e cerebellum (O- (D) Cryosections th ro u g h th e heart uncovered 0-galactosidase staining in cardiomyocytes but n o t in interstitial cells. (E-K) Staining of E9.0 embryos revealed expression o f 2-kinase in m ultiple tissues. Intense staining was de tec ted in th e neural tu b e, notochord, embryonic brain, somites, heart, and yolk sac. (E) W hole-m ount staining showing expression in regions corresponding to th e neural tu b e /n o to ch o rd (black arrow head) and som ites (white asterisk). (F) T ransverse section th ro u g h th e tru n k show ing staining in th e neural tu b e, notochord, and regions o f th e somite. W ithin th e neural tu b e , staining is seen in th e ventricular zone and in m igrating neuroblasts (white arrow). (G) Frontal section dem onstrating specific staining in th e ventricular zone o f th e neural tu b e and m yotom e of th e somite. (H) /3-Galactosidase staining is present th ro u g h o u t th e ventricular zone o f th e em bryonic brain. (/ and S) In th e yolk sac, staining is de tec ted in epithelial and endothelial cells but is ab sen t from blood islands (J). (K) Sagittal section th ro u g h th e h ea rt showing staining in th e atrial and ventricular chambers. (Inset) W ithin th e ventricle, staining is seen in th e m yocardium (white asterisks) and endocardium (white arrow head). Bl, blood island; BV, blood vessel; DG, d e n ta te gyrus; dm , derm om yotom e; FB, forebrain; HB, hindbrain; MB, m idbrain; My, m yotom e; NC, notochord; NT, neural tub e; sc, sclerotom e; So, somite.

Verbsky etal. PNAS | June 14, 2005 | vol. 102 j no. 24 | 8451

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s2 1

©lns(1,4,5)Pj

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lns(1,4,5,6)P4

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Fig. 4. Pathw ays for synthesis o f lnsP6 in d iffe re n t organism s. (A) Hom o sapiens. (B) Saccharom yces cerevis'tae. Drosophila m elanogaster. and Arabidopsis thaiiana. A d ap ted from ref. 13.

mount staining of E9.0 embryos demonstrated robust staining in regions corresponding to the neural tube, notochord, and somites (Fig. 3E). Transverse sections through the neural tube showed staining in the ventricular zone and in migrating neuroblasts (Fig. 3F). Intense /3-galactosidase activity was also present in the notochord and specific regions of the somite. To further examine expression in the somite, frontal sections through the trunk were stained. These frontal sections contained strong staining in the myotome (future skeletal muscle) of the somite. In addition to the neural tube, staining was present in the ventricular zone and migrating neuroblasts throughout the embryonic brain.

Prominent /3-galactosidase activity was also detected in the yolk sac and embryonic heart. Within the yolk sac, staining was present in both the epithelial and endothelial layers (blood vessels) but absent from the blood islands. In the heart, staining was detected in both the atrial and ventricular chambers. High magnification of the ventricular myocardium revealed staining in both the endocardium and myocardium. Other sites of expression included the cardinal vein, aorta, digestive tract, and pharyngeal arches (data not shown).

DiscussionHere, we describe the mouse line generated from the XA232 ES cell line from a BayGenomics gene trap screen. We show that the pGTOpfs construct has successfully inserted into intron 1 of the mouse 2-kinase gene on chromosome 13, where it splices to exon 1 of the 2-kinase gene and disrupts its activity; MEFs that are heterozygous for the construct show decreased 2-kinase activity, as shown by a partial block in the conversion of InsPs to InsPg. Furthermore, mice homozygous for this construct die before E8.5, indicating that loss of 2-kinase is lethal in mice.

The accumulation of PP-InsP4 in these lines is consistent with the result from the yeast 2-kinase deletions (2) and is thought to be derived from the excess InsPs. The InsPg levels were equal in the two labelings. We have noted in studies of the human 2-kinase that cells seek to maintain InsPg levels (13), consistent with these results. O ther isomers in the area of InsP2 were also increased, but the columns were run without

internal standards of InsP2 isomers, so the identity of the isomers was not confirmed.

The eukaryotic pathway for the production of InsPg has been worked out by a number of laboratories in yeast and in tissue culture cell lines (Fig. 4) (2, 13, 14). InsPg is produced by phosphorylation of InsPs at the D2 position by 2-kinase (Fig. 4). InsPs is produced from the phosphorylation of an InsP4 isomer by a single enzyme named IPK2, IPMK, or Ins(l,3,4,6) 5-kinase, depending on which laboratory cloned the gene or the organism involved. Some debate remains as to the identity of the InsP4 isomer on which this protein works, although the identity of the protein itself is not in question, because these proteins are homologous. There is general agreement with respect to the substrate of 2-kinase and, thus, the lone pathway to InsPg from InsPs. Because only one enzyme makes InsPs (IPK2/IPM K/5- kinase), loss of this enzyme also depletes InsP6 levels (13).

The companion paper describes work by Frederick et al. (15) in which the researchers took a targeted approach to knocking out the IPK2 (5-kinase) gene. Because it is responsible for the production of InsPs, the substrate of 2-kinase, it should share some phenotypes with the 2-kinase mouse. IPK2 (5-kinase) knockout embryos die between E8.5 and E9.5; day-8.5 embryos were able to be recovered and labeled with [3H]inositol, showing that the homozygous mutant embryos are lacking InsPs and InsP6. In the case of 2-kinase, mutants die before day 8.5, an interesting result considering that 2-kinase lies downstream of IPK2 (5-kinase) in the pathway for the production of InsP6. In humans, the half-life of 2-kinase protein is approximately one- half that of 5-kinase protein (data not shown). Because homozygous embryos have no functional enzyme, it must be carried over from maternal stores in the egg. If there is a difference in half-lives in mice, this difference may suggest that the InsP6 stores in the 2-kinase mutants may be depleted earlier than in the IPK2 knockouts. Taken together, the two knockout mice suggest that the higher inositol phosphates are required for life, although they do not necessarily pinpoint which one. Loss of 2-kinase would result in loss of InsPg and its downstream metabolites, InsP? and InsPg, and the accumulation of InsPs and PP-InsP4. The alteration of the levels of any of these inositol phosphates

8452 | www.pnas.org/cgi/doi/10.1073/pnas.0503656102 Verbsky etal.



may be responsible for the observed lethality. Future in vivo work would be required to define the required inositol phosphate.

The gross anatomy of the IPK2 knockout suggests that there is a defect in migration of neural crest cells. Although we did not acquire embryos from day 8.5, the strong staining in the neural precursors at this stage supports this hypothesis. Specifically, expression in the notochord is consistent with the IPK2 phenotype. The notochord is involved in neural induction and morphogenesis, and therefore a defect in its function could affect neural crest development and migration. Additional neural defects could be suggested by the /3-galactosidase staining in neuroblasts. Because 2-kinase embryos die before neural tube closure, and because embryos with severe neural defects can survive until later embryonic stages, it is likely that the embryonic lethality of 2-kinase knockout embryos in not a result of defects in neurogenesis.

One pattern of expression that would suggest a cause of death in these embryos is the strong staining of the visceral endoderm of the yolk sac. This extraembryonic cell layer performs a number of functions during the period in which the 2-kinase nulls die: specifically, nutrient absorption and delivery to the developing embryo and production of factors involved in embryo development and anterior patterning (16). Efficient endocytosis and exocytosis would be required for nutrient delivery. The proposed roles of InsPg in endocytosis and exocytosis suggest that it is necessary for these functions in the visceral endoderm. This period of development is also necessary for formation of blood cells and the vasculature of the placenta and embryo. The strong expression around blood vessels and blood islands may

indicate a non-cell-autonomous role of InsPg in angioblast differentiation or blood vessel formation.

The /3-galactosidase staining results are also consistent with the Northern blot analysis published previously (4). Expression of 2-kinase in the somatic myotome, embryonic nervous system, and heart is supported by Northern blots demonstrating 2-kinase mRNA in adult skeletal muscle, brain, and cardiac muscle. These results not only show that our /3-galactosidase staining corresponds to known sites of 2-kinase expression, but also indicate that 2-kinase expression patterns are conserved between adult tissues and their developmental progenitors.

The staining of the adult brain is significant with respect to previously reported functions of InsPg. InsPg mass was reported to be highest in the hippocampus and the most variable when stimulated by electric shock, consistent with the strong staining in the hippocampus that we observe (9). Here, it may function to regulate the activity of calcium channels. The 2-kinase expression looks identical to in situ staining of type IV phosphatidylinositol 5 phosphatase (17), which differs in the pattern of staining from other phosphatidyl inositol 5 phosphatases (e.g., synaptojanin). The significance of this pattern of staining remains to be seen, although its does suggest some specific function for InsPg in the hippocampus.

This work was supported by National Institutes of Health Grants HL55272 and HL16634. The ES cell line was obtained from the National Heart, Lung, and Blood Institute-funded Program for Genomics Applications at BayGenomics, which is supported by Grants HL66621, HL66600, and HL66590.

1. Posternak, S. (1919) C. R. Acad. Sci. 169, 138-140.2. York, J. D., Odom, A. R., Murphy, R., Ives, E. B. & Wente, S. R. (1999) Science

285, 96-100.3. Feng, Y., Wente, S. R. & Majerus, P. W. (2001) Proc. Nall. Acad. Sci. USA 98,

875-879.4. Verbsky, J. W., Wilson, M. P., Kisseleva, M. V., Majerus, P. W. & Wente, S. R.

(2 0 0 2 )/ Biol. Chem. 277, 31857-31862.5. Hanakahi, L. A., Bartlet-Jones, M., Chappell, C., Pappin, D. & West, S. C.

(2000) Cell 102, 721-729.6. Hoy, M., Efanov, A. M., Bertorello, A. M., Zaitsev, S. V., Olsen, H. L., Bokvist,

K., Leibiger, B., Leibiger, I. B., Zwiller, J., Berggren, P. O. & Gromada, J. (2002) Proc. Natl. Acad. Sci. USA 99, 6773-6777.

7. Norris, F. A., Ungewickell, E. & Majerus, P. W. (1 9 9 5 )/ Biol. Chem. 270, 214-217.

8. Larsson, O., Barker, C. J., Sj-oholm, A., Carlqvist, H., Michell, R. H., Bertorello, A., Nilsson, T., Honkancn, R. E., Mayr, G. W., Zwiller, J. & Berggren, P. O. (1997) Science i n , 471-474.

9. Yang, S. N., Yu, J., Mayr, G. W., Hofmann, F., Larsson, O. & Berggren, P. O.(2001) FASEBJ. 15, 1753-1763.

10. Goldstein, J. L. (2001) Nai. Med. 7, 1079-1080.11. Austin, C. P., Battey, J. F., Bradley, A., Bucan, M., Capecchi, M., Collins, F. S.,

Dove, W. F., Duyk, G., Dymecki, S., Eppig, J. T., et al. (2004) Nat. Genet. 36, 921-924.

12. Lobe, C. G., Koop, K. E., Kreppner, W., Lomeli, H., Gcrtscnstein, M. & Nagy,A. (1999) Dev. Biol. 208, 281-292.

13. Verbsky, J. W., Chang, S. C., Wilson, M. P., Mochizuki, Y. & Majerus, P. W. (2005)/ Biol. Chem. 280, 1911-1920.

14. Fugii, M. & York, J. W. (2005)/ Biol. Chem. 2 8 0 ,1156-1164.15. Frederick, J. P., Mattiske, D., Wofford, J. A., Megosh, L.C., Drake, L. Y.,

Chiou, S.-T., Hogan, B. L. M. & York, J. D. (2005) Proc. Natl. Acad. Sci. USA 102, 8454-8459.

16. Bielinska, M., Narita, N. & Wilson, D. B. (1999) Int. J. Dev. Biol. 43, 183-205.

17. Kisseleva, M. V. (2001) Dissertation (Washington University, St. Louis).

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Chapter 5 : INCREASED LEVELS OF INOSITOL HEXAKISPHOSPHATE (lnsP6) RESULT IN AN INCREASED AMOUNT OF RIP AND PROTECTION OF HEK293 CELLS FROM TNFcc AND FAS INDUCED APOPTOSIS.

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T h e J o u r n a l o f B io l o g ic a l C h e m ist r y

© 2005 by The American Society for Biochemistry and Molecular Biology, Inc.Vol. 280, No. 32, Issue of August 12, pp. 29263-29268, 2005

Printed in U.SA.

Increased L evels o f Inositol H exakisphosphate (InsP6) P rotect HEK293 Cells from Tumor N ecrosis Factor a - and Fas-induced Apoptosis*

Received for publication, March 28, 2005, and in revised form, May 31, 2005 Published, JBC Papers in Press, June 20, 2005, DOI 10.1074/jbc.M503366200

J o h n V erb sk y an d P h ilip W. M ajerustFrom the Department o f Internal Medicine, Division o f Hematology, Washington University School o f Medicine,St. Louis, Missouri 63110

The overexpression o f inosito l 1,3,4-trisphosphate 5/6- kinase has recently been show n to protect HEK293 cells from tum or n ecrosis factor a (TNFa)-induced apoptosis. This overexpression lead s to an in crease in the levels o f both inosito l 1,3,4,5,6-pentakisphosphate (InsP5) and inosito l 1,2,3,4,5,6-hexakisphosphate (InsP6). C ells that overexpress InsP0 2-kinase have increased lev e ls o f InsP6 and are a lso p rotected from TN Fa-induced apoptosis; furtherm ore, ce lls that express an RNA interference construct to the 2-kinase are d eficien t in InsPe and are sensitized to TNFa-induced apoptosis. Therefore the protective effect o f 5/6-kinase on TN Fa-m ediated apop tosis is due to an in crease o f InsP6 or to a m etabolite derived from InsP6. Furtherm ore, w e find tha t th e InsP6 also protects from Fas-m ediated apoptosis. N o effect w as seen in th e endocytic rate o f transferrin receptor, caspase 8 activity, or TNF receptor num ber at the cell surface. C ells that overexpress 2-kinase do show an in crease in th e am ount o f receptor-interacting protein (RIP), w h ile ce lls w ith reduced InsP6 lev e ls show rela tive ly less RIP, provid ing a possib le m echanism for the effect on apoptosis.

The pathways th a t produce the higher soluble inositol phosphates in hum an cells have been elucidated recently (1-3). The action of phospholipase C on phosphatidylinositol 4,5-bisphosphate, yields inositol 1,4,5-trisphosphate (InsP3)1 and diacylglycerol. In mammalian cells, InsP3 is phosphorylated to inositol1.3.4.5-tetrakisphosphate by an InsP3 3-kinase and dephosphorylated to inositol 1,3,4-trisphosphate (Ins(l,3,4)P3) by a 5-phosphatase (4). Ins(l,3,4)P3 is then phosphorylated to inositol1.3.4.6-tetrakisphosphate (Ins(l,3,4,6)P4) by the Ins(l,3,4)P3 5/6-kinase, to inositol 1,3,4,5,6-pentakisphosphate (InsP5) by Ins(l,3,4,6)P4 5-kinase, and to inositol 1,2,3,4,5,6-hexakisphosphate (InsP6) by InsP5 2-kinase (1, 5). In cell culture, production of InsP6 is regulated by the activity of 5/6-kinase, which is

* This work was supported by National Institutes of Health Grants HL 55272 and HL 16634. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

t To whom correspondence should be addressed: Box 8125, Washington University School of Medicine, 660 S. Euclid, St. Louis, MO 63110. E-mail: [email protected].

1 The abbreviations used are: InsP3, inositol 1,4,5-trisphosphate; Ins(l,3,4)P3, inositol 1,3,4-trisphosphate; Ins(l,3,4,6)P4, inositol 1,3,4,6-tetrakisphosphate; InsP6, inositol 1,3,4,5,6-pentakisphosphate; InsP6, inositol 1,2,3,4,5,6-hexakisphosphate; HPLC, high performance liquid chromatography; mAb, monoclonal antibody; PBS, phosphate- buffered saline; PARP, poly(ADP-ribose) polymerase; cPARP, cleaved PARP; TNF, tumor necrosis factor; RIP, receptor-interacting protein; RNAi, RNA interference.


rate-limiting (6) producing the committed isomer in the synthesis of InsP6, Ins(l,3,4,6)P4. Overexpression of 5/6-kinase results in an increase in InsP5 and InsP6, while depletion of 5/6-kinase results in the loss of InsP5 and InsP6.

Another function for the inositol (1,3,4)P3 5/6-kinase other than phosphorylating inositol phosphates was recently discovered. Wilson et al. (7) have shown th a t 5/6-kinase co-purifies with the COP9 signalosome from cow brain. This complex of nine proteins has been shown to have the ability to phosphorylate c-Jun, IkBq:, and p53 (8-10). Wilson et al. (7) subsequently showed th a t 5/6-kinase purified from insect cells also phosphorylates c-Jun, p53, and IxBa, making it likely th a t in part the protein kinase activity of the COP9 signalosome may be attributed to 5/6-kinase.

c-Jun and IkB are both involved in TNFa signaling and apoptosis. TNFa is involved in numerous processes including cell death and development and oncogenesis and immune, inflammatory, and stress responses (11). TNFa acts in opposing ways with regards to apoptosis. Through one arm of the pathway it can activate transcription by NFkB; TRADD is recruited to the TNF receptor through its death domains, and it in turn recruits RIP, a death domain-containing kinase, and TRAF2. Together TRAF2 and RIP recruit and activate IkB kinase complex, which phosphorylates IkBo, signaling it for ubiquitination and destruction. NFkB is then translocated to the nucleus to stim ulate transcription. Two of the many transcriptional targets of NFkB are FLIP and cIAP, proteins th a t inhibit apoptosis, and thus NFkB activity is considered anti-apoptotic. The second of the pathways it shares with Fas; this pathway stimulates apoptosis by recruiting FADD and caspase 8, which results in the cleavage and activation of caspase 8, initiating the caspase cascade. The pro-apoptotic action of TNFa cannot overcome its anti-apoptotic activity through NFkB in most cells; thus, to induce apoptosis in cells, protein synthesis has to be inhibited or NFkB signaling has to be blocked.

The ability of 5/6-kinase to phosphorylate proteins involved in TNFa-mediated apoptosis led Sun et al. (12) to determine whether the overexpression of 5/6-kinase had an effect on apoptosis. They found th a t HEK293 cells were protected from TNFa-induced apoptosis when there were elevated amounts of 5/6-kinase. They investigated whether this protection was due to increased NFkB signaling, but they found no difference in IkBo stability or NFkB activity by gel shifts of 5/6-kinase- expressing lines. This led to the possibility th a t the protection from apoptosis was due to inhibition of caspase activation ra ther than NFkB stimulation. It also suggested th a t the protection from apoptosis may not be due to the associated protein kinase activity of 5/6-kinase but rather through its inositol phosphate kinase activity and thus to the soluble, more highly phosphorylated inositol phosphates.

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http://www.jbc.org

29264 lnsP6 Protects from TNFa- and Fas-induced Apoptosis

We recently have shown th a t an increase in 5/6-kinase activity in cells results in a concomitant increase of InsPe and InsP6. Thus activities ascribed to the inositol phosphate kinase function of 5/6-kinase may be due to actions of InsP5 or InsP6 or to another downstream metabolite. Here we show th a t expression of 2-kinase and an increase of InsP6 can protect cells from TNFa-mediated apoptosis, and we attribute the protective effect of 5/6-kinase on TNFa-mediated apoptosis to the production of InsP6 itself. In addition, we find th a t expression of 2-kinase also results in a protection from Fas mediated apoptosis, and thus InsP6 may be a general regulator of apoptosis. Furthermore, we notice th a t altered InsP6 levels in cells result in altered levels of the protein RIP, which may provide a mechanism for the role of InsP6 in apoptosis.

MATERIALS AND METHODS

All chemicals were reagent grade or better. Restriction endonucleases, DNA modifying enzymes, and general reagents were from Amer- sham Biosciences, Roche Applied Sciences, Fisher, Invitrogen, New England Biolabs, Promega Corp., Sigma, and Stratagene unless stated otherwise. Acrylamide solution, Bio-Safe Coomassie Blue stain, and Bradford protein assay kit used for protein work were purchased from Bio-Rad. The SuperSignal West Pico kit used for detection of Western transfer blots was from Pierce. Radiolabeled [3H]inositol and [3H]InsP6 were purchased from American Radiolabeled Chemicals (St. Louis, MO) and Amersham Biosciences, respectively. TNFa was obtained from Peprotech and activating Fas antibody from BD Biosciences. Anti- caspase 8 (c20) goat polyclonal antibody was obtained from Santa Cruz Biotechnology, anti-caspase 8 monoclonal antibody (mAb) from Cell Signaling, and anti-RIP mAb from BD Biosciences. Protein G was obtained from Sigma.

Strains, Plasmids, and Growth Conditions—Methods for Escherichia coli growth and selection were described previously (13, 14). E. coli strain XL-lBlue (Stratagene) was used as the bacterial host for all plasmids unless stated otherwise. Bacterial strains were cultured in LB (1% tryptone, 0.5% yeast extract, 1% NaCl, pH 7.4) medium supplemented with ampicillin (100 /xg/ml) where appropriate and transformed by standard methods. All bacterial strains were grown at 37 °C

Cloning, Production, and Maintenance o f Cell Litres—Stable cell lines expressing 2-kinase were reported previously (6). The stably transfected 2-kinase RNAi line was produced as follows. Oligonucleotides containing the antisense target to the 2-kinase (5'-GAAGAC- CTCGGAAGAGATA-3') or to luciferase (5' -CTTACGCTGAGTACT- TCGA-3') were annealed and ligated in the pSUPER vector (a gift from Dr. Reuven Agami). Equal amounts of either the 2-kinase or the luciferase RNAi construct were mixed 7:1 with pBabe containing a puromycin resistance gene and transfected into the same parent cells as the overexpression lines, TRex HEK293(Invitrogen), using Lipofectamine as per the manufacturer’s protocol. Cells were allowed to recover for 1 day and serially diluted, and clones were selected with 1 pg/ml puromycin. Individual clones were obtained and maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 1 /xg/ml puromycin.

High Performance Liquid Chromatography (HPLC)—HPLC was conducted as follows. Cell were grown in the presence of [3H] inositol (10 p,Ci/ml) for 4 days. Cells were lysed in methanol/0.5 n HC1 (2:1) and extracted with chloroform. The aqueous phase was separated, dried, and resuspended in distilled water. Samples were applied to a Whatman Partisphere SAX strong anion exchange column (4.6 mm xl25 mm) running a 30 min gradient from 0 to 1.7 M ammonium phosphate, pH 3.5, followed by a 30-min isocratic elution with 1.7 M ammonium phosphate, pH 3.5. Radioactivity was measured using the inline detector 0-RAM (IN/US System Inc.), and the identity of the individual inositol phosphates was assigned on the basis of co-elution with known standards.

Apoptotic Cell Staining—Cells were grown on polylysine-treated cov- erslips to ~80% confluence, then treated with 1 ng/ml TNFa (Peprotech) and cycloheximide (1 pg/ml) for 18 h. Apoptotic cells were detected using the APOPercentage apoptotic kit (Accurate Chemical, Westbury, NY).

Western Blot Analysis—Specified tissue culture cells were removed from the plates by gentle aspiration, washed with PBS, and lysed in 20 mM HEPES, pH 7 .6 ,140 mM NaCl, 10% glycerol, 0.2% Nonidet P-40 plus protease inhibitors (Complete Mini EDTA-free, Roche Applied Science). Cells were treated to two freeze-thaw cycles in an ethanol-dry ice bath, and particulate debris was removed by centrifugation at 10,000 x g in an Eppendorf centrifuge at 4 ®C. The protein concentration of the clarified

lysate was determined using the Bradford assay (Bio-Rad protein assay) as per the manufacturer’s protocol. Using standard techniques, 25 pg of protein of each sample was loaded onto a 10% gel for SDS-PAGE and subsequently electroblotted onto polyvinylidene difluoride membrane (Immobilon-P, Millipore). Western analysis was conducted using anti- PARP polyclonal antibody (Cell Signaling) using standard techniques. The appropriate horseradish peroxidase-conjugated secondary antibody and the SuperSignal West Pico chemiluminescent substrate (Pierce) were used to visualize the appropriate bands. Where indicated, bands were compared by densitometry of Western blots using an Eastman Kodak Co. Image Station 440 CF, and the data were analyzed using Kodak ID V.3.5.4 (Scientific Imaging System).

TNFa or Fas treatment o f HEK293 Cells—2 x 10B cells were plated in 12-well plates in the presence (for the 2-kinase-expressing lines or vector control) or absence (for 2-kinase RNAi or luciferase control lines) of tetracycline for 24 h and then treated with TNFa (1 ng/ml) plus cycloheximide (1 pg/ml) or the stated amount of Fas antibody plus protein G (a 4:1 ratio of Fas to protein G) for the indicated amount of time. Extracts were processed for Western blots as described above.

2-Kinase RNAi Construct Expression in 516-Kinase-expressing Cells—5/6-Kinase cells lines were transfected with the same 2-kinase RNAi construct used to generate stable cell lines, grown for 1 day to recover, then split into 12-well plates and grown for 24 h in the presence of tetracycline to induce 5/6-kinase expression. These cells were then treated for various times with TNFa and cycloheximide as above.

Transferrin Uptake—Cells were incubated with 126I-transferrin (0.25 Mg/ml) in binding medium (0.1% bovine serum albumin in Dulbecco’s modified Eagle’s medium) at 37 °C for 1-8 min. At the end of the incubation, the medium was aspirated, and the monolayers were rapidly washed three times with cold PBS to remove unbound ligand. The cells were then incubated for 5 min with 0.2 M acetic acid, pH 2.8, containing 0.5 M NaCl at 4 °C. The acid wash was combined with a short rinse in the same buffer and used to determine the amount of surface- bound 12BI-ligand. The cells were lysed in 1 n NaOH to determine the intracellular (internalized) radioactivity. The ratio of internalized to surface radioactivity was plotted against time. At 10 min, a 100-fold molar excess of unlabeled ligand was added, and the cells were treated as before to determine the background binding.

Immunoprecipitation—For caspase 8 co-immunoprecipitations, two 90% confluent pl50 tissue culture plates treated as indicated were used for each immunoprecipitation. Cells were washed in PBS and lysed in 1.5 ml of lysis buffer (20 mM HEPES, pH 7.6, 140 mM NaCl, 10% glycerol, 0.2% Nonidet P-40 plus protease inhibitors (Complete Mini EDTA-free, Roche)) for 30 min on ice. An equal mass of each lysate was precleared on protein G-agarose for 1 h, and the caspase 8 complex was precipitated with 2 pg of anti-caspase 8 antibody and 50 pi of protein G-agarose a t 4 °C overnight. Beads were recovered by centrifugation, washed twice with lysis buffer, and subjected to Western analysis as above. To ensure that equal amounts of caspase 8 were being precipitated, 5% of the precipitated protein was run on a separate gel and blotted with mAb against caspase 8. Total cellular RIP was immunoprecipitated as follows: 0.5 x 10® cells were plated on 6-well plates and grown for 24 h. Cells from each well were lysed as for the caspase 8 immunoprecipitations, and RIP was precipitated with 1 pg of anti-RIP mAb for 1 h. The complexes were precipitated with 25 pi of protein G-agarose for 30 min and washed three times with PBS, and the pellets were subjected to Western blot analysis as above.

RESULTS

HEK293 Cells Expressing a Stably Transfected RNAi Construct to the 2-Kinase Result in Altered InsP6 Profiles— HEK293 cell lines constructed with a tetracycline inducible 2-kinase gene show an altered inositol phosphate profile as reported previously; specifically, expression of the 2-kinase resulted in an increase in InsP6 and a loss of InsP5. We also generated HEK293 cells stably transfected with an RNAi construct to the 2-kinase. As a control we produced cell lines transfected with the pSUPER construct containing an RNAi insert to the luciferase gene. When these cells were labeled with [3H]inositol, and their soluble inositol phosphates purified and separated by HPLC, there is a decrease of InsP6 relative to the controls by about 70% {cf. Fig. 1, B to A) and an increase in InsP6 and pyrophosphorylated InsP4. This result is consistent with inositol labeling of HEK293 cells transiently transfected with siRNAs to 2-kinase as reported previously (1).

53


InsPg Protects from TNFa- and Fas-induced Apoptosis 29265A. HEK-293 + Luciferase RNAi B. HEK-293 * 2-kinase RNAi

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Fig. 1. HPLC profiles o f [3H]inositol-labeled HEK293 cells expressing a RNAi co n stru c t to th e 2-kinase: 2-kinase- o r luciferase RNAi-expressing cells w ere grow n in th e presence of te tracycline and tr i tia ted inositol for 4 days and th e ir soluble inositols ex trac ted and sepa ra ted by HPLC. The identity of the labeled peaks was confirmed by internal standards (data not shown).

2-Kinase-expressing Cells Are Resistant to TNFa-mediated Apoptosis, whereas 2-Kinase RNAi Lines Are More Susceptible to Apoptosis—5/6-Kinase-overexpressing cells show an increase of InsP5 and InsP6 and a protection from TNFa-induced apoptosis. 2-Kinase-overexpressing cells show increased InsP6 and a loss of InsP6. When treated with TNFa 2-kinase-overexpressing cells (Fig. 2B) show a relative decrease in the number of apoptotic cells, as determined by APOPercentage staining, when compared w ith vector cells (Fig. 2A). Similarly, relative to the luciferase lines (Fig. 2C) the 2-kinase RNAi lines (Fig. 2D) showed more apoptotic staining when treated with TNFa.

The results shown in Fig. 2 were confirmed by W estern blot analysis. Cells expressing 2-kinase and the vector control were treated with TNFa and cycloheximide for 7 and 24 h and their extracts blotted with a polyclonal antibody against PARP, a target of caspase 3. Consistent with the results from the cell staining, cells overexpressing 2-kinase showed a decrease in the amount of apoptosis as determined by the amount of cleaved PARP relative to the vector lines a t both time points (Fig. 3A). We also looked a t cleaved PARP accumulation in 2-kinase RNAi lines. Compared with the luciferase lines, the 2-kinase RNAi line showed more cleaved PARP at 7 and 24 h, and is therefore more susceptible to TNFa mediated apoptosis (Fig. 3B).

In both sets of experiments, the amount of apoptosis seen in the tetracycline-induced vector lines was greater than tha t seen in the luciferase RNAi control lines. Since the vector lines were treated with tetracycline, the transcription machinery was induced by the addition of tetracycline, as it is in the 2-kinase- expressing lines. The RNAi lines were not treated with tetracycline. This may account for the difference between the control lines. Therefore, it is necessary to consider the increase or decrease of apoptosis relative to their respective control.

The 2-Kinase RNAi Construct Can Overcome the Protection from Apoptosis o f 5 /6-Kinase Expression—To address whether the protective effect was solely due to the presence of InsP6 and does not involve other products resulting from 5/6-kinase and its associated protein kinase activity, we expressed the 2-kinase RNAi construct in cells overexpressing 5/6-kinase. There was an increase in apoptosis in the cells transfected w ith the 2-kinase RNAi relative to those transfected with the luciferase RNAi (Fig. 4). Taken with the above results, we conclude tha t the protective effect from TNFa-mediated apoptosis is due to the presence of InsP6 or a downstream metabolite.

Increased InsP6 Protects against FAS-mediated Apoptosis, while Decreased InsP6 Levels Render Cells More Susceptible to FAS-mediated Apoptosis—In addition to protection from TNFa-mediated apoptosis, we also found th a t 2-kinase overexpression could protect cells from Fas-induced apoptosis as shown in Fig. 5. Cell lines were treated with increasing amounts of Fas antibody for 20 h, and their cell extracts were analyzed by W estern blotting for PARP. Cells th a t were overexpressing the 2-kinase showed less PARP cleavage a t 1 and 3 /ig/ml as compared with the vector control lines (Fig. 5A). Furthermore, the 2-kinase RNAi line showed more apoptosis as compared with the luciferase control (Fig. 5B). The results of PARP cleavage for the 6 /xg/ml Fas trea tm ent were analyzed by densitom etry to normalize loading. W hereas the ratio of full-length PARP to tubulin were sim ilar for RNAi and Luciferase lines (1.7 to 1.6, respectively), the ratio of cleaved PARP to tubulin was almost three tim es greater in the RNAi line th an luciferase line (0.41 to 0.15, respectively). Therefore, relatively more PARP is cleaved in the RNAi line than the luciferase line.

Alterations in InsP6 Levels Do Not Affect Receptor Internalization, Caspase 8 Activity, or TNF Receptor Number—Since it is unlikely th a t the protective effect of the 2-kinase on apoptosis is due to NFkB activation, we looked for other cellular alterations th a t would explain the protective effect. Receptor internalization is required for TNF induced apoptosis and is mediated by a canonical YXXW motif known to mediate receptor internalization by clathrin-coated vesicles (15); when mutated, TNF receptor internalization is inhibited, and the death inducing signaling complex is not formed, thus blocking apoptosis. Since InsP6 has been implicated in endocytosis, we measured transferrin internalization in cells overexpressing the 2-kinase. As seen in Fig. 6, 2-kinase overexpression does not affect transferrin internalization compared with vector control. The regression coefficient of four experiments, denoting the internalization constant (K d ) , was averaged and showed no difference between the 2-kinase or vector lines. Furthermore, we saw no difference in internalization rates between 2-kinase and luciferase lines (data not shown). We also measured the TNF and Fas receptor number a t the cell surface by FACS analysis and found no difference between the cell lines containing increased amounts of InsP6 and those with depleted levels of InsP6. Finally, caspase 8 activity was assayed with increasing amounts of InsP6 or inositol hexakissulfate to control for

54


29266 InsP6 Protects from TNFa- and Fas-induced Apoptosis

B. 2-Wnas*

F ig . 2. A PO Percentage s ta in ing of cells expressing th e 2-kinase o r th e 2-kinase RNAi. TRex vector cells and 2-kinase-expressing cells were grown in the presence of tetracycline. All cell lines were treated with TNFa and cycloheximide for 16 h. 2-Kinase lines (B) show fewer apoptotic cells compared with the vector lines 04). Consistently, the 2-kinase RNAi lines (D) show more apoptotic cells than luciferase RNAi controls (C).

C. Luctfsrtt* RNAi

A.

cleaved PARP— I

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B. Luciferase 2-K RNAiTime in " " T TNF/CHX 0 (hours)

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cleaved PARP— mF ig . 3. PARP W estern blo ts of TN F-treated 2-kinase (2-K) lines.

2-Kinase-expressing or vector lines (A), and luciferase or 2-kinase RNAi lines (B), were treated for 0, 7, or 24 h with TNFa/cycloheximide and their extracts blotted and probed with anti-PARP antibody.

Luciferase 2-kinase

uq/ml Fas

cleaved PARP

tubulin

lu c ife ra se 2-kmase RNAij jg /m l Fas 0

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tubulin

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F ig . 4. 2-Kinase RNAi construct expression overcom es the p ro tective effect of 5/6-kinase expression in HEK293 cells. 5/6- Kinase lines were transiently transfected with a luciferase RNAi construct or a RNAi construct to the 2-kinase. These cells were then treated with tetracycline to induce 5/6-kinase expression. 24 h post-transfection, the cells were treated with TNFa/cycloheximide for 0, 3, or 6 h and their extracts blotted with PARP antibody.

nonspecific charge effect, and no effect w as seen on caspase 8 activ ity up to 100 /xM In sP 6 or inositol hexak issu lfa te .

2-Kinase-overexpre8sing Cells an d 2-Kinase R N A i Lines Show ed A ltered Levels o f the Anti-apoptotic Protein R IP —W este rn b lo tting of 25 /xg of cell ly sate showed a n increase in th e level of R IP in th e 2-kinase-expressing lines as com pared w ith th e vector control and a decrease in th e RNAi s tab le lines as com pared w ith th e luciferase control (Fig. 7). W hen norm alized to th e tu bu lin control, R IP levels w ere increased by abou t 50% in th e 2 -kinase-expressing lines a n d decreased by abou t 50% in th e RNAi lines.

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F ig . 5. Expression of th e 2-km ase affords p ro tec tion from Fas- m ediated apoptosis, w hereas depletion o f In sP 6 re su lts in an increased suscep tib ility to Fas m ediated apoptosis. A, 2-kinase- expressing or vector lines were grown in tetracycline for 24 h and then treated with activating Fas antibody as indicated and blotted against anti-PARP as for TNFa treatment. B, luciferase or 2-kinase RNAi stable cell lines were treated with activating Fas antibody as indicated and blotted against PARP. C, PARP, cPARP, and tubulin bands from the 6 mg/ml Fas lane in B were subject to densitometry on a Eastman Kodak Co. Image Station 440 CF, and the ratio of PARP to tubulin and cPARP to tubulin was plotted for luciferase and RNAi lines.

T he elevated am ount o f R IP in cellu lar ex trac ts is reflected in an increase in co-im m unoprecipitations of R IP w ith anti- caspase 8. T he m ethod of M icheau and Tschopp (16) w as em ployed to address th e effect of th e 2 -k inase on th e form ation of th e caspase 8/TRADD/RIP complex w ith th e TNF receptor. In teresting ly , i t w as unnecessary to s tim u la te cells w ith TNF to co-im m unoprecipitate R IP w ith caspase 8, suggesting th a t

55


InsP6 Protects from TNFa- and Fas-induced Apoptosis 29267

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F ig . 6. A, transferrin uptake assays in 2-kinase-overexpressing cell lines or vector controls in the presence of tetracycline. B, th e average linear regression coefficient for the first three tim e points in A from four different experim ents representing the coefficient of internalization.

F ig. 7. Cell lines expressing the 2-kinase contain relatively higher levels o f RIP, whereas cells deficient in InsP6 contain relatively lower levels o f RIP. Western blots of 2-kinase-expressing, 2-kinase RNAi, or vector control cell extracts probed with anti-RIP mAb antibody. Tubulin was used as a loading control.

the complex forms to a small degree in cells unstim ulated with TNFa. When HEK293 cells expressed 2-kinase, more RIP protein was precipitated w ith caspase 8 as compared with the vector controls (Fig. 8). T reatm ent of these cells with the proteosome inhibitor MG 132 restored the level of RIP in the immunoprecipitations in the vector lines equal to th a t of the 2-kinase lines. These results would suggest th a t in these cells 2-kinase overexpression affects the turnover ra te of the caspase 8, FADD, TRADD, and RIP co-complex.

DISCUSSION

The overexpression of 5/6-kinase has been shown by Sun et al. (12) to protect against TNFa-mediated apoptosis, although the mechanism was unknown. Since the overexpression of 5/6- kinase also results in an elevation in the amounts of InsP5 and InsP6 (1), it is possible th a t this protective effect is due to one or more of these isomers of inositol phosphate. Cells overexpressing 2-kinase accumulate InsP6, deplete InsP5, and show protection from apoptosis relative to vector controls. Furthermore, HEK293 cells stably transfected with an RNAi construct to 2-kinase deplete InsP6 and accumulate InsP5 and show an increased susceptibility to apoptosis. In addition, expression of 5/6-kinase was not able to overcome the susceptibility to apoptosis caused by expression of the 2-kinase RNAi construct. This result confirms the data of Sun et al. (12) th a t suggested th a t the protective effect of the 5/6-kinase on apoptosis is not due to its protein kinase activity but rather to its inositol kinase activity. Thus, the protection from apoptosis is due to InsP6 or possibly some more highly phosphorylated inositol phosphate. In addition, we have shown th a t overexpression of 2-kinase protects from Fas-induced apoptosis, while 2-kinase RNAi expression renders the cells more sensitive to Fas-induced apoptosis. Taken together, the two studies support each other in their implication th a t soluble inositol phosphates are regulators of apoptosis.

As mentioned above, our results do not conclusively implicate InsP6 itself in the protection from apoptosis; we can only say th a t the isomer involved has to lie downstream of InsP6. InsP6 is metabolized to the higher inositol pyrophosphates

F ig. 8. The increase in RIP levels is correlated with an increase in RIP in caspase 8 co-immunoprecipitations. Caspase 8 was immunoprecipitated from HEK293 cells as stated with goat anti- caspase 8 antibody, and RIP was detected using a monoclonal RIP antibody. 5% of each immunoprecipitation was run on a separate gel and blotted with caspase 8 monoclonal antibody to control for the efficiency of the immunoprecipitation of caspase 8.

InsP7 and InsP8. We, though, have seen no increase in an InsP7 isomer when expressing the 2-kinase in metabolically labeled cells (data not shown), and there is some evidence th a t the inositol pyrophosphates may stim ulate apoptosis. Morrison et al. (18) used an antisense technique to identify genes th a t are involved in interferon /31-induced apoptosis in an ovarian carcinoma cell line. One gene th a t was shown to sensitize these cells to interferon /3-induced apoptosis was the inositol hexakisphosphate kinase 2. This protein converts InsP6 to the pyrophosphate InsP7. Interferon /3 acts by stim ulating tran scription through the JAK/STAT pathway. Interestingly, the transcription of a number of targets stim ulated by INF3 are involved in TNFa-mediated apoptosis {e.g. caspases 8) or are analogous to TNF apoptosis, e.g. TRAIL (TNF-related apoptosis-inducing ligand). Since InsP6 kinases phosphorylate InsP6, their expression may result in the depletion of cellular levels of InsP6, which we show here to sensitize cells to apoptosis. Thus the balance between InsP6 and the inositol pyrophosphates may act as a switch controlling apoptosis. Nonetheless, such speculation requires experiments w ith the InsP6 kinases to determine definitively the inositol isomer involved.

The protection from apoptosis afforded by expression of the 2-kinase probably is not due to the known anti-apoptotic consequences of TNFa, namely through NFkB activation. No decrease in I kB stability or increase in N FkB activity is seen in 5/6-kinase-overexpressing cells. The protection from Fas-mediated apoptosis would argue against the role of N FkB activation. Also, the TNFa assays are done in the presence of cycloheximide, where protein synthesis is inhibited. The protective effect may work on the apoptotic branch of TNFa signaling.

We addressed a number of the possible steps in the activation of the apoptotic branch of TNFa signaling. We looked a t cell surface expression of the TNF receptor between lines th a t had altered levels of InsP6 and saw no difference nor did we see an effect of InsP6 on caspase 8 activity in vitro. TNF activation has recently been shown to require the internalization of the TNF receptor (19). InsPe has been implicated in endocytosis and could delay activation of caspase 8 by altering the endocytic ra te of the receptor. Yet cells w ith altered levels of InsP6 showed no defect in the uptake of transferrin, and it is unlikely th a t th is would provide the mechanism for the protection from apoptosis. Furtherm ore, our results provide the first in vivo description of the affect of altered InsP6 levels on endocytosis. Although it has been implicated in endocytosis by a number of studies, we did not see an effect on endocytosis when InsP6 levels were elevated (Fig. 6) or depleted (data not shown).

The increase in InsP6 is correlated with an increase in RIP and may provide a mechanism for the protection afforded by an increase in InsP6 levels. RIP—/ — mouse embryonic fibroblasts are sensitive to TNFa-mediated apoptosis (20), likely due to its role recruiting and activating the IkB kinase complex, resulting in the activation of NFkB. Since th is does not seem to be

56


29268 InsP6 Protects from TNFa- and Fas-induced Apoptosis

involved in the current study, other roles for RIP m ust be considered. RIP is cleaved by caspase 8 upon TNFa engagem ent of its receptor, resulting in a C-terminal and N-terminal fragment, and consequently RIP levels drop. Expression of the C-terminal fragment of RIP in cells stim ulates the FADD and TRADD interaction, while expression of the full-length RIP inhibits the FADD to TRADD interaction (21). If full-length RIP can compete for binding with cRIP to TRADD, this may result in a delay of recruitm ent of FADD to TRADD and thus delay caspase activation. Our results are consistent with the work by Sun et al. (12) th a t suggested th a t the protection from TNFa-mediated apoptosis was due to an inhibition of the recruitment of FADD to TRADD.

It is not clear how InsP6 affects RIP levels. Prelim inary experiments to determine the effect of InsP6 itself on the half- life of RIP did show th a t cells deficient in InsP6 lost RIP more quickly when treated w ith cycloheximide; a t 3 h after trea tment, RIP levels dropped to 40% of the original level of RIP in the 2-kinase RNAi lines, while they remained a t about 90% of the original levels in the luciferase control lines (data not shown). The role of InsP6 is well known in mRNA export. Since RIP is a protein with a relatively short half-life, it may require more efficient export of mRNAs to m aintain a level of message for efficient translation of protein. Alternatively, InsP6 may affect its turnover rate by affecting the proteosome or its ta rgeting for degradation by ubiquitination.

Acknowledgment—We thank Shao Chang for his time and intelligent input during discussions regarding this work.

REFERENCES1. Verbsky, J . W., Wilson, M. P., Kisseleva, M. V., Majerus, P. W., and Wente,

S. R. (2002) J. Biol. Chem. 277, 31857-318622. Chang, S. C., Miller, A. L., Feng, Y., Wente, S. R., and Majerus, P. W. (2002)

J. Biol. Chem. 277, 43836-438433. Wilson, M. P., and Majerus, P. W. (1996) J. Biol. Chem. 271, 11904-119104. Majerus, P. W. (1992) Annu. Rev. Biochem. 61, 225-2505. Fujii, M., and York, J. D. (2005) J. Biol. Chem. 2 8 0 ,1156-11646. Verbsky, J . W., Chang, S. C., Wilson, M. P., Mochizuki, Y., and Majerus, P. W.

(2005) J. Biol. Chem. 280, 1911-19207. Wilson, M. P., Sun, Y., Cao, L., and Majerus, P. W. (2001) J . Biol. Chem. 276,

40998-410048. Naumann, M., Bech-Otschir, D., Huang, X ., Ferrell, K., and Dubiel, W. (1999)

J. Biol. Chem. 274, 35297-353009. Bech-Otschir, D., Kraft, R., Huang, X ., Henklein, P., Kapelari, B., Pollmann,

C., and Dubiel, W. (2001) EMBO J. 2 0 ,1630-163910. Seeger, M., Kraft, R., Ferrell, K., Bech-Otschir, D., Dumdey, R., Schade, R.,

Gordon, C., Naumann, M., and Dubiel, W. (1998) FASE BJ. 12, 469-47811. Chen, G. Q., and Goeddel, D. V. (2002) Science 296, 1634-163512. Sun, Y., Mochizuki, Y., and Majerus, P. W. (2003) J. Biol. Chem. 278,

43645-4365313. Chanda, V. B. (ed) (1997) Current Protocols in Molecular Biology, John Wiley

& Sons, Inc., New York14. Sambrook, J ., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A

Laboratory M anual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

15. Bonifacino, J. S., and Traub, L. M. (2003) Annu. Rev. Biochem. 72, 395-44716. Micheau, O., and Tschopp, J. (2003) Cell 114, 181-19017. Deleted in proof18. Morrison, B. H., Bauer, J . A., Kalvakolanu, D. V., and Lindner, D. J. (2001)

J. Biol. Chem. 276, 24965-2497019. Schneider-Brachert, W., Tchikov, V., Neumeyer, J ., Jakob, M., Winoto-Mor-

bach, S., Held-Feindt, J ., Heinrich, M., Merkel, O., Ehrenschwender, M., and Adam, D. (2004) Immunity 21, 415-428

20. Kelliher, M. A., Grimm, S., Ishida, Y., Kuo, F., Stanger, B. Z., and Leder, P.(1998) Immunity 8, 297-303

21. Lin, Y., Devin, A., Rodriguez, Y., and Liu, Z. G. (1999) Genes Dev. 13,2514-2526

57


Chapter 6 : CONCLUSIONS AND FUTURE DIRECTIONS

58


This thesis has made a number o f advancements in the understanding o f the synthesis and

physiological role InsP6. First o f all, it reported the cloning and in vitro biochemical

characterization o f human InsPs 2-kinase, the first non-fungal 2-kinase reported.

Secondly it described the pathway o f the synthesis o f InsP6 in vivo starting from

Ins(l,3,4)P3, and described cell lines that were constructed in which InsP6 levels were

altered. Next, it described an effective null 2-kinase mouse, and showed that loss o f the

2-kinase was embryonic lethal. Finally, it addressed the role o f InsP6 in apoptosis and

endocytosis using these cell lines. A few issues have arisen during these studies.

The synthesis pathway o f inositol phosphates in humans is clear. 5/6-kinase is

necessary and sufficient for the production o f InsP6 and the pathway proceeds from

Ins(l,3,4)P3 to Ins(l,3,4,6)P4 to Ins(l,3,4,5,6)Ps to InsPg. Some controversy does remain

with respect to the synthesis pathways in other mammals. Fugii et al.{ 1) could produce

InsPs from InsP3 when IP O was over-expressed in Rat-1 cells. They argue that this is

due to phosphorylation o f Ins( 1,3,4,5)P3 to (1,3,4,5,6)P5. We expressed the 5-kinase in

HEK293 cells and did not see an increase in InsPs or InsPg, though over-expression o f

5/6-kinase resulted in increases in both. This difference is difficult to reconcile

considering the high degree o f homology between the two proteins. An increase in InsP5

was not seen when rat I P O was expressed in human cells(John York, personal

communication), consistent with a difference in activities o f 3-kinase or 5-phosphatase,

or with the activity o f the rat 5-kinase itself. Fugii et al. did not provide kinetic data for

the rat 5-kinase, and therefore it cannot be compared to the kinetics reported for the

human protein. In human cells, the kinase activity o f 5-kinase on Ins(l,3,4,5)P4 is

insufficient to overcome 5-phosphatase activity giving rise to Ins(l,3,4)P3. Therefore no

59


increase is seen when 5-kinase is over-expressed, because the action o f 5/6-kinase is rate

limiting. Rat cells may have decreased 5-phosphatase activity compared to humans; an

increase in Ins(l,3,4,5)P4 could then be used as substrate to make InsP5. Alternatively,

5/6-kinase may be more active in Rat-1 cells, and the pathway may still proceed through

Ins(l,3,4)P3. Fugii et al. could not rule out the possibility that the increase in InsP5

occurs because o f synthesis through the Ins(l,3,4)P3 isomer, because they did not look at

5/6-kinase in Rat-1 cells. This debate is important for any study that looks at the

expression o f the proteins involved in the synthesis o f InsP6, because InsP6 levels may be

affected accordingly. It is also relevant to the options we have for studying 2-kinase in

mice.

The embryonic lethality in mice lacking 2-kinase and the lack o f an obvious

phenotype in heterozygous mice, should not hinder further inquiry into InsP6 biology. A

number o f tools remain available. If the production o f InsP6 is regulated by 5/6-kinase in

mice as it is in humans, double 2-kinase, 5/6-kinase heterozygotes may drop the levels o f

InsP6 to a point where a phenotype is uncovered. This is likely because the results from

the labeling o f MEFs suggest that there is not a surplus capacity o f 2-kinase activity in

heterozygotes; MEFs did accumulate InsPs during labeling experiments in order to

maintain the levels o f InsP6. The Majerus lab is currently addressing this with two mice

lines that have a gene trap construct inserted downstream o f exons o f the 5/6-kinase gene.

As these lines are better developed, one could easily carry out this experiment.

Work can also be conducted to determine the cause o f death in 2-kinase null mice.

Our (3-galactosidase staining was strong in the visceral endoderm o f the yolk sack. This

organ has been implicated in nutrient uptake and delivery, as well as involvement in

60


embryonic development and anterior patteming(2). Bielinska et al. describe two

strategies for studying gene products required for the function o f the visceral endoderm:

tetraploid aggregation chimeras in which tetraploid embryos are mixed with embryonic

stem cells and implanted in a pseudopregnant female, or injections o f wild type blastocyts

with 2-kinase null embryonic stem cells. In both cases, the wild type tissue contributes to

the visceral endoderm, while the embryo is mutant. If the reason for the death o f the

embryos is starvation due to disrupted function o f the visceral endoderm, the resulting

embryos should survive to a later stage. Homozygous null embryos die before day 8.5

p ost coitum. Before day 8.5, organogenesis is limited, and thus the cause o f lethality may

not be due to loss o f 2-kinase in neural or heart precursors. The visceral endoderm is an

attractive target because hematopoiesis is induced by products o f the visceral endoderm

starting around day 6 or 6.5. Loss o f these factors may also be a contributing factor.

Some o f the proposed roles for InsP6 can now be addressed using some o f the

tools we have produced. For instance, the reports o f InsP6 binding to Ku should be

examined, especially with respect to telomere maintenance. In yeast, the pyrophosphate

PP-InsP4 is needed to inhibit telomere lengthening. Since the heterozygote MEF’s from

the mice we have bred show an increase in PP-InsP4, one could look at telomere length in

these mice. This is not a trival task, and it is necessary to breed multiple generations to

see defects in telomere maintenance. One could also breed the 2-kinase heterozygote

mutants with Ku heterozygotes, and look for genetic interactions or defects in telomere

maintenance.

We do find a significant effect o f altering InsP6 levels on the progression o f Fas

and TNFa induced apoptosis, specifically by altering RIP levels, although other protein

61


levels may be affected also. Metabolic inhibitors like cycloheximide result in a

sensitization o f cells to Fas and TNFa induced apoptosis. Fulda et al. (3)suggested that it

was the loss o f RIP and FLIP, two proteins whose level drops quickly in response

cycloheximide, that is responsible for the sensitization. Since RIP levels are increased in

cells expressing 2-kinase, FLIP levels may also be altered by 2-kinase, and would

provide another mechanism for the protection. FLIP levels should be addressed in these

cell lines.

It is not known how InsP6 affect the protein levels o f RIP. Preliminary

experiment suggested that high InsP6 increased the half-life o f RIP in cells treated with

cycloheximide, and low levels o f InsP6 decreased it (data not shown). Further study

would be required to confirm these results. InsP6 may inhibit the function o f the

proteosome, or it may alter the function o f the proteins responsible for targeting RIP for

degradation. Recently, the zinc finger containing protein A20 has been shown to remove

lysine-63 linked ubiquitin from RIP, and subsequently add lysine-48 linked ubiquitin to

RIP(4). This conversion signals its degradation. It would be interesting to see whether

InsP6 can alter this process. In support o f this, we co-purified a de-ubiquitinating

enzyme, Ubp6, also known as USP14 in humans, during attempts to purify 2-kinase from

soy beans on an affinity column by using an InsP6 elution (data not shown). This de-

ubiquitinating enzyme may be binding InsP6, or InsPg may disrupt its interaction with a

2-kinase containing complex, and therefore it was seen in a very late stage o f purification.

The work on A20 suggested that lysine-63 ubiquitination may act as a scaffold to recruit

signaling molecules. It mentioned that some IKK members have ubiquitin like domains

which they hypothesize could be involved in their recruitment to the ubiquitinated RIP.

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Interesting, the de-ubiquitinating enzyme we purified also contains ubiquitin like

domains, and may be involved in their recruitment to such a complex to regulate the

ubiquitinated state o f proteins like RIP.

Recently, RIP has been implicated in the cellular response to DNA damaging

agents. NFkB is induced by DNA damage, but the mechanism was unknown. Now it

seems that RIP initiates signaling though NFkB by recruiting IKK by itself, without

requiring TRADD or TRAF2(5), which are needed for TNFa induced signaling through

the TNF receptor. Furthermore, an increase o f RIP resulted in an increase o f NFkB

activation. The 2-kinase then may be induced by DNA damage, resulting in increased

levels o f RIP, and thus increased NFkB activation. Also, InsP6 has been shown to

stimulate non-homologous end joining o f double strand DNA breaks through its binding

to Ku. This would be a clever way in which the cell would coordinate the activation o f

the signaling pathway in response to DNA damage with an increase in the metabolite

which helps repair the DNA, i.e. InsPg. The authors note that RIP itself does not shuttle

to the nucleus, and thus can’t be responsible for sensing the damaged DNA. The 2-

kinase protein may be a candidate for this, because we know the 2-kinase protein does

shuttle through the nucleus (data not shown). It would be interesting to look at the

cellular response to DNA damaging agents in our cell lines, and see whether 2-kinase is

induced in response to DNA damage.

The elevation or depletion o f InsPg levels in HEK293 cells did not alter the rate o f

transferrin uptake in cell culture lines. Since it has been reported that InsPg binds AP-2

and API 80 and could compete with PIP2 for binding, the lack o f an effect suggests that in

vivo InsPg does not play a role in endocytosis. Localized increases o f InsPg may be more

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important the total cellular increases in InsP6, but since we are increasing 2-kinase protein

itself, and not simply manipulating InsP6 levels, presumably it would be able deliver

InsPg where it was needed. Other factors may play a role along with 2-kinase, or InsPg

may act as a substrate for the production o f the pyrophosphates, which have also been

reported to be involved in vesicular traffic(6). Nonetheless, the initial attempt to link

InsP6 and endocytosis was not successful.

This in vivo study should also raise some questions about the work that confers an

anti-cancer effect on InsPg. It is hard to reconcile those conclusions, reached by bathing

cells in an acidic highly charged molecule, with these which show the opposite effect. It

is unlikely, given these in vivo results, that elevated levels o f InsPg outside the tumor cells

stimulate the cells to apoptose, when the elevation o f InsPg inside the cells blocks

apoptosis. As for the endocytic studies, these studies demonstrate the need for in vivo

experiments to address the physiological role o f InsPg.

The role o f InsPg in exocytosis was not addressed directly by this work, though he

staining o f the hippocampus in the heterozygous mice does support the work implicating

InsPg in exocytosis through activation o f L-type calcium channels found in the

hippocampus. The heterozygous mouse may be a candidate to assess potential defects in

exocytosis through electophysiologic recording o f neurotransmission. Alternatively, the

cloning o f 2-kinase allows the inquiry into potential binding partners for 2-kinase protein,

which may elucidate further its role in exocytosis.

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References:

1. Fujii, M., and York, J. D. (2005) J Biol Chem 280, 1156-11642. Bielinska, M., Narita, N., and Wilson, D. B. (1999) In tJ D ev Biol 43, 183-2053. Fulda, S., Meyer, E., and Debatin, K. M. (2000) Cancer Res 60, 3947-39564. Wertz, I. E., O'Rourke, K. M., Zhou, H., Eby, M., Aravind, L., Seshagiri, S., Wu,

P., Wiesmann, C., Baker, R., Boone, D. L., Ma, A., Koonin, E. V., and Dixit, V. M. (2004) Nature 430, 694-699

5. Hur, G. M., Lewis, J., Yang, Q., Lin, Y., Nakano, H., Nedospasov, S., and Liu, Z. G. (2003) Genes D ev 17, 873-882

6. Saiardi, A., Sciambi, C., McCaffery, J. M., Wendland, B., and Snyder, S. H. (2002) Proc Natl Acad Sci U S A 9 9 , 14206-14211

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