Protein phosphatase 2A and phosphoprotein SET regulate ... · granulosa cells, pregnenolone is...

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Protein phosphatase 2A and phosphoprotein SET regulateandrogen production by P450c17*

Amit V. Pandey‡, Synthia H. Mellon§¶ and Walter L. Miller‡¶†

From the ‡Departments of Pediatrics and of §Obstetrics, Gynecology andReproductive Sciences, ¶The Metabolic Research Unit and ¶The Center forReproductive Sciences, University of California San Francisco, SanFrancisco, CA 94143-0978

†Address for Correspondence:

Prof. Walter L. MillerDept. of Pediatrics,Bldg MR4 Room 209University of California, San FranciscoSan Francisco, CA 94143-0978

Tel.: 415-476-2598Fax.: 415-476-6286E-mail: [email protected]

Running title: PP2A and regulation of androgens

Key words: Adrenarche, polycystic ovary syndrome, sex steroids, serinekinase, PP2A

Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on November 19, 2002 as Manuscript M209527200 by guest on July 3, 2018

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Abstract

Cytochrome P450c17 catalyzes 17a-hydroxylation needed for cortisol synthesis

and 17,20 lyase activity needed to produce sex steroids. Serine phosphorylation of

P450c17 specifically increases 17,20 lyase activity, but the physiological factors

regulating this effect remain unknown. Treating human adrenal NCI-H295A cells with

the phosphatase inhibitors okadaic acid, fostriecin and cantharidin increased 17,20

lyase activity, suggesting involvement of protein phosphatase 2A1 (PP2A) or 4 (PP4).

PP2A but not PP4 inhibited 17,20 lyase activity in microsomes from cultured cells but

neither affected 17a-hydroxylation. Inhibition of 17,20 lyase activity by PP2A was

concentration-dependent, could be inhibited by okadaic acid and was restored by

endogenous protein kinases. PP2A but not PP4 coimmunoprecipitated with P450c17

and suppression of PP2A by siRNA increased 17,20 lyase activity. Phosphoprotein SET

found in adrenals inhibited PP2A but not PP4 and fostered 17,20 lyase activity. The

identification of PP2A and SET as posttranslational regulators of androgen biosynthesis

suggests potential additional mechanisms contributing to adrenarche and

hyperandrogenic disorders such as PCOS.

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Introduction

Steroid hormones are synthesized by a set of pathways that begin with the

conversion of cholesterol to pregnenolone by mitochondrial cytochrome P450scc, the

quantitative regulator of steroidogenesis. Pregnenolone can then be directed to one of

three principal pathways by microsomal P450c17, the qualitative regulator of

steroidogenesis, which catalyzes both 17a-hydroxylase and 17,20 lyase activities (1-4)

(Fig. 1). In the absence of P450c17, e.g. in adrenal zona glomerulosa and ovarian

granulosa cells, pregnenolone is converted to 17-desoxy, C21 steroids including

progesterone, corticosterone and aldosterone. In the presence of the 17a-hydroxylase

activity of P450c17, the adrenal zona fasciculata produces C21 17a-hydroxy steroids

including the glucocorticoid cortisol. When both 17a-hydroxylase and 17,20 lyase

activities are present, the adrenal zona reticularis and gonads produce the C19 17-

ketosteroid dehydroepiandrosterone (DHEA), which is the precursor of androgens and

estrogens.

The ratio of the 17a-hydroxylase to 17,20 lyase activities of human P450c17 is

developmentally regulated. Adjusted for body size, the human adrenal produces nearly

constant amounts of cortisol throughout life, indicating relatively constant 17a-

hydroxylase activity (5). By contrast, the production of DHEA is minimal in childhood,

rises 100-fold to levels that exceed the production of cortisol in young adulthood, then

gradually decreases with advancing age (6). The mechanisms by which human adrenal

C19 steroid synthesis is turned on (adrenarche) and turned off (adrenopause) remain

unclear. Recent clinical observations suggest a link between premature exaggerated


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adrenarche and the polycystic ovary syndrome (7-10). Adrenarche is difficult to study

because it occurs only in higher primates (10-12), and no cellular model exists; instead,

a productive approach has centered on the biochemistry of P450c17.

P450c17 is a single enzyme encoded by a single gene that is expressed in both

the adrenals and gonads (13,14). Like other microsomal P450 enzymes, P450c17

catalyzes several chemical reactions on a single active site by receiving electrons from

the flavoprotein P450 oxidoreductase (OR). Human P450c17 can catalyze the 17a-

hydroxylation of pregnenolone to 17aOH-pregnonolone (17OH-Preg) or of progesterone

to 17aOH progesterone (17OH-Prog). However, the 17,20 lyase reaction almost

exclusively converts 17OH-Preg to DHEA; human P450c17 catalyzes the conversion of

17OH-Prog to androstenedione with only 3% of the efficiency of the reaction with 17OH-

Preg (15); thus most human sex steroids are made from DHEA. At least three factors

influence the ratio of 17,20 lyase to 17a-hydroxylase activities at a post-translational

level. First, high molar ratios of OR to P450c17 favor lyase activity (2,15,16). Second,

cytochrome b5 acts allosterically to foster the interaction between P450c17 and OR to

promote the lyase reaction, but b5 does not function as an electron donor (2,15,17).

Third, the phosphorylation of P450c17 on serine and threonine, but not tyrosine

residues increases 17,20 lyase activity through as-yet unidentified mechanisms, which

may involve increasing its affinity for b5 and/or OR (18). The precise residues of

P450c17 that are phosphorylated and the responsible kinase(s) remain unknown.

However, when a protein is activated by phosphorylation there generally is an

equilibrium between phosphorylation by a kinase and dephosphorylation by a


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phosphatase (19). We now provide evidence that PP2A serves as the principal

phosphatase regulating P450c17 phosphorylation and 17,20 lyase activity.

MATERIALS AND METHODS

Cells and transfection - The NCI-H295A adherent population (20) of human

adrenocortical NCI-H295 tumor cells (21,22) was grown on 150 mm Petri plates as

described (20). Cells at 60-80% confluence were treated with okadaic acid, fostriecin

and cantharidin (Calbiochem, www.calbiochem.com). NCI-H295A cells in 100 mm

dishes were transfected with 5mg of the plasmids pBJF, pBJF-Flag-PP2A, pBJF-Flag-

PP4 or pBJF-Flag-PP6 (23) using Lipofectamine 2000 (Invitrogen, www.invitrogen.com)

following the manufacturer’s protocol. For in vitro labeling with 32P, NCI-H295A cells

were transferred to phosphate free medium for 1 h and labeled with 1 mCi/ml [32P]

orthophosphate for 4 h at 37°C.

Microsome preparation and enzyme assays- NCI-H295A cells were harvested

and lysed by sonication (six times for 10s at 30 KCs-1) in 50mM potassium phosphate

buffer (pH 7.4), containing 100 mM KCl and 0.1 mM EDTA. Unbroken cells, the nuclear

fraction and mitochondria were separated at 12,000xg for 20 min and microsomes were

pelleted from the 12,000xg supernatant at 100,000xg for 90 min. Microsomes were

resuspended in same buffer containing 20% glycerol and used immediately for enzyme

assay. 17a-hydroxylase and 17,20 lyase activity assays were as described (15,24).

Briefly, microsomes (20 mg protein) were incubated at 37°C with 50,000 cpm of [3H ]


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progesterone or [3H ]17OH-Preg in 50 mM potassium phosphate buffer (pH 7.4) and

catalysis was initiated by addition of 1 mM NADPH. After the appropriate time (30-60

min), steroids were extracted in 500 ml of 1:1 ethyl acetate : isooctane and concentrated

by evaporation under nitrogen. Concentrated steroids were dissolved in 20 ml of

trichloromethane and analyzed by thin layer chromatography over silica gel plates (PE

SIL G/UV, Whatman) using a 3:1 mixture of chloroform/ethyl acetate as mobile phase.

Radiolabeled steroids were quantitated using a Storm 860 phosphorimager (Amersham

Biosciences www.amershambiosciences.com).

Coimmunoprecipitation and western blotting - Antibodies against PP2A catalytic

subunit (Upstate Biotech, www.upstatebiotech.com), PP4 (25) and SET (26) were

covalently linked to Protein-A-Sepharose using Disuccinimidyl suberate (DSS) (Pierce,

www.piercenet.com) and mixed with lysates of NCI-H295A cells (4mg of antibody / ml of

lysate). The bound proteins were eluted, separated by SDS/4-20% PAGE and

immunoblotted with P450c17 antiserum (16) (1:3000 dilution) and detected by

enhanced chemiluminescence (Amersham Biosciences).

Phosphatase assays - NCI-H295A cells were lysed in buffer containing 50 mM

Tris-HCl (pH 8.0), 1% Nonidet P-40, 120 mM NaCl, 1mM EDTA, 5 mM EGTA, 1mM

dithiothreitol, 1mM PMSF, and 1mg/ml each of aprotinin and leupeptin. Recombinant

catalytic subunit of PP2A was from Promega (www.promega.com) ; PP4 was prepared

by immunoprecipitation from NCI-H295A lysates using antibody provided by Dr. T.H.

Tan (25). The activities of these phosphatases were confirmed against phosphorylated


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microsomal proteins by monitoring the release of free phosphate. PP4 and PP2A were

immunoprecipitated and washed three times with 50 mM HEPES buffer (pH 7.4)

containing 0.1% Triton X-100 and 500 mM NaCl. Immunoprecipitates were incubated in

100 ml of 50 mM HEPES (pH 7.0), 0.1 mM DTT, 0.1 mM CaCl2, 1mM MgCl2 and 1mM

MnCl2 at 25°C for 30 min, pelleted by centrifugation, and the supernatant was

transferred to fresh tubes. The assay was terminated with 500 ml of Biomol Green

reagent (Biomol laboratories, www.biomol.com), a phosphate detection reagent based

on malachite green, incubated for 20 min at room temperature and read at 620 nm. One

unit of phosphatase activity was defined as amount of enzyme required to release 1

nmol of phosphate per hour at 30 °C. For the in vitro PP2A experiments, microsomes

were treated with 12.5 U/ml of pure recombinant PP2A catalytic subunit (Promega) at

25°C in 50 mM Tris-HCl (pH 7.4) containing 20 mM MgCl2. The reaction was terminated

by addition of 1mM okadaic acid and 10 mM NaF and chilling on ice for 10 min.

siRNA construction and transfection – Small interfering RNAs (siRNAs) to the

catalytic subunits of human PP2A, and PP4, were designed with 3' overhanging

thymidine dimers as described (27). Target sequences were aligned to the human

genome database in a BLAST search (www.ncbi.nlm.nih.gov/blast) to eliminate those

with significant similarity to other genes. Web based siRNA design software from

Ambion (http://www.ambion.com/techlib/misc/siRNA_finder.html) was used for selecting

siRNA sequences. Three target sequences for each gene corresponding to sequences

located in the 5', 3', or middle regions of each transcript were synthesized and used for

transfection (Table 1). The siRNAs were synthesized using a transcription-based


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SilencerTM siRNA synthesis kit (Ambion, www.ambion.com). Transfection was carried

out using the Oligofectamine transfection reagent (Invitrogen) as described (28). In

brief, NCI-H295A cells were grown to 50-70% confluency in complete medium without

antibiotics in 100 mM plates. Cells were washed with serum-free medium prior to

transfection. All the siRNA duplexes (2.0 mg/100 mm plate) were diluted in separate

tubes with 100 ml of Opti-MEM™ (Invitrogen). In separate tube Oligofectamine reagent

was diluted 1:5 with Opti-MEM™ medium and incubated for 10 min at room

temperature. Diluted Oligofectamine reagent was added to siRNA duplexes (50 ml

diluted Oligofectamine reagent / mg of siRNA) and the mixture was incubated for 20 min

at room temperature. The volume of medium overlaying the cells was adjusted to 7.5

ml and the siRNA transfection complexes (200 ml) were added; six hours later the

medium was supplemented with serum to make a final serum concentration of 2%.

Because of the slow doubling time of NCI-H295A cells (20,22) the cells were then

incubated for an additional 72 hr before harvesting for analysis.

Preparation and transfection of rat SETb protein- Full length rat SETb cDNA (29)

was sub-cloned into pBluebac His2Sf9 (Invitrogen), and transfected into Spodoptera Sf9

cells for 48 h. Cells (50 ml culture) were collected, washed and lysed in 4 ml of 500 mM

NaCl, 25 mM Hepes, pH 7.5 (buffer B) containing 1% Tween 20, 10% glycerol, 1mM

pefabloc and a mixture of protease inhibitors. The cell lysate was applied to a 250 ml Ni-

NTA column previously equilibrated with buffer B containing 1% Tween 20. The column

was first washed with 5ml of buffer B containing 1% Tween 20, then with 5 ml of buffer

B, and finally with 5 ml of buffer B containing 5 mM imidazole. The bound SETb was


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eluted with 5 ml of buffer B containing 250 mM imidazole. Protein transfection into NCI-

H295A cells was performed with Chariot reagent (Active Motif, www.activemotif.com).

NCI-H295A cells were plated in 6 well plates and grown to 40-60% confluency. Purified

SET protein was diluted with PBS (1.0 mg/ml) and Chariot reagent was diluted with

water (1:20). In a separate tube, 100 ml of diluted SET protein was mixed with 100 ml of

diluted Chariot reagent and incubated at room temperature for 30 min. Growth medium

from the cells was aspirated and cells were washed with PBS. Transfection complex

(200 ml/well) was added to cells, the volume was adjusted to 600 ml with serum-free

RPMI-1640 medium, and the cells were incubated for one hour (37 °C, 5% CO2). Cells

were supplemented with 1.0 ml complete growth medium and incubation was continued

for additional 4 hours after which the cells were harvested and used for experiments.

PCR of human SET and phosphatases - Total RNA (1.0 mg) from NCI-H295A

cells was converted to cDNA using Superscript II reverse transcriptase (Invitrogen) for

30 min at 50°C, followed by 35 cycles of PCR amplification (30s at 94°C, 30s at 60°C

and 90s at 72°C). Primer pairs are described in Table 2.

Preparation of endogenous kinases and in vitro phosphorylation - Soluble

intracellular kinases were enriched using an ATP-Sepharose matrix (Upstate Biotech)

as described (30). ATP-Sepharose matrix (0.25 ml) was washed three times with 1 ml of

25 mM HEPES, pH 7.4, 150 mM NaCl; 1mM NADH; 1 mM NAD; 1 mM ADP; 1mM

AMP; 1 mM DTT and 60 mM MgCl2 (buffer A). NCI-H295A cells were lysed in 50 mM

Tris-HCl pH 7.4 containing 1% NP-40; 5 mM EDTA; 2 mM EGTA; 150 mM NaCl; 1 mM


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PMSF; 1mg/ml each of aprotinin, leupeptin and pepstatin; 1 mM DTT; 0.15 mM Na3VO4;

10 mM NaF; 500 mM cantharidin, 1mM NADH; 1mM NAD; 1mM ADP and 1mM AMP,

centrifuged at 100,000 x g for 90 min at 4°C and 1 ml of the soluble fraction (1 mg

protein) was added to the washed ATP-Sepharose beads. After incubation for 4 h with

agitation at 4°C, the beads were washed 4 times with 0.5 ml of buffer A containing 500

mM NaCl, suspended in 0.5 ml of buffer A containing 10 mM ATP and incubated at

room temperature for 60 min to elute bound kinases. Bacterially expressed human

P450c17 bound to Ni-NTA-Sepharose was incubated with 1-2 mg of protein from the

kinase-enriched fraction of NCI-H295A cells in the presence of 10 mM Mg-ATP. NTA-

Sepharose containing 0.25-0.5 mg of bound P450c17 in a volume of 50 ml was

incubated with 1mM [32P] ATP (1.0 mCi) and 25 mM MgCl2, and washed 5 times with 0.5

ml of 50 mM Tris-HCl, pH 7.4 containing 500 mM NaCl. Bound [32P]P450c17 was

denatured by boiling in SDS gel sample buffer, separated by SDS/ 12% PAGE, and

analyzed by phosphorimager.

Bacterially-expressed human P450c17 - The pCWH17-mod(His)4 expression

plasmid containing the cDNA for modified human P450c17 (31), was transformed into

E. coli JM109. Ampicillin-resistant colonies were grown at 37°C to OD600 0.4-0.6,

P450c17 expression was induced by 0.4 mM IPTG at 25°C for 48 h, and P450c17 was

purified as described (31). In brief, spheroplasts prepared by lysozyme treatment of

bacteria were lysed by sonication for 3 min at 30 kCs-1, centrifuged at 4,000 x g for 10

min, and the pellet containing P450c17 was extracted with 1% Triton X-114

(Calbiochem) and ultracentrifuged at 100,000 x g for 30 min. A reddish-brown


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detergent-rich supernatant fraction containing P450c17 was isolated and passed over a

Ni-NTA-Sepharose column. The column was washed with 20 mM histidine to remove

non-specific binding and eluted with 200 mM histidine. Further purification was carried

out by hydroxyapatite chromatography to remove histidine and some other protein

contaminants.

RESULTS

Regulation of 17,20 lyase activity by phosphatases in NCI-H295A cells -

Serine/threonine phosphorylation of P450c17 fosters 17,20 lyase activity, and

dephosphorylation of P450c17 in vitro with alkaline phosphatase decreases 17,20 lyase

activity (18). To determine if a phosphatase participates in the physiological regulation

of 17,20 lyase activity in vivo, we treated NCI-H295A cells with phosphatase inhibitors

and measured 17,20 lyase activity (Table 3). Okadaic acid (32,33), cantharidin (34) and

fostriecin (35) are inhibitors of protein phosphatases 2A (PP2A) and 4 (PP4) (36). Low

concentrations of okadaic acid and cantharidin that are relatively specific for PP2A and

PP4 increased 17,20 lyase activity 4-fold. Okadaic acid and cantharidin have some

activity against protein phosphatase 1 (PP1) (35) but fostriecin has IC50 values of 1.5

nM for PP2A (35,36), 3.0 nM for PP4 (36) and 131 mM for PP1 (35), making it an

essentially pure inhibitor of PP2A and PP4 (35,36). Concentrations of either 5 or 25 nM

fostriecin again increased 17,20 lyase activity 4 fold, implicating PP2A and/or PP4 as

the relevant phosphatases.


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The action of okadaic acid on NCI-H295A cells was dose-dependent with a half

maximal effect on lyase activity at 10 nM and a maximal effect at 100 nM, but there was

no effect on 17a hydroxylase activity (Fig 2A). While it is logical to presume that the

action of PP2A to inhibit 17,20 lyase activity was directly attributable to

dephosphorylation of P450c17, it could also have resulted from an indirect protein-

protein interaction. To discriminate between these two possibilities, PP2A was

preincubated with various concentrations of okadaic acid. The okadaic acid-treated

PP2A was added to NCI-H295A microsomes and the 17,20 lyase activity was measured

(Fig 2B). The half maximal inhibitory concentration of okadaic acid was ~0.5 nM,

suggesting that catalytic activity of PP2A is required for its inhibition of 17,20 lyase

activity. Preincubation of PP2A with 100 nM okadaic acid for 15 min before addition to

microsomal preparations neutralized the effect of PP2A (data not shown), consistent

with PP2A inhibiting 17,20 lyase activity by dephosphorylating P450c17. These values

are consistent with the IC50 value of okadaic acid (0.1 nM) on purified preparations of

PP2A or PP4, and those observed in cell culture treatments (10 nM) (32). Thus PP2A or

PP4 appear to function physiologically as intracellular factors that de-phosphorylate

P450c17, resulting in suppression of 17,20 lyase activity and sex steroid synthesis.

To determine whether the induction of 17,20 lyase activity by phosphatase

inhibitors correlated with the degree of P450c17 phosphorylation, we grew NCI-H295A

cells in 32P and okadaic acid and estimated 32P incorporation into P450c17.

Phosphorimaging of equivalent amounts of immunoprecipitable P450c17 showed that

10 nM okadaic acid promoted the incorporation of 32P (Fig 2C). Therefore,


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phosphatases affected by okadaic acid (PP2A/PP4) appear to play a role in the

reversible phosphorylation of P450c17.

Effect of PP2A and PP4 on P450c17 activities in NCI-H295A microsomes- To

identify the specific phosphatases responsible for the effects of the inhibitor treatments,

we treated steroidogenically active microsomes from NCI-H295A cells with PP2A and

PP4 and assayed 17,20 lyase activity. PP2A (12.5 U/ml) inhibited 17,20 lyase activity to

the same extent as alkaline phosphatase (12.5 U/ml), but PP4 (25 U/ml) had no effect

(Fig 3A). This action of PP2A could be partially overcome by pre-treating the PP2A with

the phosphoprotein SET (7.8 nM), an inhibitor of PP2A (37). SET alone did not affect

17,20 lyase activity (not shown). To determine if PP2A was sufficient for removing the

physiologically relevant phosphate groups, we treated NCI-H295A microsomes with

PP2A (Fig 3B). Preincubation of microsomes with up to 12.5 U/ml of PP2A had no effect

on the conversion of progesterone to 17OH-Prog (an index of 17a-hydroxylase activity)

but PP2A decreased 17,20 lyase activity (conversion of 17OH-Preg to DHEA ) in a

dose-dependent manner with a half maximal effect at about 0.5 U/ml, and complete

inhibition at 12.5 U/ml. Thus PP2A exerts the same selective effect on the steroid 17,20

lyase activity of P450c17 that we previously observed with non-specific calf intestinal

alkaline phosphatase (18), but PP4 does not exert this effect. The presence of PP2A,

PP4 and SET in NCI-H295A cells was confirmed by western blotting (data not shown).

PP4 and PP6 cannot mimic the action of PP2A- PP2A shares 66% sequence

identity with PP4 and 58% identity with PP6, indicating they belong to a related family of


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phosphatases (38), and some inhibitors of PP2A also affect PP4 (36,38) and might

potentially inhibit PP6. To determine if the action of PP2A on P450c17 was specific or

simply representative of this family of phosphatases, we transfected NCI-H295A cells

with expression vectors for the catalytic subunits of PP2A, PP4 or PP6 (23), verified the

expression of these proteins by western blotting, and measured 17,20 lyase activity (Fig

4A). Compared to cells transfected with an empty vector, the 17,20 lyase activity of cells

expressing PP2A was reduced, whereas the 17,20 lyase activity of cells expressing

PP4 and PP6 was unchanged. Thus PP4 and PP6 were unable to dephosphorylate the

relevant residues of P450c17.

To determine if PP2A interacts directly with P450c17, we immunoprecipitated

PP2A, PP4 and SET from NCI-H295A cells under non-denaturing conditions and

confirmed the immunoprecipitation of each by western blotting (not shown). Probing

with antisera to P450c17 showed that P450c17 co-immunoprecipitated with PP2A but

not with PP4 or SET (Fig 4B), indicating that the action of PP2A is to dephosphorylate

P450c17 itself, and not some other protein that influences P450c17 activity.

Suppression of PP2A by siRNA – To determine whether the effects of PP2A on

the 17,20 lyase activity that we had documented with the biochemical assays in vitro

were relevant to the regulation of 17,20 lyase activity in cells in vivo we used RNA

interference to suppress the expression of the catalytic subunits of PP2A and PP4 in

NCI-H295A cells. Three 21 nt siRNA segments directed against the 5’ end, the middle,

or the 3’ end of the mRNAs for PP2A and PP4 were transfected into NCI-H295A cells


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and the cells were examined 72 hr later. Western blotting showed that both PP2A and

PP4 were reduced by half compared to cells transfected with control 21 nt RNAs (Fig

5A). Neither transfection with the control RNA or the siRNA directed against PP4

affected 17,20 lyase activity, but transfection with siRNA against PP2A increased 17,20

lyase activity by 250% (Fig 5 B, C). Thus PP2A regulates 17,20 lyase activity in vivo as

well as in vitro.

Role of SET in regulating 17,20 lyase activity - The presence of both a kinase

and PP2A in NCI-H295A cells indicates the presence of conflicting activities, suggesting

that each activity may be regulated by a cascade of additional factors. The

phosphoprotein SET, a highly specific inhibitor of PP2A (37), was tested as an attractive

candidate for such a factor. SET exerts other activities, including inhibition of cell cycle

(39,40), promoting expression of the gene for P450c17 in mouse testis MA-10 Leydig

cells (29,41) and modifying the substrate specificity of PP1 (42). SET (7.8 nM) inhibited

the activity of PP2A but not PP4 using phosphorylated NCI-H295A microsomal proteins

as substrate in vitro (Fig 6A). Because transfection of SET expression vectors inhibited

cell cycle, we assayed SET’s activity on P450c17 in vivo using a liposome-mediated

protein transfection procedure to introduce recombinant rat SETb into NCI-H295A cells

(65-75% transfection efficiency; data not shown). Transfection with 25 ng of SET for 4 h

increased 17,20 lyase activity to the same degree as treating the cells with 10 nM

okadaic acid (Fig 6B) but had no effect on total amount of P450c17 protein detectable

by western blot (Fig 6C). Analysis of NCI-H295A cells by RT-PCR shows they express a

wide variety of protein phosphatases (Fig 6D), and both SETa and SETb (Fig. 6E),


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which differ only at their amino termini, due to alternate first exon choice (43); both

SETa and SETb can inhibit PP2A activity equally in vitro (44).

Reactivation of 17,20 lyase activity of NCI-H295A microsomes treated with PP2A

- To confirm that the action of PP2A on 17,20 lyase activity is mediated by

dephosphorylating P450c17 rather than by working on some other upstream target, we

determined if the 17,20 lyase activity that had been lost to the action of PP2A could be

restored by re-phosphorylating P450c17. We prepared NCI-H295A cytoplasmic extract

and enriched it for protein kinase activity by affinity chromatography on ATP-Sepharose

(30). The retained fraction lacked phosphatase activity but was enriched for ATP-

dependent kinase activity. NCI-H295A microsomes were dephosphorylated with PP2A,

then the PP2A was inactivated with 100 nM okadaic acid and 10 mM NaF. Under these

conditions the microsomes retained 17a-hydroxylase activity but had lost almost all

17,20 lyase activity (Fig. 7A). Re-phosphorylation of these PP2A-treated microsomes

using 10 mM Mg-ATP and the cytosolic fraction enriched for protein kinases (5-10 mg

protein) fully restored 17,20 lyase activity (Fig 7A). Neither the kinase preparation nor

the cytosolic fraction of NCI-H295A cells contained significant 17,20 lyase activity (Fig

7B). Thus one or more ATP-dependent protein kinases present endogenously in NCI-

H295A cell cytoplasm is sufficient to restore full 17,20 lyase activity to dephosphorylated

P450c17 in NCI-H295A microsomes.

To determine whether NCI-H295A microsomes contain other unidentified factors

required for the phosphorylation of P450c17, we examined the ability of the NCI-H295A


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cytosolic kinases to phosphorylate purified human P450c17 expressed in bacteria (31).

Human P450c17 containing N-terminal modifications that confer solubility without

affecting activity (31) was expressed in E.coli JM109 cells and purified by Ni-NTA

sepharose chromatography. P450c17 (0.25-0.5 mg), still attached to Ni-NTA sepharose

through a 4X His linker, was incubated at 25 °C for 30 min with 1mCi [32P]ATP (1 mM);

25 mM MgCl2 and the cytoplasmic kinase fraction prepared from NCI-H295A cells (1-2

mg protein). The Ni-NTA sepharose-P450c17 beads were washed to remove other

proteins, separated by 12% SDS-PAGE and 32P incorporation in P450c17 bands was

detected by phosphorimager analysis. The endogenous kinases present in NCI-H295A

cytoplasm could phosphorylate P450c17 (Fig 7C). Comparison of the amount of 32P

incorporated with the amount of P450c17 protein indicated that an average of 5.7

phosphates were incorporated. Treatment with PP2A diminished the acquired

radioactivity confirming that PP2A acts to dephosphorylate P450c17. Equivalent results

were obtained using P450c17 immunoprecipitated from NCI-H295A microsomes (Fig.

7D).


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DISCUSSION

Reversible protein phosphorylation by protein kinases and phosphatases

regulates numerous cellular processes. About 30% of proteins undergo phosphorylation

that influences their conformation and biological activity (38,45). Although most studies

of protein phosphorylation have focused on protein kinases, recent data suggest that

phosphatases are equally important components of reversible regulatory cycles of

phosphorylation and dephosphorylation (19,45). Based on primary amino acid

sequence and three-dimensional structures, there are three main families of protein

phosphatases, termed PPP, PPM and PTP (38). The PPP and PPM families are

serine/threonine phosphatases while the PTP phosphatases can dephosphorylate

tyrosine as well as serine and threonine. PP1, PP2A (also called PP2), PP2B (also

called PP3), PP4, PP5, PP6 and PP7 are the principal members of the PPP family.

PP2A is a heterotrimer of A, B and C subunits (46). The 36 kDa catalytic C subunit

attaches to one of over 50 different B subunits ranging from 50 to 130 kDa, with the help

of the 65 kDa A subunit (19). Different PP2A holoenzymes form at various phases of the

cell cycle and during metabolic processes (19,46,47). Okadaic acid, microcystin LR,

tautomycin, cantharidin, calyculin A and fostriecin are highly specific inhibitors of the

PPP family (36,38,48). The sensitivity of specific cellular process to different

concentrations of these inhibitors can facilitate the identification of physiologically

relevant phosphatases. Okadaic acid and fostriecin are particularly useful as they can

permeate membranes, inhibiting PP1, PP2A and PP4 in intact cells (33,34,38). The

sensitivity of the 17,20 lyase reaction to very low concentrations of fostriecin and


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okadaic acid, both in vivo and in vitro, the sensitivity to SET protein, the inability of PP4

and PP6 to dephosphorylate and inhibit the 17,20 lyase reaction, and the specific effect

of suppression of PP2A, but not PP4, by siRNA all strongly suggest a role for PP2A in

the regulation of P450c17 phosphorylation and 17,20 lyase activity.

PP2A activity can be regulated by a tyrosine kinase that phosphorylates and

inactivates the catalytic subunit (49) and by interaction with SET (37). SET is also a

nuclear protein involved in cell proliferation and inhibiting cyclins (39), and is also known

as TAF-1, which regulates adenovirus DNA replication (50). SET also acts as a

transcription factor that regulates mouse testicular P450c17 (29,41). SET appears to be

an endogenous regulator of PP2A’s action on P450c17. SET has been identified as the

heat-stable cytoplasmic peptide inhibitor of PP2A termed I2PP2A (37). NCI-H295A cells

express SET endogenously; transfection of these cells with recombinant SET protein

enhanced 17,20 lyase activity similarly to the effect of okadaic acid, and SET did not

inhibit PP4, implicating the PP2A/SET system as regulating the 17,20 lyase activity of

P450c17. SET itself is a phosphoprotein. It is possible that

phosphorylation/dephosphorylation of SET by other protein kinases and phosphatases

govern its function as an inhibitor of PP2A, providing another site for control of the 17,20

lyase activity of P450c17. It is not yet clear if SET is involved in the transcriptional

regulation of the human gene for P450c17 as it is with mouse gene. We found no

change in the amount of P450c17 protein in our experiments, consistent with post-

translational action. The regulation of 17,20 lyase activity at both the transcriptional and

post-translational level by a single protein would make it an attractive candidate for a


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factor involved in adrenarche and the hyperandrogenism of the polycystic ovary

syndrome.

The 17,20 lyase activity lost to treatment with PP2A could be restored by re-

phosphorylating P450c17 with endogenous kinases found in NCI-H295A cytoplasm.

Thus one or more endogenous kinases and phosphatases appear to be in dynamic

equilibrium, suggesting there may be multiple control points for the regulation of 17,20

lyase by P450c17 phosphorylation. It is likely that a cascade of other factors regulates

both the positive action of the kinase and the negative action of the phosphatase. The

demonstration that SET is present in NCI-H295A cells and can foster 17,20 lyase

activity by inhibiting PP2A suggests that it is the first of these factors to be identified.

Similarly, many protein kinases are themselves regulated by complex cascades of

phosphorylation and dephosphorylation. Thus we propose that the regulation of the

17,20 lyase activity of P450c17 is positively regulated by a kinase pathway and

negatively regulated by a phosphatase pathway, both of which contain multiple

components, each of which represent a potential site of regulation (Fig 8). We

previously suggested that IGF-1 is associated with the induction of adrenarche (18) as

disorders of insulin signal transduction appear to be associated with PCOS, hence we

propose that the pathways regulating the phosphorylation and dephosphorylation of

P450c17 are linked to the IGF-1 and insulin signal transduction pathways. Thus future

elucidation of PP2A regulatory subunits and kinases involved in the P450c17

phosphorylation or dephosphorylation pathways should reveal candidate factors that

may play key roles in PCOS.


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Acknowledgments – We thank Dr. T.H. Tan (Baylor College, Houston, TX) for anti-

PP4 and Dr. K. Nagata (Tsukuba U, Tsukuba, Japan) for the anti-SET antibodies; Dr.

M.R. Waterman (Vanderbilt U, Nashville, TN) for the pCwh17-mod(his)4, Dr. J. Chen (U

Illinois, Urbana, IL) for Plasmids pBJF and pBJF-Flag-PP2A, and Dr. Stuart L Schreiber

(Harvard U, Cambridge, MA) for Plasmids pBJF-Flag-PP4 and pBJF-Flag-PP6; and Dr.

J.W.M. Martens for help with the NCI-H295A cells and Dr. C.E. Flück for preparing NCI-

H295A RNA.

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FOOTNOTES

* This work was supported by National Institutes of Health Grants HD34449 (WLM),

HD41958 (WLM) and HD27970 (SHM).

†To whom correspondence should be addressed: Dept. of Pediatrics,Bldg MR4 Room 209, University of California, San FranciscoSan Francisco, CA 94143-0978. Tel.: 415-476-2598 Fax.: 415-476-6286E-mail: [email protected]

1The abbreviations used are: PP2A, Protein phosphatase 2A; PP4, Proteinphosphatase 4; PP6, Protein phosphatase 6; PCOS, Polycystic ovary syndrome, DHEA,Dehydroepiandrosterone; OR, P450 oxidoreductase; 17OH-Preg, 17OH-Pregnonolone;17OH-Prog, 17OH-Progesterone; DSS, Disuccinimidyl suberate; siRNA, smallinterfering RNA.


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Table 1siRNA target sequences and siRNA duplexes. Three target sequences ( base numbersin parenthesis correspond to the position in mRNA sequence) were identified for PP2A

and PP4; the synthesized sense and antisense siRNAs are shown below each DNAtarget.

____________________________________________________________PP2A

(129) 5’-AAATGTGCAAGAGGTTCGTTG-3’(S) 5’-AUGUGCAAGAGGUUCGUUGtt-3'(AS) 3'-ttUACACGUUCUCCAAGCAAC-5'

(395) 5’-AATGTCTGCGAAAGTATGGGA-3’(S) 5'-UGUCUGCGAAAGUAUGGGAtt-3'(AS) 3'-ttACAGACGCUUUCAUACCCU-5'

(745) 5’-AATTGGTGTCATGATCGGAAT-3’(S) 5'-UUGGUGUCAUGAUCGGAAUtt-3'(AS) 3'-ttAACCACAGUACUAGCCUUA-5'

____________________________________________________________

PP4(91) 5’-AAGGCCAGAGAGATCTTGGTA-3’(S) 5'-GGCCAGAGAGAUCUUGGUAtt-3'(AS) 3'-ttCCGGUCUCUCUAGAACCAU-5'

(537) 5’-AATCGACCGAAAGCAAGAGGT-3’(S) 5'-UCGACCGAAAGCAAGAGGUtt-3'(AS) 3'-ttAGCUGGCUUUCGUUCUCCA-5'

(746) 5’-AAGTGGCACTTCAATGAGACG-3’(S) 5'-GUGGCACUUCAAUGAGACGtt-3'(AS) 3'-ttCACCGUGAAGUUACUCUGC-5'

_________________________________________________________


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Table 2

RT-PCR primer pairs

------------------------------------------------------------------------------------------------------------PP1 (S) 5’-ATGGCGGACGGGGAGCTGAA-3’

(AS)5’-TCACCTTTTCTTCGGCGGATTA-3’

PP2A (S) 5’-ARGGACGACAAGGCGTTCA-3’(AS)5’-TTATAGGAAGTAGTCTGGGGTG-3’

PP4 (S) 5’-ATGGCGGAGATCAGCGACC-3’(AS)5’-CACAGGAAGTAGTCGGCCAC-3’

PP5 (S) 5’-ATGGCGATGGCGGAGGGCGAGA-3’(AS)5’-TCACATCATTCCTAGCTGCAG-3’

PP6 (S) 5’-ATGGCGCCGCTAGACCTGGACA-3’(AS)5’-TCAAAGGAAATATGGCGTTGTC-3’

PP7 (S) 5’-ATGGGATGCAGCAGTTCTTCAA-3’(AS)5’-TTAGCCAAGGTTGGTGACATCA-3’

SETa (S) 5’-GGCAGCCATATGGCCCCTAAACGCCAGTCTCCA-3’(AS)5’-CGCGGATCCTTCGTCATCTTCTCCTTCATC-3’

SETb (S) 5’-GGCAGCCATATGTCGGCGCCGGCGGCCAAAGTC-3’(AS)5’-CGCGGATCCTTCGTCATCTTCTCCTTCATC-3’


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Table 3

Effect of PP2A inhibitors on 17,20 lyase activity in NCI-H295A microsomes

-----------------------------------------------------------------------------------------------------------

Inhibitor % 17,20 Lyase activity ± SD

-----------------------------------------------------------------------------------------------------------

Control 100

Fostriecin (5 nM) 401 ±75

Fostriecin (25 nM) 477 ± 87

Cantharidin (50 nM) 389 ± 64

Okadaic Acid (10 nM) 450 ± 97

bvPhen 127 ± 34

Na-Orthovanadate 110 ± 11

-----------------------------------------------------------------------------------------------------------


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Legends to the figures:

Figure 1 Early steps of sex steroid biosynthesis. P450scc converts cholesterol

to pregnenolone, a C21 D5-steroid. Human P450c17 performs the 17a-

hydroxylase reaction equally well using pregnenolone and progesterone

as substrates, but the 17,20-lyase reaction occurs 50-100 times more

efficiently using 17OH-Preg as substrate rather than 17OH-Prog. Thus

conversion of 17OH-Prog to androstenedione is minimal, and DHEA is the

principal precursor of sex steroid synthesis.

Figure 2 Okadaic acid and phosphatase treatment of NCI-H295A cell

microsomes. (A) Okadaic acid increases 17,20 lyase activity in NCI-

H295A cells. After incubating cells for 60 min with the indicated

concentrations of okadaic acid, cells were harvested and microsomes

were prepared. 17a-hydroxylase activity was measured as the conversion

of [3H] Progesterone to 17OH-Prog (open bars) and 17,20 lyase activity

was measured as the conversion of [3H] 17OH-Preg to DHEA (closed

bars), assayed by thin layer chromatography. The data are shown

normalized to controls with no okadaic acid. The specific activity for the

control 17a-hydroxylase reaction was 0.25 pmol 17OH Preg produced per

microgram of protein per minute and for 17,20 lyase reaction it was 0.35

pmol of DHEA produced per microgram of protein per minute. (B) PP2A

treatment of microsomes in the presence of okadaic acid. Microsomes


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isolated from NCI-H295A cells were treated with 5.0 U/ml of PP2A in the

presence of the indicated concentrations of okadaic acid. Data are

presented as % of activity seen in the absence of okadaic acid; black bars,

without PP2A treatment; white bars, with PP2A treatment. (C) Effect of

okadaic acid on phosphorylation of P450c17. NCI-H295A cells grown in

32P were treated with okadaic acid and the labeled P450c17 was

immunoprecipitated with antiserum bound to Protein A-Sepharose beads

and analyzed on SDS/12%/PAGE by western blotting (top) and

phosphorimaging (bottom).

Figure 3 Effect of protein phosphatases on P450c17 activities. (A) Effect of

phosphatases on 17,20 lyase activity. NCI-H295A cells treated as

indicated and conversion of 17-OH Preg to DHEA was assessed by thin

layer chromatograpgy. PP4 did not inhibit 17,20 lyase activity, whereas

both alkaline phosphatase and PP2A did. The effect of PP2A could be

partially overcome by preincubating PP2A with SET. PP2A and PP4 used

could dephosphorylate other microsomal proteins in vitro (not shown). The

activity of SET was confirmed by its ability to inhibit PP2A. The numbers in

parenthesis indicate activity as a percentage of control from quantitation

by phosphorimager. The specific activity for the 17,20 lyase reaction in

control cells was 0.35 pmol of DHEA produced per microgram of protein

per minute. (B) Response of P450c17 enzyme activities to treatment with

PP2A. Treatment of microsomes from NCI-H295A cells with the indicated


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amounts of PP2A had no detectable effect on 17a-hydroxylase activity

(conversion of Progesterone to 17OH-Prog), but PP2A inhibited 17,20

lyase activity in a dose dependent manner (conversion of 17OH-Preg to

DHEA) with an IC50 value of 2.0 U/ml (mean of three experiments). The

numbers in parenthesis indicate activity as a percentage of control from

the quantitation of data by phosphorimager. The specific activity for the

17,20 lyase reaction was 0.35 pmol of DHEA produced per microgram of

protein per minute, and for the 17a-hydroxylase reaction, 0.25 pmol of

17OH Preg produced per microgram of protein per minute.

Figure 4 Effect of phosphatases on P450c17. (A) Effect of PP2A, PP4 and PP6

on 17,20 lyase activity. NCI-H295A cells were transfected for 36h with

expression vectors for the catalytic subunits of PP2A, PP4 PP6 or empty

vector control and 17,20 lyase activity was measured in isolated

microsomes. PP2A but not PP4 and PP6, reduced 17,20 lyase activity.

The numbers in parenthesis indicate activity as a percentage of control

from the quantitation of data by phosphorimager. (B) Co-

immunoprecipitation of P450c17 with phosphatases. NCI-H295A cell

lysates were immunoprecipitated with antisera to PP2A, PP4 and SET,

separated by SDS/12% PAGE and probed by western blotting with

antiserum to P450c17. NCI-H295A cell lysate contains immunodetectable

P450c17, confirmed by the co-migration with purified recombinant

P450c17. Cell lysates immunoprecipitated with antiserum to PP4 or SET


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did not coimmunoprecipitate P450c17, but lysates immunoprecipitated

with antisera to PP2A did. Western blot analysis of immunoprecipitates

show that antisera against PP2A, PP4 and SET bring down these

proteins.

Figure 5 Effect of PP2A and PP4 suppression by siRNA on 17,20 lyase

activity. (A) Western blots of PP2A and PP4 in NCI-H295A cells 72 hr

after transfection with siRNAs. (B) Thin layer chromatogram showing

increased 17,20 lyase activity of NCI-H295A cells transfected with siRNA

against PP2A as compared to untransfected cells and cells transfected

with a control siRNA of same length. (C) Suppression of PP2A but not

PP4 leads to increased 17,20 lyase activity in NCI-H295A cells. NCI-

H295A cells were transfected with siRNAs against PP2A or PP4 and

17,20 lyase activity was measured in isolated microsomes. Suppression of

PP2A but not PP4 resulted in increased 17,20 lyase activity.

Figure 6 Effect of SET protein on P450c17. (A) SET inhibits PP2A but not PP4.

Purified preparations of PP2A and PP4 were preincubated with or without

SET for 15 min, phosphorylated NCI-H295A microsomal proteins were

added and the amount of free phosphate released into solution was

quantitated at 620 nm using an assay system based on malachite green.

SET inhibited the activity of PP2A but not PP4. Data are mean ± SD of

triplicate assays. (B) SET fosters 17,20 lyase activity. Equal masses of


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protein (SET or BSA) were introduced into NCI-H295A cells by protein

transfection, then microsomes were isolated and assayed for 17,20 lyase

activity. Data are mean ± SD from three different experiments. Specific

activity for the 17,20 lyase reaction in control cells was 0.35 pmol of DHEA

produced per microgram of protein per minute. (C) Western blot showing

that the amount of P450c17 did not change in cells transfected with SET

as compared to controls or cells treated with OA. (D) RT-PCR of

phosphatases in NCI-H295A cells. (E) RT-PCR of SET isoforms in NCI-

H295A cells. RT-PCR was performed using 1mg of total RNA from NCI-

H295A cells for cDNA synthesis using gene specific primers, followed by

35 cycles of amplification and displayed by agarose gel electrophoresis.

Figure 7 Effect of endogenous kinase(s) and PP2A on P450c17. (A)

Endogenous kinases in NCI-H295A cells can foster 17,20 lyase activity.

NCI-H295A microsomes were assayed for 17,20-lyase activity after

treatment with the preparations indicated; activity is shown as the percent

of activity seen with untreated microsomes (left hand bar). The specific

activity for the 17,20 lyase reaction in control cells was 0.35 pmol of DHEA

produced per microgram of protein per minute. ‘Kinase’ refers to the

kinase-enriched fraction eluted from the ATP-Sepharose matrix.

Endogenous kinases were able to reverse the effect of PP2A on 17,20-

lyase activity, confirming the reversible nature protein phosphorylation in

P450c17. (B) The lyase activity of NCI-H295A is confined to microsomes,


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and is not found in cytosol or the kinase fraction. (C) In vitro

phosphorylation of bacterially expressed human P450c17. Bacterially

expressed modified human P450c17 bound to Ni-NTA Sepharose was

incubated with the kinase fraction from NCI-H295A cells plus [32P] ATP.

Controls of P450c17 plus ATP without the kinase fraction or of the kinase

fraction plus ATP without P450c17 showed low incorporation or binding of

32P (Lane 3). Incubation of P450c17 with the kinase fraction showed

substantial incorporation of 32P (Lane 2), a significant part of which could

be removed with PP2A (Lane 4). Lane 1 shows molecular weight markers.

(D) In vitro phosphorylation of P450c17 immunoprecipitated from NCI-

H295A cells. Immunoprecipitated P450c17 was incubated with the kinase

fraction from NCI-H295A cells plus [32P] ATP. Incorporation of 32P into

P450c17 was increased in presence of the kinase fraction from NCI-

H295A cells.

Figure 8 Proposal for the regulation of 17,20 lyase activity by reversible

phosphorylation of P450c17. Arrows indicate stimulation, blocked lines

indicate inhibition. It is known that a cAMP dependent serine/threonine

(S/T) kinase stimulates the 17,20 lyase activity of P450c17 and a tyrosine

(Y) kinase, SET and okadaic acid (OA) inhibit PP2A. The nature of the

kinases that regulate SET and that presumably regulate S/T kinase are

unknown.


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Amit V. Pandey, Synthia H. Mellon and Walter L. MillerP450c17

Protein phosphatase 2A and phosphoprotein SET regulate androgen production by

published online November 19, 2002J. Biol. Chem.

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Protein phosphatase 2A and phosphoprotein SET regulate ... · granulosa cells, pregnenolone is...

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