The Pennsylvania State University

The Graduate School

Department of Biochemistry and Molecular Biology

3,5-BISTRIFLUOROMETHYL PYRAZOLE (BTP)

COMPOUNDS AND REGULATION OF STORE-

OPERATED CALCIUM CHANNELS BY THE ACTIN-

BINDING PROTEIN DREBRIN

A Thesis in

Biochemistry, Microbiology, and Molecular Biology

by

Jason C. Mercer

© 2005 Jason C. Mercer

Submitted in Partial Fulfillment of the Requirements

for the Degree of

Doctor of Philosophy

May 2005

ii

The thesis of Jason C. Mercer has been reviewed and approved* by the following:

Avery August Associate Professor of Immunology Thesis Advisor Chair of Committee Pamela H. Correll Associate Professor of Veterinary Science Richard J. Frisque Professor of Molecular Virology Andrew J. Henderson Associate Professor of Veterinary Science Blake R. Peterson Associate Professor of Chemistry Robert A. Schlegel Professor of Biochemistry and Molecular Biology Head of the Department of Biochemistry and Molecular Biology *Signatures are on file in the Graduate School

iii

ABSTRACT

Stimulation of tyrosine kinase coupled receptors, such as the T cell receptor in T

lymphocytes, results in activation of phospholipase-Cγ (PLCγ) which hydrolyzes

phosphoinositol 4,5, bisphosphate (PIP2) to generate the second messengers inositol 1,4,5

trisphosphate (IP3) and diacylglycerol (DAG). Similarly, IP3 and DAG are generated by

phospholipase-Cβ (PLCβ) downstream of G-protein coupled receptors in other cell types.

This results in increased intracellular calcium concentration [Ca2+]i due to the action of

IP3 on IP3 receptors (IP3R) in the endoplasmic reticulum (ER) membrane which, when

activated, stimulate the release of ER calcium stores into the cytoplasm. Emptying of the

ER calcium stores stimulates entry of extracellular calcium through store-operated

channels (SOCs), thus maintaining the higher concentration of intracellular Ca2+. [Ca2+]i

increases play a critical role in a variety of cellular processes such as transcription factor

activation and cytoskeletal reorganization.

Although very little is known about the regulation of SOCs, actin cytoskeletal

changes have been suggested to be essential for their operation. However, actin

cytoskeletal changes appear to be dispensable for ER calcium release. A recent model

for activation of SOCS has been proposed wherein the signal between the ER and the

plasma membrane that activates SOCs involves a secretion-like mechanism that is

blocked by thick cortical actin.

Recently, a class of compounds called BTPs (3,5-bistrifluoromethyl pyrazoles)

was found to inhibit activation of the calcium regulated transcription factor Nuclear

Factor of Activated T cells . We have determined that BTP acts by blocking store-

operated calcium entry following Ca2+ store depletion by ionomycin in Jurkat T cells.

iv

Utilizing an affinity purification approach we have identified the actin

reorganizing protein drebrin as a likely target of BTP. Drebrin is a member of the

ADF/cofilin family of actin binding proteins and has been implicated in actin

rearrangements driving dendritic spine outgrowth in neurons. We demonstrate that

drebrin expression is essential for activation of store-operated calcium entry in Jurkat T

cells as reduction in drebrin expression by siRNA treatment results in a block in SOC

mediated [Ca2+]i increase but not ER Ca2+ release, similar to BTP treatment.

Additionally, we show that BTP is able to block drebrin dependent actin rearrangement.

Based on our findings, we propose that BTP blocks SOC activation by preventing drebrin

mediated cytoskeletal changes that are necessary for activation of store-operated

channels.

v

TABLE OF CONTENTS

LIST OF FIGURES…………...…………………………………………………...vii LIST OF TABLES………………………………………………………………… x

ABREVIATIONS………………………………………………………………….. xi ACKNOWLEDGEMENTS………………………………………………………... xiii CHAPTER 1. Introduction…………..………………………….…...……………..1

Regulation of intracellular Ca2+ homeostasis……………………………… 2 Store-operated Ca2+ entry….………………………………………………. 4 The TRP family of ion channels……..……….…………………..………... 7 Activation of store-operated channels……….…………………………….. 9 Regulation of NFAT family transcription factors.…………..…………....... 16 Actin cytoskeleton and cellular processes…………………………………. 20 Drebrin……………………………………………………………………... 22 3,5-bistrifluoromethyl pyrazole (BTP)…………………………………….. 25 Aims of this study……………………………………..…………………….28 Hypothesis………………………………………………….……………… 28 Specific Aims………………………………………………….……...……. 28 CHAPTER 2. Materials and Methods…………………….………………………. 30

Cells, antibodies, plasmids, and reagents………………….……………......31 Western Blot……………………………………………………………….. 32

Fluorescent calcium measurement….……………………………………... 32 siRNA knock-down…………………………………..……………………. 33 Coomasie stain………………………………………………………………33

In-gel digest and mass spectrometry……………………………………..… 34 Immunofluorescence analysis……………………………………..……….. 35 ELISA…………………………………………………….………….…….. 35 CHAPTER 3. Effects of BTP……………………………………………………... 37

Rationale…………………………………………………………………… 38

BTP inhibits NFAT activation…………………………………………….. 38 BTP blocks calcium regulated store-operated channels…………………... 44 BTP inhibits dynamic cytoskeletal changes in response to calcium ionophore…………………………………….…………………………… 48 BTP does not inhibit tyrosine kinase activation…………………………... 50 BTP blunts MAP kinase signaling…………………………………………. 52

Discussion………………………………………………………………….. 55

vi

CHAPTER 4 Purification and characterization of potential targets of BTP………. 58 Rationale…………………………………………………………………… 59

Purification and mass spec identification of BTP-binding proteins……….. 59 BTP binds to the N-terminal portion of drebrin…………………….…….. 77

BTP blocks drebrin function………………………………………………. 79 BTP does not affect drebrin protein expression…………………………… 82

Drebrin expression is required for SOC activation………….……………... 84 Drebrin expression is necessary for NFAT activation…………..………..... 89 Discussion………………………………………………………………….. 91 CHAPTER 5. Discussion………………………………………………………….. 95 CHAPTER 6. Future Directions…………………………………………………... 108 APPENDIX A. Characterization of the serine/threonine kinase…………………… 114

Introduction………………………………………………………………... 115 Effects on TCR signaling……………………………………….................. 119 Discussion (Part I)…………………………………………………………. 135 Effects on cell-cycle……………………………………………….............. 136 Discussion (Part II)………………………………………………………… 142

BIBLIOGRAPHY………………………………………………………………….. 143

vii

LIST OF FIGURES Figure 1.1. Activation of Calcium Signaling ………………………………… 3 Figure 1.2. Calcium Influx Factor (CIF) model for store-operated calcium

activation…………………………………………………………. 10

Figure 1.3. Conformational Coupling model for SOC activation……………… 12

Figure 1.4. Secretion-like coupling model for SOC activation………………… 14 Figure 1.5. Domain organization of NFAT…………………………………….. 18 Figure 1.6. Activation of NFAT by calcium…...………………………………. 19 Figure 1.7. Domain organization of Drebrin………………………………….. 23

Figure 1.8. Structure of BTP2………………………………………………….. 27 Figure 3.1. BTP inhibits NFAT in multiple cell types…………………………. 40

Figure 3.2. Constitutively active calcineurin overcomes BTP inhibition……… 41 Figure 3.3. BTP inhibits NFAT nuclear translocation……...………………….. 42

Figure 3.4. BTP blocks intracellular calcium mobilization….………………….46

Figure 3.5. BTP inhibits entry of extracellular calcium……………………….. 47 Figure 3.6. BTP inhibits dynamic actin rearrangement following ionomycin

treatment………………………………………………….………. 49

Figure 3.7. BTP does not affect tyrosine phosphorylation of cellular proteins, PLCγ1, or ITK activation…………………………..……………… 51

Figure 3.8. BTP inhibits MAP kinase activation………………………………..53

Figure 4.1. Synthesis of BTP-biotin…………………………………………… 61 Figure 4.2. BTP-biotin retains inhibitory activity………………………..……. 62

Figure 4.3 Structure of Estrone-BTP…………………………………………... 63 Figure 4.4. Estrone-BTP retains inhibitory activity towards IL-2 production…. 64

Figure 4.5. Schematic representation of BTPBP affinity purification………..... 67

viii

Figure 4.5. Purification of BTP-binding proteins……………………………….68 Figure 4.6. BTP binds drebrin………………………………………………….. 74

Figure 4.7. BTP/drebrin interaction remains intact when BTP is expressed in

bacteria…………………………………………………………….. 76

Figure 4.8. Mapping of BTP/drebrin interaction……………………………….. 78

Figure 4.9. BTP inhibits drebrin function……..……………………………….. 80

Figure 4.10 Quantification of filopodia-like extensions………………………... 81 Figure 4.11. BTP does not affect drebrin protein expression…………………… 83

Figure 4.12. Time-course of drebrin knock-down by siRNA…………………… 85

Figure 4.13. Loss of drebrin expression prevents calcium flux…………………. 87

Figure 4.14. Drebrin is essential for store-operated channel function………….. 88

Figure 4.15. Drebrin is essential for NFAT activation………………………….. 90 Figure 4.16. Structure of BTPs…………………………………………………. 92

Figure 5.1. Possible role for drebrin in the CIF model………………………… 102 Figure 5.2. Possible role for drebrin in the conformational coupling model….. 103 Figure 5.3. Possible role for drebrin in the secretion-like coupling model…….. 104 Figure 5.4. Model for drebrin involvement in SOC activation…..……………. 105 Figure 6.1. Structure of BTP derivatives for determining active portion of BTP

molecule…………………………………………………………… 110 Figure A.1 Schematic representation of the Ste20 Group of serine/threonine

kinases………………………………………………………………116

Figure A.2 Schematic representation of LOK………………………………….. 117 Figure A.3 LOK kinase domain inhibits antigen induced IL-2 production

in Jurkat T cells…………………………………………………….. 120

Figure A.4 LOK downregulates MEKK1 induced activation of the CD28RE transcriptional activity in Jurkat T cells……………………………. 122

ix

Figure A.5 LOK downregulates MEKK1 induced activation of AP-1 transcriptional activation in Jurkat T cells…………………………. 124

Figure A.6 LOK downregulates MEKK1 induced activation of NFκB transcriptional activity in Jurkat T cells. ………………………….. 125

Figure A.7 LOK kinase domain inhibits NFAT activation…………………….. 126 Figure A.8 LOKK decreases tyrosine phosphorylation following

TCR stimulation……………………………………………………. 128

Figure A.9 LOKK inhibits TCR-ζ chain and ZAP-70 phosphorylation……….. 129 Figure A.10 CD28 costimulation does not rescue tyrosine phosphorylation

in LOKK cells……………………………………………………… 130

Figure A.11 Jurkat-LOKK cells are deficient in lipid raft associated tyrosine phosphorylation…………………………………………… 132

Figure A.12 Normal localization of Lck to lipid rafts in Jurkat LOKK cells…….133 Figure A.13 LOK kinase domain does not localize to lipid rafts............... ……... 134 Figure A.14 LOK coiled-coil region exhibits a unique sub-cellular localization

pattern……………………………………………………………… 137

Figure A.15 LOKK causes cells to arrest in G2/M phase following serum starvation…………………………………………………………… 138

Figure A.16 Serum allows LOKK expressing cells to overcome G2/M phase Arrest………………………………………………………………. 139

Figure A.17 Serum allows LOKK expressing cells to overcome G2/M phase arrest (20 hrs)………………………………………………………. 140

Figure A.18 Serum allows LOKK expressing cells to overcome G2/M phase arrest (24 hrs)………………………………………………………. 141

x

LIST OF TABLES

Table 4.1 Peptide sequences obtained for p120 (Yale)…………….................. 67

Table 4.2 Peptide sequences obtained for p120 (Penn State)………………… 68

Table 4.3 Peptide sequences obtained for p75………………………………... 69

Table 4.4 Peptide sequences obtained for p40………………………………... 70

Table A.1 Amino acid homology between LOK and other Ste20 family Member……………………………………………………………..118

xi

ABREVIATIONS

3,5-bistrifluoromethyl pyrazole (BTP) phospholipase-Cγ (PLCγ) phosphoinositol 4,5, bisphosphate (PIP2) 1,4,5 trisphosphate (IP3) endoplasmic reticulum (ER) Plasma membrane (PM) intracellular calcium concentration ([Ca2+]i) IP3 receptors (IP3R) store-operated channels (SOCs) transient receptor potential (TRP) transient receptor potential-canonical (TRPC) transient receptor potential-melastatin (TRPM) transient receptor potential-vanilloid (TRPV) small interfering RNA (siRNA) RNA interference (RNAi) calcium-release activated channel (CRAC) calcium-release activated channel current (ICRAC) Calcium Inducible Factor (CIF) Guanosine triphosphate (GTP) latrunculinB (LatB) Nuclear Factor of Activated T cells (NFAT) calcineurin (CN)

xii

constitutively active calcineurin (CA-CN) filamentous actin (F-actin) globular actin (G-actin) T cell Receptor (TcR) cytochalasin D (cytD) THelper type 1 (TH1) THelper type 2 (TH2) cyclosporin A (CsA) Fetal Calf Serum (FCS) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) Phorbol-12-myristic acid (PMA) PMA/ionomycin stimulation (P/I) c-jun N terminal kinase (JNK) extracellular related kinase (ERK) Green Fluorescent Protein (GFP) estrone-BTP (E-BTP) Lymphocyte-Oriented Kinase (LOK) LOK kinase domain (LOKK)

xiii

ACKNOWLEDGEMENTS

I wish to thank my advisor, Avery August, for all of his support and advice

throughout my graduate career. He has been a wonderful mentor and friend to me in my

years at Penn State. He has also provided stimulating scientific discussion about my

thesis project and about other general scientific topics. I also wish to thank each of my

committee members, Pamela Correll, Dick Frisque, Andrew Henderson, and Blake

Peterson for their support and advice. I also thank Dr. Blake Peterson and his graduate

student Laurie Mottram for a productive and fruitful collaboration and for providing me

with the various derivatives of BTP used in this study. Additionally I would like to thank

members past and present of the August lab and the other Immunology Research labs at

Penn State for their technical help, discussion, and friendship. Finally, I would further

like to thank my wife, Robyn, for her support and patience.

1

CHAPTER 1

Introduction

2

Cells use a wide variety of signaling mechanisms in order to relay information

between different sites within the cell. These mechanisms often combine the actions of

many different proteins that serve both enzymatic and structural roles in the process. The

end result of such signaling cascades can range from changes in cell shape to

transcriptional activation. Often one signaling pathway will serve to activate many

different functional outcomes. Signals initiated by changes in intracellular Ca2+

concentration are a prime example of one type of signal which activates many different

functional outcomes. Ca2+ signaling is a highly conserved process in most non-excitable

cells yet the processes that regulate Ca2+ signaling are still poorly understood.

Regulation of intracellular Ca2+ homeostasis

Increases in intracellular calcium concentration play a critical role in a number of

cellular processes such as transcriptional activation and cytoskeletal rearrangement.

Under resting conditions intracellular calcium concentration is approximately 100 nM

and upon stimulation can rise as high as 1-10 µM (1). Calcium mobilization following

receptor stimulation is achieved via two distinct phases (Fig. 1.1). First, stimulation of

tyrosine kinase coupled receptors or G-protein coupled receptors, results in activation of

phospholipase-Cγ (PLCγ) or phospholipase-Cβ (PLCβ) respectively. The PLCs

hydrolyze phosphoinositol 4,5, bisphosphate (PIP2) in the plasma membrane to generate

the second messengers inositol 1,4,5 trisphosphate (IP3) and diacylglycerol (DAG) (2).

IP3 then diffuses from the plasma membrane to the endoplasmic reticulum (ER) where it

acts on IP3 receptors (IP3R) which, when activated, stimulate the release of ER calcium

stores

3

Figure 1.1. Activation of cell surface receptors coupled to PLCs results in hydrolysis of

membrane PIP2 to generate the second messengers DAG and IP3. IP3 binds to IP3Rs in

the ER membrane resulting in release of ER Ca2+ stores. Emptying of the ER stores

triggers activation of plasma membrane associated Ca2+ channels (SOCs) that allow

Ca2+ entry from the extracellular space. ER calcium stores can also be depleted by

inhibition of SERCA pumps located in the ER membrane, or by action of the IP3R

agonist ionomycin.

TCR/BCRCa2+

SOC

PLCγPIP2

DAG

IP3

ER

IP3R

Ca2+

?

?Ca2+

Ca2+

SERCACa2+

thapsigargin

ionomycin

Activation of Ca2+ signaling

4

into the cytoplasm, resulting in a rapid, but transient spike in intracellular calcium

concentration [Ca2+]i (3, 4). The second phase of calcium mobilization is triggered by

emptying of the ER calcium stores. The filling status of the ER store is relayed via an

unknown mechanism to unidentified channels within the plasma membrane known as

store-operated channels (SOC). These channels open in response to ER store depletion

and allow Ca2+ to enter the cell from the extracellular space (for review see (5)). SOC

activation causes a stable increase in [Ca2+]i that can last for several minutes to hours

depending on the cell type. While the process of IP3 signaling to IP3Rs in the ER to

cause release of intracellular calcium stores is well established, little is known about the

mechanism by which SOCs are activated.

Store-operated Calcium entry

The phenomenon of store-operated calcium entry was first inferred from

observations using 86Rb+ efflux as an indicator of Ca2+ activated K+ channel activity to

infer the intracellular Ca2+ concentration. Since the K+ channels responsible for 86Rb+

efflux are activated by high [Ca2+]i, this served as an indirect measure of the [Ca2+]i.

These initial experiments indicated that the intracellular pool, once filled, was resistant to

extracellular calcium chelation, but extracellular Ca2+ was required for refilling the stores

following depletion (6). Once initiated, continued receptor stimulation was not required

for store repletion (6). Importantly, [Ca2+]i did not appear to increase during this process

(7). This idea was first proposed by J.W. Putney in an influential review where he

proposed that because the pool appeared to be replenished from extracellular sources, but

did not require continued receptor stimulation, that the trigger for opening the plasma

5

membrane channels was in fact the emptying of the intracellular stores. He also believed

that because [Ca2+]i did not change that the extracellular Ca2+ would have to be shunted

directly into the store without entering the cytoplasm (8). Thus, IP3’s activity toward the

ER stores was an intermediary step causing store-depletion, however it was the process of

store-depletion itself that triggered the opening of the plasma membrane channels. With

the introduction of the calcium sensitive intracellular dye Fura-2AM, parts of this original

model needed to be revised. First, one could now detect obvious increases in [Ca2+]i

during store refilling. This of course partially negates the idea that the stores are filled

directly without Ca2+ entering the cytoplasm (7). The second problem that arose was that

experiments had shown that when La3+, which enters the pore of Ca2+ channels but

cannot be released, was used to block Ca2+ efflux from the plasma membrane the stores

could be replenished even in the absence of extracellular Ca2+, indicating that

extracellular Ca2+ is not necessarily required for replenishment, and that the stores could

refill from Ca2+ within the cytoplasm (7, 9). This was also confirmed using the

cytoplasmic Ca2+ chelator Bapta-AM. When cytoplasmic calcium is chelated, this

inhibits the rate of refilling of the pools (10). Therefore, the original capacitative model

stands with some correction. However, the idea that an IP3 sensitive intracellular pool

regulates plasma membrane Ca2+ permeability stands. In fact we now know that this pool

is contained within the endoplasmic reticulum and is activated by binding of IP3 to IP3

receptors located on the ER membrane (11-13). However, the idea that somehow the

plasma membrane is able to directly fill the ER without Ca2+ entering the cytoplasm

seems to be refuted. These studies established a set of criteria by which potential store-

operated channel candidates should be assessed. 1) It must be activated by depletion of

6

the IP3 sensitive intracellular Ca2+ store. Note, however, that IP3 is not required for store-

depletion as Ca2+-ATPase inhibitors such as thapsigargin are capable of producing the

same results without increasing the levels of phosphoinositols, indicating that

phosphoinositols are not required for SOC activation (14, 15). 2) It cannot be directly

activated by IP3 or diacylglycerol.

The definition of the SOC was further refined with the first electrophysiological

measurement of a store-operated calcium influx by Hoth and Penner (16). This was no

small task as the amount of Ca2+ that enters the cell during store replenishment is

relatively small and produces a current that is below the level of background noise in

most electrophysiological measurements. They overcame this limitation by using mast

cells which have very little background current, and by increasing the extracellular Ca2+

from 1µM to 10µM. This new Ca2+ current was termed “calcium release-activated

current” (ICRAC). ICRAC was found to be highly selective for Ca2+ over Ba2+, Sr2+, or Mn2+

(16). Additionally, it was determined that ICRAC has very little fluctuation in current (i.e.

noise), indicating that current amplitude through any one channel is low (16).

Electrophysiological measurements have allowed the refinement of the definition of

store-operated calcium current. However, although ICRAC has been described in several

types, mostly hematopoeitic cells, it has not been found in all cells. This may reflect

either the presence of high background current in the cell preventing analysis, or it could

indicate that store-operated channels are regulated differently in different cell types,

perhaps by heterodimerization of the ion-channel proteins, differential surface expression

of the channels, different regulatory protein binding, or a combination of any of these

factors. Nevertheless, these initial electrophysiological characterizations have provided

7

an electrical fingerprint with which to match potential ion-channels. However, these

criteria have also fueled controversy in the field whenever new ion-channels are proposed

to be the elusive SOC. To date, no single channel has been described which matches all

of the electrophysiological characteristics of an SOC. The major caveat to experiments

used to verify potential SOC candidates has been the use of ectopic or overexpression

studies which could create artifacts such as favoring homodimerization of the

overexpressed protein when it would normally heterodimerize with a less abundant

subunit. Even in knockout experiments one must consider the possibility of

compensation by other subunits. As a result of these limitations no ion-channel has been

verified as the SOC. However, as discussed below, one family of ion-channels has

emerged as the most likely candidates.

The TRP family of ion-channels

The term “TRP” stands for transient receptor potential and was first used to

describe a mutant phenotype in D. melanogaster characterized by altered depolarization

of the photoreceptor cells in the eye when exposed to light (17). Normally, exposure of

photoreceptor cells results in activation of a G-protein coupled receptor which leads to

production of IP3 and subsequent elevation in [Ca2+]i. This increase in [Ca2+]i is

prolonged in response to continued light exposure (17, 18). However, TRP mutants

respond by initially depolarizing, but the depolarization is transient (19). An additional

mutant was also identified and called TRP-like (TRPL) (20). The proteins responsible

for these mutations were thought to be Ca2+ channels because of their high degree of

structural homology to voltage-gated Ca2+ channels (21, 22). Their identity as Ca2+

8

channels was later confirmed by expressing them in Sf9 cells, which lead to increased

plasma membrane permeability of Ca2+ following stimulation of receptors coupled to

PLC (22-25). Expression of TRPL extended the increases in [Ca2+]i caused by receptor

stimulation and appeared to be somehow activated by the phosphatidylinositol response

(23, 26). On the other hand, expression of TRP lead to increased [Ca2+]i following

treatment with thapsigargin (25). Thus TRP became a candidate for a store-operated

channel. This sparked the search for a mammalian homologue of the TRP channel.

To date, at least 28 mammalian TRP channels have been cloned. These have been

divided into six related protein families, the TRPC, TRPV, TRPM, TRPP, TRPML

families, and ANKTM1 comprises its own family (27). All TRP channels are six-

transmembrane polypeptide subunits that assemble as tetramers to form cation-permeable

pores (27). Although TRP genes are widely expressed, different subunits are expressed

in different combinations in a tissue-specific manner (28).

The TRPC family shares the most homology with Drosophila TRP. They are

activated in response to stimulation of G-protein coupled of tyrosine kinase coupled

receptors (27). The TRPCs have been shown to form heteromultimers with unique

properties from those of their homomultimer counterparts, as is the case with

TRPC1/TRPC4 or TRPC1/TRPC5 multimers (27, 29, 30). TRPC4-/- mice exhibit

decreased lung microvascular permeability as a result of a defect in store-operated

calcium entry in the lung endothelial cells (31, 32). Additionally, knockdown of TRPC4

expression in mouse mesangial cells produced a block in SOC activation (33). TRPC3

has been shown to be activated both in a store-dependent and store-independent manner

depending on the level of protein expression (34). When TRPC3 is expressed at lower

9

levels, such as in the DT40 chicken B cell line, it is activated by store depletion.

However, when TRPC3 expression is increased by transfection with a plasmid encoding

TRPC3 under a strong promoter, it can then be activated directly by DAG (34, 35).

Additionally, TRPC3 was shown to be essential for T cell receptor induced Ca2+ entry in

Jurkat T cells as mutants deficient in TRPC3 expression exhibited only a transient

increase in intracellular calcium. This deficiency was characterized as a lack of Ca2+

entry from the extracellular space (36). Despite this evidence, none of the TRPC family

members have been unequivocally demonstrated to be store-operated channels. This

problem likely stems from the shortcomings of heterologous expression systems or

compensation mechanisms set in place upon total protein loss. Also, store-operated

channels in different cell types probably utilize different channel subunits and regulate

them in different ways so that in any cell type store-operated calcium entry exhibits

slightly different characteristics.

Activation of store-operated channels

Following the general acceptance that intracellular store depletion leads to activation of

plasma membrane channels, the next major question to arise was: how is the filling

status of the ER relayed to the plasma membrane? This question has yet to be answered

satisfactorily. There are, however, three main hypotheses that attempt to explain this

process.

The initial hypothesis for communication between the ER and plasma membrane

involved production of a diffusible second messenger that would activate channels in the

plasma membrane, similar to the action of IP3 on IP3 receptors in the ER (Fig. 1.2). Two

10

Figure 1.2. The calcium inducible factor (CIF) model postulates that a diffusible second

messenger is released from the ER when ER Ca2+ stores are depleted. The CIF then

diffuses to the plasma membrane where it activates the opening of store-operated

channels.

TCR/BCRCa2+

SOC

PLCγPIP2

DAG

IP3PKC

Ras

ER

IP3R

Ca2+

Ca2+

Ca2+

CIF

Calcium influx factor (CIF) model

11

groups have reported the possible existence of this factor. One group prepared extracts

from stimulated Jurkat T cells and were able to increase [Ca2+]i in several cell lines (37).

They partially purified this factor, which they termed calcium influx factor (CIF), and

reported that it was less than 500 Da and contained a phosphate group that, when

removed, inactivated the molecule (38). However, when a different group attempted to

confirm these results they found that the CIF prep procedure described had different

effects on different cell lines. In Jurkat T cells and in an astrocytoma cell line the extract

evoked Ca2+ entry without intracellular Ca2+ release, as previously described (39). When

the extract was applied to mouse lacrimal cells, hepatocytes, and X. laevis oocytes, all

known to exhibit store-operated calcium entry, the results were inconsistent with the

presence of a CIF. In the lacrimal cells and oocytes, the extract evoked a biphasic Ca2+

response consistent with activation of a receptor (39). In the hepatocytes, no detectable

Ca2+ signal was produced (39). Additionally, this study concluded that several factors

were present in the extract that could affect Ca2+ signaling, most likely through receptor

activation rather than activation of SOCs (39). In contrast, Thomas and Hanley reported

that CIF was able to activate SOCs in X. laevis oocytes, consistent with the original study

(40). However, in a follow-up study they revealed that CIF actually synergizes with IP3

to increase calcium release from intracellular stores (41). They suggest that CIF probably

helps to increase sensitivity of the IP3 receptors thus allowing lower concentrations of IP3

to activate store depletion (41).

The other major study reporting a calcium influx factor came from a yeast mutant

defective in the ER Ca2+-ATPase pumps that maintain the ER calcium store. Extracts

12

Figure 1.3 The conformational coupling model hypothesizes that regions of the ER are

in direct contact with or in very close proximity to regions of the plasma membrane that

contain store-operated channels. When ER stores are depleted, physical interaction

between the ER and PM takes place to activate store-operated channels.

TCR/BCRCa2+

SOC

PLCγPIP2

DAG

PKC

Ras

IP3

ER

IP3R

Ca2+

Ca2+Ca2+

Conformational coupling model

13

from these cells were shown to activate ICRAC in Jurkat T cells, Ca2+ influx in Xenopus

oocytes, and an apparently store-sensitive channel in smooth muscle cells (42, 43).

While the CIF model continues to be explored, there have been no further reports

of the possible factor responsible for this action. This is quite significant because CIF

was first reported 12 years ago and at that time was partially purified and appeared to be

relatively stable even when heated (37, 38, 40). While the CIF model remains plausible,

it is troublesome that the factor has not been identified such a long time after its initial

description.

The second model is known as the conformational coupling model. This model

hypothesizes that the channels are somehow coupled to receptors in the ER that change

conformation in response to store-depletion, thus activating the plasma membrane SOC

(Fig. 1.3). The most likely candidate for the ER receptor is the IP3R, which has

demonstrated dose dependent activity towards IP3 that could be explained by

conformational changes induced by Ca2+ binding (44). This model is similar to a

confirmed mechanism in skeletal muscle cells where the ryanodine receptors, which

share significant homology to the IP3Rs, in the sarcoplasmic reticulum couple to

dihydropyridine receptors in the plasma membrane to allow extracellular calcium entry

(45). In this light it was promising when the TRPC1 cation channel was reported to be

coupled to IP3R by the adapter protein, Homer1, and that this controlled its activation

(46). However, this seems to be an unlikely mechanism for controlling SOCs since DT40

B cells lacking all three IP3Rs are still able to mobilize calcium entry in response to

thapsigargin, indicating that IP3Rs are dispensable for calcium entry (47-49).

14

Figure 1.4. The secretion-like coupling model hypothesizes that store-operated channels

or subunits of them are sequestered in vesicles in the cytoplasm. When ER stores are

depleted, these vesicles are trafficked to the plasma membrane where they can either

integrate into the membrane, or associate via a more transient interaction.

TCR/BCRCa2+

SOC

PLCγPIP2

DAG

PKC

Ras

IP3

ER

IP3R

Ca2+

Ca2+

Ca2+

SOC

Rac

Actin

Secretion-like coupling model

15

The caveat to this apparent flaw in the model is that the IP3R-/- DT40 cells still express

the ryanodine receptor which could, in the absence of IP3Rs, provide partial

compensation in this regard.

The third model is called the secretion-like coupling model. This model raises the

possibility that store-operated channels are controlled via a mechanism that resembles

regulated exocytosis (Fig. 1.4)(50, 51). The demonstration that SNAP-25, a protein

involved in vesicle docking to the cell membrane, is essential for store-operated calcium

entry makes this a particularly plausible argument (50). This is also in general agreement

with reports from several groups demonstrating a role for GTP and Rho family small G

proteins in calcium entry and in vesicle trafficking (52-54). Recently, members of the

TRPC family of cation channels have been shown to be regulated by differential surface

expression in a Rac dependent manner, consistent with a secretion-like model (55). One

of the more attractive aspects of this model is that it suggests the involvement of the actin

cytoskeleton in calcium entry. Calcium entry can be facilitated in cells that have a thick

cortical actin layer, such as lymphocytes, simply by treatment with actin depolymerizing

agents such as latrunculinB (LatB) (56). Conversely, treatment with agents that drive

actin into the polymerized state such as jasplakinolide prevent the activation of calcium

entry (51). Interestingly, manipulation of the actin cytoskeleton does not affect calcium

release from the ER (51). Presumably, a large vesicle containing either the SOCs

themselves or factors that activate them would be prevented from interacting with the

plasma membrane if a “wall” of cortical actin were present. A small factor such as IP3

would be able to pass through unhindered (51). This model could alternatively be

explained if physical interaction between the ER and plasma membrane were required to

16

facilitate calcium entry. In this scenario, a thick cortical actin layer might prevent such

an interaction. Indeed, evidence exists to support this idea. First, the direct coupling

model presented earlier supports the possibility of direct interaction of the ER and PM

(44). Additionally, sites of IP3R induced Ca2+ depletion have been observed

microscopically at sites of Ca2+ entry (57). This would suggest that there is at least a

close spatial relationship between sites of store depletion and Ca2+ entry. Perhaps one of

the more interesting developments for the secretion-like coupling model is the discovery

that TRPC channels, which have been identified as possible SOCs, are sequestered in

intracellular vesicles in resting cells and upon stimulation are rapidly transported to the

plasma membrane (55). This process was determined to be dependent upon Rac

activation as expression of a dominant negative form of Rac blocked translocation of the

vesicles (55). Yet, as discussed below, the TRPC channels have not been categorically

confirmed as SOCs and considerable debate exists as to their role in store-operated

calcium entry.

It should be noted that the three models for SOC activation presented here are not

necessarily mutually exclusive. For instance, CIF could cause a conformational change

in the IP3R in order to activate SOCs in the plasma membrane. This would combine

elements of both the CIF and conformational-coupling models. Particularly in the case of

secretion-like coupling, this model could in fact be a more accurate representation of the

mechanism by which the conformational coupling model works. Conformational

changes in the IP3R may be what causes release of the SOC vesicles that get “secreted” to

the plasma membrane.

17

Regulation of NFAT family transcription factors

Transcriptional activation by the transcription factor, nuclear factor of activated T

cells (NFAT) is an example of how sustained elevated [Ca2+]i affects cellular processes.

NFAT is essential for transcription of many cytokine genes that are involved in

regulation of an immune response. In the resting state NFAT is highly phosphorylated on

serine residues that fall within four conserved serine-rich motifs (SRR-1, SPxx, SRR-2,

and KTS motifs(58). In response to intracellular Ca2+ concentration increase, the

phosphatase calcineurin(CN), which is activated by binding of the small Ca2+-binding

protein calmodulin, binds to PxIxIT consensus sequences located at the N- and C-

terminal ends of the regulatory region and dephosphorylates the serine residues located

within the SRR-1, SP-1, SP-2, and SP-3 regions (58). These serines make up 13 of the

14 identified phosphorylated serines within NFAT1)(Fig. 1.5). Dephosphorylation of the

regulatory region exposes the nuclear localization sequence of NFAT allowing it to be

transported to the nucleus where it is transcriptionally active (Fig. 1.6). One particularly

important aspect of NFAT activation is the requirement for sustained elevated [Ca2+]i to

maintain NFAT in the activated state (59). Following chelation of extracellular Ca2+,

NFAT is rapidly phosphorylated and exported from the nucleus (59).

18

Figure 1.5. NFAT contains N- and C-terminal activation domains, a DNA binding

domain, and a highly phosphorylated regulatory domain. The regulatory domain is

flanked by PxIxIT sequences that the phosphatase calcineurin binds to. Four highly

serine phosphorylated regions within the regulatory domain are dephosphorylated by

calcineurin to expose the nuclear localization sequence (NLS) allowing NFAT to

translocate to the nucleus upon activation.

AD Regulatorydomain DNA-binding domain

SRR-1 SP-1 SP-2 SP-3NLS

Calcineurin binding domain (PxIxIT)

phosphorylated serines

NFAT

Domain organization of NFAT

19

Figure 1.6. In the resting state NFAT is highly phosphorylated and kept in the

cytoplasm. Upon stimulation, PLC is activated and cleaves PIP2 to produce IP3. IP3

acts on IP3-receptors (IP3R) in the ER and causes release of intracellular calcium stores.

Depletion of intracellular signals the SOCs in the PM, through an unknown mechanism,

to open. This causes an overall increase in intracellular calcium concentration, which

activates calmodulin to bind and activate calcineurin that in turn dephosphorylates and

activates NFAT.

Calmodulin

TCR/BCRCa2+

SOC

PLCγPIP2

DAG

IP3 PKC

Ras

ER

IP3R

Ca2+

?

?Ca2+

Ca2+

calcineurin

NFAT

NFATP P P P

PPPPPPP

PP

Activation of NFAT by calcineurin

20

Actin cytoskeleton and cellular processes

Actin is maintained in two forms in the cell, globular (G-actin) and filamentous

(F-actin). G-actin is the monomeric form of actin. In order to form a microfilament G-

actin is polymerized in an ATP-dependent fashion. The process of actin fiber growth is

highly regulated. Actin filaments can form long, linear bundles as seen in structures such

as stress fibers, highly branched sheets such as with lamellapodia, or thick networks of

branched fibers as in the cortical actin network that maintain the overall shape of the cell

membrane, and a variety of structures that combine different types of actin fibers. While

the major functions of the actin cytoskeleton appear to be in maintenance of cell shape

and motility, a number of specialized cell processes have evolved to utilize the actin

cytoskeleton. For instance, actin rearrangements following T cell receptor stimulation

are essential for full activation of the cell. This effect of actin reorganization is due

mainly to formation of an actin cap at the site of antigen contact, also known as the

immunological synapse (60). Overexpression of the GTP exchange factor vav-1 activates

the small G-protein Rac which in turn induces cytoskeletal changes. Overexpression of

vav-1 is sufficient for activation of the transcription factor NFAT following stimulation

through the costimulatory molecule CD28 even in the absence of TCR stimulation,

indirectly indicating a role for actin rearrangement in TCR signaling (60). Further

confirmation of this concept has been observed in the DT40 chicken B cell line, which

shares the same basic signal transduction mechanism downstream of the B cell receptor.

When these cells were treated with the actin destabilizing agents latrunculin B (latB) or

cytochalasin D (cytD) they were spontaneously activated. In the presence of these

21

agents, stimulation through the B cell receptor leads to enhanced and prolonged

activation (56).

Small G-proteins of the Rho family play a crucial role in regulation of the actin

cytoskeleton. In fibroblasts, activation of Rho family members induces specific

cytoskeletal structures to form. Activation of Rho by lysophosphatidic acid induces

stress fiber formation and focal adhesion formation. Expression of constitutively active

Rac induces lamellipodia formation. Activation of cdc42 induces filopodia formation.

Additionally, since cdc42 activates Rac, activation of filopodia formation by cdc42 also

causes lamellipodia formation (for review see (61)).

Regulation of the actin cytoskeleton is intimately tied to calcium regulation. For

instance the actin severing proteins gelsolin and profilin are inhibited by phosphoinositol

4,5 bisphosphate, which is present when PLCs are inactive (62, 63). Gelsolin is also

regulated directly by binding to calcium, which activates its actin severing activity (63).

Actin stabilizing proteins such as α-actinin tend to be inhibited by higher Ca2+

concentrations (64). Finally certain actin stabilizing interactions such as that between

caldesmon and tropomyosin are induced at low Ca2+ concentration and inhibited at high

Ca2+ concentrations (64). Thus the actin cytoskeleton is highly sensitive to localized

changes in [Ca2+]i. A good illustration of this point comes from the fact that during

chemotaxis cells exhibit a gradient of calcium such that the leading edge of the cell has

high Ca2+ concentration and the trailing edge has relatively low Ca2+ concentration (65).

This allows activation of proteins that break down actin and allow the cytoplasm to flow

forward at the leading edge, while allowing maintenance of adhesion structures and

overall cell shape toward the rear of the cell.

22

Drebrin

The protein developmentally regulated brain protein (drebrin) was first identified

as a brain specific protein that is important for dendritic spine morphology, apparently

through its effects on actin (66, 67). Drebrin is expressed as two splice-variant isoforms

in mammals termed drebrin E or A (68). In the developing brain, these splice-variants

are developmentally regulated such that the embryonic (E) isoform is expressed in the

embryonic brain and is suppressed in the adult brain whereas the adult (A) isoform is

expressed throughout adulthood (66, 69). These two variants differ in the insertion of a

46 amino acid sequence following the actin binding domain that is found in drebrin A but

not drebrin E. An additional splice variant, termed Drebrin E1, that lacks an internal 43

amino acid sequence common to drebrin A and drebrin E (E2 in chicken) was found in

the chicken, however no evidence of this variant has been found in mammals to date (70).

Drebrin is also expressed in a variety of other cell types including certain epithelial cells

(71, 72). Drebrin is a member of the ADF-H/cofilin family of actin binding proteins. In

addition to an ADF-H domain it contains an actin binding domain, a proline rich region

that may serve as an SH3 binding motif, two homer ligand motifs, and a putative SH2

binding motif (Fig. 1.7). Little is known about the regulation of drebrin. Although

expression of the different splice variants appears to be tightly controlled, no specific

function has been attributed to any of the isoforms.

Drebrin has been suggested to play a crucial role in actin rearrangements in the

neuronal dendritic spine. When drebrin was overexpressed in neurons, it was shown to

change the shape of dendritic spines (73). When drebrin expression was suppressed by

23

Figure 1.7 The actin binding protein drebrin is contains an N-terminal ADF-H domain, a

central actin binding domain, a small proline rich (P rich) region, two homer binding

domains, and a putative SH2 binding domain. In mammals drebrin is expressed as two

splice variants differing by the insertion of a 46 amino acid insertion following the actin

binding domain of drebrin A that is absent in drebrin E. Additionally, a short splice

variant of drebrin A has been described that is truncated following the insertion sequence.

ADF-H Actin bindingdomain

P rich

Drebrin

A-specificinsertion

Homerbinding

SH2binding

Domain organization of drebrin

24

transfection of anti-sense oligos specific for drebrin, neurite outgrowth was abolished

further demonstrating the role of drebrin in the dendritic spine (74). The dendritic spine

appears to be unique in its actin structure in that actin throughout the entire spine remains

dynamic and turns over rapidly (75). By comparison, other actin rich extensions such as

filopodia contain only one growing end of the actin fibers and the other end is capped and

protected. Thus, as the extension becomes longer, more of the filament becomes stable

and only actin at the growing end is dynamic. This appears to be the result of the

increased concentration of actin severing proteins such as gelsolin and the decreased

occurrence of stabilizing proteins like tropomyosin within the dendritic spine.

Interestingly, drebrin has been shown to compete with tropomyosin for actin binding and

has been found in complexes containing gelsolin, which severs actin making the

filaments more dynamic (76, 77).

Drebrin appears to facilitate a more dynamic actin cytoskeleton. When drebrin is

overexpressed in fibroblasts, it causes the formation of thick, curved actin bundles and

the projection of many neurite like outgrowths (67). This activity is attributed to the N-

terminal portion of the protein, specifically within the first 366 amino acids (78). This

portion of drebrin contains the ADF-H domain as well as the actin binding domain.

Recently, it has been reported that drebrin associates with specific pools of actin

on the Golgi membrane (79, 80). ARF1 is a GTP binding protein that regulates the

assembly of transport vesicles on the Golgi membrane. Fucini et al determined that ARF

induces assembly of two distinct pools on the Golgi membrane, one that cofractionates

with COPI vesicles following salt extraction and is sensitive to actin depolymerization by

cytochalasin D, and the other that remains on the Golgi following salt extraction and is

25

insensitive to cytochalasin D (79). Interestingly, drebrin was found associated with the

cytochalasin D sensitive actin pool, and that cytochalasin D treatment increases the rate

of budding of COPI coated vesicles (79). Unfortunately, this study did not explore the

role of drebrin associated with the Golgi.

Decreased drebrin expression has been associated with two disease states. Both

Down syndrome and Alzheimer’s patients exhibit decreased drebrin protein expression

(81, 82). The effect of reduced drebrin expression is reduced synaptic plasticity due to

the inability to reorganize the dendritic spines, which presumably leads to the

pathological effects observed in these patients (83).

3,5-bistrifluoromethyl pyrazole (BTP)

Recently, a class of compounds called BTPs (3,5-bistrifluoromethyl pyrazoles)

was found to inhibit both TH1 and TH2 cytokine production in T cells (84-86) (Fig. 1.8).

Specifically, it was found that BTPs prevented activation of NFAT and did not seem to

affect other transcription factors such as NFκB or AP-1 (84, 86) BTPs are unique in

comparison to the more widely studied NFAT inhibitors FK506 and Cyclosporin A

(CsA) in that BTPs do not directly inhibit CN phosphatase activity (86, 87). However,

BTP treatment blocks NFAT dephosphorylation and nuclear import similar to FK506 and

CsA (84).

Based on BTPs effects on NFAT activation, it is possible that it inhibits NFAT

directly, or that it inhibits a process upstream of NFAT activation. Therefore, in order to

understand the mechanism of action of BTP, we sought to characterize the effects of BTP

26

treatment on cells. Additionally, we sought to identify possible protein targets of BTP in

order to elucidate its mechanism of action.

27

.

Figure 1.8 BTP2 is a member of the 3,5-bis(trifluoromethyl)pyrazole class of

compounds. The class of compounds share the core structure outlined including the 3,5-

bis(trifluoromethyl)pyrazole ring, but differ in the excluded ring structure

Structure of BTP2

28

Aims of this study

The focus of this study was to understand how the small molecule immuno-

suppressant BTP inhibits activation of the transcription factor NFAT. This information

should provide further insight into the biological processes regulating T cell activation,

and perhaps identify new players in the signaling pathway leading to NFAT activation.

Hypothesis: BTP binds to a protein involved in NFAT activation and effects its function

Specific Aim 1: To characterize the effects of BTP treatment on cells. BTP was

previously demonstrated to inhibit NFAT activation, however it did not directly inhibit

the upstream phosphatase responsible for activating NFAT as do the drugs FK506 and

cyclosporin A. At present it is not known how BTP prevents NFAT activation. The

experiments in this aim are designed to assess the effects of BTP on cellular processes,

particularly those known to be upstream of NFAT activation.

Specific Aim 2: To identify potential target proteins of BTP. Proteins that bind to BTP

are potential targets of the drug. We first attempted to identify proteins that bind to BTP

in order to identify potential targets of the drug. Next we verified the interaction of the

most promising candidate of three identified binding proteins in order to validate the

interaction.

Specific Aim 3: To examine the role of drebrin in calcium signaling. Drebrin has been

studied in the context of neuronal dendrite outgrowth and its effects on neuronal dendrite

29

morphology. Nothing is known about drebrin’s role in calcium signaling. BTP inhibits

calcium influx and drebrin binds to BTP, we therefore sought to determine if drebrin

plays a role in calcium signaling. Particularly we wanted to determine if loss of drebrin

protein expression would affect the cells ability to mobilize calcium following

intracellular store depletion.

30

CHAPTER 2

Materials and Methods

31

Cells, antibodies, plasmids, and reagents. Jurkat E6-1 T cells were grown in complete

RPMI (RPMI-C) supplemented with 10% FCS. Chicken DT40 B cells were maintained

in RPMI-C supplemented with 10% FCS and 1% chicken serum. HEK293T and CHO

cells were grown in complete DMEM supplemented with 5% FCS. Polyclonal anti-

drebrin was from Sigma (St. Louis, MO), anti-GFP and anti-actin antibodies were from

Santa Cruz Biotech. (Santa Cruz, CA). Alexa-fluor 568 conjugated phalloidin was from

Molecular Probes (Eugene, OR). Constitutively active calcineurin plasmid was a kind gift

of Dr. Neil Clipstone, Northwestern University, Chicago, IL (88). pEGFPC1-rat-

drebrinA plasmid and pEGFPC1-drebrin fragment plasmids were kind gifts of Dr.

Tomoaki Shirao (Gunma University, Japan) (78). pCDNA3-NFAT4-GFP plasmid was a

kind gift of Dr. Frank McKeon , Harvard Medical School, Boston, MA (89). GST-

drebrin bacterial expression vector was constructed by removing the drebrinA cDNA

from EGFPC1-DrebrinA using the 5’-BglII and 3’-BamHI sites followed by inserted into

the BamHI site of pGex2TK (Amersham Biosciences, Piscataway, NJ). Recombinants

were tested for orientation by cutting with PstI which yields 2.9 kb and 4.2 kb fragments

in the correct orientation. BTP2 (N-[4-[3,5-bis(trifluoromethyl)-1H-pyrazole-1-yl]

phenyl]-4-methyl-1,2,3-thiadiazol) was a kind gift of Drs. James Trevillyan and Stevan

Djuric, Abbott Laboratories, Chicago, IL and for later studies was purchased from

Calbiochem (San Diego, CA). Other BTP derivatives were synthesized by Laurie

Mottram, Penn State University (University Park, PA).

32

Western Blot. Samples for western blot were first separated by size using SDS-PAGE.

Briefly, samples were boiled at 100°C for 10 minutes in 2X SDS-PAGE reducing buffer

(0.499 M Tris, 20% glycerol, 4% sodium dodecyl-sulfate, 2% 2-mercapto-ethanol,

0.025% bromophenol blue). Proteins were then separated by electrophoresis through

polyacrylamide/SDS gel and transferred to PVDF membrane prior to blocking in 5%

milk/Tris-buffered Saline + 0.1% Tween-20 (TBS-T) for 1 hour at room temperature with

rocking. Membranes were then incubated in 5 ml of 5% milk/TBS-T plus primary

antibody, normally diluted 1:500-1:2000, for 1 hour at room temperature with rocking.

Following three 15 minute washes in TBS-T, secondary antibody solution (5 ml 5%

milk/TBS-T plus HRP linked secondary antibody (1:10,000-1:100,000 dilution)) was

added and incubated for 30 minutes at room temperature with rocking. Following three

final 15 minute washes in TBS-T, ECL plus substrate (Amersham Biosciences,

Piscataway, NJ) was added in order to visualize protein bands that reacted with the

antibodies. The membrane was then exposed to blue sensitive X-ray film to visualize

bands.

Fluorescent calcium measurement. Changes in [Ca2+]i were monitored by loading 107

cell/ml Jurkat T cells with 1 µM Fura-2AM (Sigma St. Louis, MO) as described in (90)

except that cells were loaded and assayed in Ringer’s solution (155 mM NaCl, 4.5 mM

KCl, 2 mM MgCl2, 10 mM dextrose, 5 mM Hepes, pH 7.4). Briefly, 107 cells/ml were

loaded in Ringer’s solution containing 1 mM CaCl2 for 1 hour at room temperature and

then washed two times with Ringer’s solution without CaCl2 and cell concentration

adjusted to 106 cells/ml in the appropriate buffer (+/- CaCl2) prior to assay. For BTP

33

treatment, cells were treated for 1 hr with 1 µM BTP and then loaded with fura-2AM for

prior to assay. Intracellular calcium changes were monitored using 5 x 105 cells in 0.5 ml

buffer. The relative fluorescence of fura-2AM was measured at 510 nm when excited by

340 nm and 380 nm light using a Hitachi F-2000 fluorescence spectrophotometer

(Hitachi, San Jose, CA) at room temperature with gentle stirring. Calcium concentration

is expressed as the ratio of fura-2 fluorescence at 510 nm caused by the two excitation

wavelengths (340 nm/380 nm).

siRNA knockdown. Drebrin protein expression was knocked down by transfecting 2.0 x

107 Jurkat T cells with 200 nM drebrin specific siRNAs (Smartpool, Dharmacon,

LaFayette, CO) or 200 nM siControl #1 non-targeting control RNAs (Dharmacon) by

electroporation using a BTX electrosquare porator 800 (Genetronics, San Diego, CA) at

300V for 20 msec in 400 µl RPMI in a 4 mm electroporation cuvette. Cells were then

cultured in RPMI-C + 10% FCS for 48-96 hours prior to assay.

Coomasie Stain. Proteins in SDS-PAGE were visualized by staining with GelCode Blue

colloidal Coomasie (Pierce Biotechnology, Rockford, IL). Briefly, gels were washed in

distilled, deionized water for 1 hour with three changes of water. The gels were then

covered in GelCode Blue solution until proteins bands were visible (15 min.-1 hr.).

Bands were further developed by washing in water for 1 hr to overnight with several

changes of water until the background became nearly clear.

34

In-gel digest and mass spectrometry. For protein identification bands were excised

from the SDS-PAGE gel and in-gel tryptic digest was performed following kit

instructions (In-gel tryptic digestion kit, Pierce Biotechnology, Rockford, IL) prior to

submission to The Proteomics and Mass Spectrometry Core Facility at Penn State

University (University Park, PA) for mass spec analysis. Briefly, 200 µl of destaining

solution (57.14% (w/v) ammonium bicarbonate, 14.29% (v/v) acetonitrile) was added to

the excised gel slice and incubated at 37°C for 30 min. This solution was then removed

and this step repeated once. The sample was then reduced by addition of 30µl reducing

buffer (25 mM ammonium bicarbonate, 50 mM Tris[2-carboxyethyl]phosphine (TCEP))

and incubation at 60°C for 10 min. Samples were then cooled to room temperature and

the reducing buffer was removed from the gel slice. Alkylation of the sample was

performed by incubating the gel slice in 30 µl alkylation buffer (25 mM ammonium

bicarbonate, 100 mM iodoacetamide) in the dark for 1 hour at room temperature.

Following removal of the alkylation buffer, the sample was washed twice in 200 µl 25

mM ammonium bicarbonate at 37°C for 15 minutes each with shaking. Following final

wash the gel slice was shrunk by adding 50 µl acetonitrile for 15 minutes at room

temperature. Following removal of acetonitrile, the samples were air dried for 5-10

minutes. Next samples were digested in 10 µl activated trypsin solution (10 ng/µl

trypsin, 25 mM ammonium bicarbonate) for 15 minutes at room temperature, followed by

addition of 25 µl 25 mM ammonium bicarbonate and further incubation at 37°C for 4

hours. The digestion mix was then collected in a clean tube. A second extraction was

then performed on the gel slice by addition of 10 µl trifluoroacetic acid for 5 minutes.

35

This solution was then added to the digestion mixture. This sample was then submitted

to the Penn State Mass Spectrometry facility for LC-ESI-MS peptide identification.

Alternatively, excised bands were sent to W.M. Keck Foundation Biotechnology

Resource Laboratory at Yale University (New Haven, CT) for in-gel digestion and mass

spec analysis (MALDI-MS). Peptide masses were used to search either the ProFound

(http://prowl.rockefeller.edu/profound_bin/WebProFound.exe) or Mascot (Matrix

Science, Boston, MA, http://www.matrixscience.com) databases for matching proteins.

Immunofluorescence analysis

CHO, HEK293T, or NIH3T3 cells were grown on glass coverslips. Prior to immuno-

staining, cells were fixed for 15 min. in PBS containing 4% para-formaldehyde and

permeabilized with PBS containing 1% triton-x 100 for 2 min. Cells were then blocked

in PBS containing 5% BSA. Cells were stained with Alexa-568 phalloidin (Molecular

Probes, Eugene, OR) to visualize F-actin. Cells were then analyzed on an Olympus

Fluoview 300 confocal laser scanning microscope (Olympus Microscope, Melville, NY) .

ELISA

ELISA for IL-2 production was performed by collecting medium from PMA/ionomycin

stimulated or unstimulated mouse primary thymocytes with and without estrone-BTP

treatment and performing ELISA according to the OptEIA IL-2 ELISA kit directions (BD

Biosciences, San Diego, CA). Briefly, 96 well ELISA plates were incubated overnight at

4°C with IL-2 capture antibody. Following two washes, the samples were then incubated

in the plate for 1 hour at room temperature. Plates were again washed to remove

36

unbound proteins. IL-2 detection antibody conjugated to horseradish-peroxidase was

then added and the plate was incubated at room temperature for 1 hour in the dark.

Following this step, the plate was washed extensively and incubated with 3,3’,5,5’-

Tetramethylbenzidine (TMB) (Sigma, St. Louis, MO) liquid substrate for 30 minutes at

room temperature. The TMB reaction was stopped by acidification with 0.5 M H2SO4.

The yellow substrate product produced from this reaction was quantified on a 96 well

spectrophotemetric plate reader by measuring absorbance of the samples at 450 nM.

37

CHAPTER 3

Effects of BTP

38

Rationale

It was previously reported that BTP inhibits cytokine production. This inhibition was

attributed to inhibition of activation of the transcription factor NFAT, which is essential

for transcription of many cytokine genes. However, BTP did not inhibit NFAT activation

as the immunosuppressive drugs FK506 and Cyclosporin A (CsA), which are currently

used to prevent organ transplant rejection, do. FK506 and CsA, inhibit the phosphatase

calcineurin (CN), which is activated by increases in intracellular calcium to

dephosphorylate NFAT. From these studies it was unclear how BTP worked to inhibit

NFAT, or what effects other than NFAT activation that BTP treatment has on cells. In

the following experiments we sought to understand the effects of BTP on cells in order to

better understand how BTP inhibits NFAT activation.

BTP inhibits NFAT activation.

In order to confirm that BTP prevents activation of NFAT as previously reported,

we utilized the luciferase reporter assay system to detect NFAT activation. This system

uses a reporter plasmid containing the firefly luciferase gene driven by a promoter

consisting of NFAT binding sites. When NFAT is active, the luciferase gene is

transcribed. Luciferase can be detected in the cell lysate by measuring light intensity

produced when the enzyme’s substrate is added to the cell lysate. When Jurkat T cells

were transfected with the NFAT-luciferase reporter plasmid, BTP2 (N-[4-[3,5-

bis(trifluoromethyl)-1H-pyrazole-1-yl] phenyl]-4-methyl-1,2,3-thiadiazol, referred to as

BTP) treatment was able to potently block its activation following treatment with PMA (a

Protein Kinase C activator) and the calcium ionophore ionomycin (Fig. 3.1 a). Similarly,

39

BTP blocked activation of NFAT in both primary thymocytes and splenocytes from a

transgenic mouse line carrying the luciferase reporter driven by NFAT binding sites as a

transgene (Fig. 3.1 b) (91). When human embryonic kidney cell line HEK293T cells,

which do not normally express NFAT were transfected with NFAT luciferase reporter

along with exogenous NFAT (89), BTP was also able to block activation of the reporter

following PMA/ionomycin treatment (Fig. 3.1 c). Since BTP does not directly inhibit

NFAT, our observation that BTP inhibits activation of exogenous NFAT in HEK293T

cells indicates that BTP acts on a target that is more ubiquitous than NFAT. It was

previously reported that BTP had no effect on the activity of calcineurin (CN), so we

addressed the possibility that it acts downstream of CN by co-transfecting Jurkat T cells

with a constitutively active form of CN along with the NFAT-luciferase reporter plasmid

(88). In the presence of constitutively active CN, BTP was unable to inhibit NFAT

activation (Fig. 3.2). This rules out the possibility that BTP blocks NFAT nuclear import

or activation downstream of CN. When HEK293T cells are transfected with NFAT-GFP

plasmid and stimulated with ionomycin, NFAT translocates from the cytoplasm to the

nucleus. NFAT-GFP subcellular location can be monitored using confocal fluorescent

microscopy following stimulation with ionomycin. In control cells, NFAT is exclusively

localized to the cytoplasm prior to ionomycin treatment. Within 30 minutes of

ionomycin treatment, NFAT can be detected in the nucleus (Fig 3.3 a) In the presence of

BTP, NFAT is localized to the cytoplasm prior to stimulation as in the control cells.

However, following ionomycin treatment NFAT remains in the cytoplasm and cannot be

detected in the nucleus (Fig. 3.3 b).

40

Figure 3.1 BTP inhibits activation of NFAT in multiple cell types. A) Jurkat T cells

were transfected with NFAT-luciferase reporter plasmid and either pretreated with 1 µM

BTP or left untreated for 1 hr prior to stimulation with PMA + ionomycin. B) Primary

splenocytes or thymocytes from NFAT-luciferase reporter transgenic mice were either

pre-treated for 1 hr with BTP or left untreated prior to 48 hr stimulation with P/I. C)

HEK293T cells were transfected with NFAT-GFP plus NFAT-luciferase reporter

plasmids and treated as above.

Jurkat cells

non P/I BTP + P/I0

25

50

75

100

125

treatment

% o

f max

imum

A)

C)293T cells

non P/I BTP+P/I0

25

50

75

100

125

stimulation

% o

f max

imum

B)NFAT-luc mouse cells

spleen thymus0

25

50

75

100

125nonP/IP/I + BTP

% o

f max

imum

tissue

41

Figure 3.2 Constitutively active calcineurin overcomes BTP inhibition. Jurkat T

cells were transfected with NFAT-luciferase reporter plasmid and constitutively active

CN or with NFAT-luciferase alone. BTP treated cells were cultured in the presence of

BTP overnight prior to assay. PMA/ionomycin treatment was carried out for 6 hrs

following overnight incubation in the presence or absence of BTP. Values normalized to

non-treated, NFAT-luc alone sample.

NFAT-luc + P/I

NFAT-luc aloneNFAT-luc + CA-calc.

non treated BTP

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

fold

act

ivat

ion

treatment

42

Vehicle 1 µµµµM BTPTime followingionomycin

0 min

15 min

30 min

43

Figure 3.3 BTP inhibits NFAT nuclear translocation. HEK293T cells were transfected with a plasmid encoding NFAT4

fused to GFP. NFAT cellular localization was followed using time-lapse confocal microscopy (still images shown) following

treatment of the cells with ionomycin. A) Control cells treated with DMSO vehicle control. B) Cells treated with 1 µM BTP

for 1 hr prior to stimulation. White arrows indicate position of nuclei. Experiment performed at room temperature.

44

BTP blocks calcium regulated store-operated channels.

Since constitutively active CN was able to rescue NFAT activation in the

presence of BTP we examined the possibility that BTP might inhibit NFAT activation by

blocking or blunting [Ca2+]i increase following stimulation. Changes in [Ca2+]i can be

monitored by loading cells with the calcium sensitive dye Fura-2AM and measuring the

fluorescent intensity of the dye at 510nm when excited at 340nm and 380nm.

Normalization of the measurements is then performed by using the ratio of the two

excitation wavelength measurements (340/380).

When Jurkat T cells were treated with BTP prior to stimulation with ionomycin,

[Ca2+]i increase was greatly reduced (Fig. 3.4a). Interestingly, the reduction in Ca2+

mobilization was more pronounced during the latter phase of Ca2+ flux rather than the

initial response. This part of the Ca2+ response is due almost entirely to uptake of

extracellular Ca2+, as sequestration of extracellular Ca2+ was able to bring [Ca2+]i back to

baseline concentration (Fig. 3.4b). This led us to examine more specifically the

operation of calcium activated store-operated channels (SOCs). SOC operation can be

separated from ER calcium release by utilizing a classic calcium add-back assay. Cells

are first stimulated in calcium-free buffer and then the [Ca2+]i is allowed to return to

baseline levels prior to the addition of exogenous Ca2+. The initial stimulation induces

the depletion of intracellular Ca2+ stores and opens the SOCs. However, since there is no

extracellular calcium available, action of the plasma membrane Ca2+ ATPase pumps

(PMCA) quickly reduces [Ca2+]i to baseline levels. SOCs remain open for several

minutes despite the absence of extracellular Ca2+, allowing cells to take up Ca2+ upon

addition of extracellular Ca2+. When we performed this experiment with BTP treated

45

Jurkat T cells we saw a significant decrease in the ability of those cells to take up

extracellular Ca2+ following addition of extracellular Ca2+, indicating that the blunted

calcium response caused by BTP is due to inhibition of SOCs (Fig. 3.5).

46

Figure 3.4 BTP blocks intracellular calcium mobilization. Jurkat T cells were treated

with DMSO or BTP and loaded with fura-2AM in order to measure intracellular calcium

concentration. A) Cells were stimulated with ionomycin in Ringer’s solution in the

presence of 1 mM extracellular calcium. B) Jurkat T cells were loaded with fura-2AM

and stimulated with ionomycin in Ringer’s solution containing 1mM CaCl2 after initial

calcium increase, 5 mM EGTA was added to the buffer in order to chelate extracellular

calcium.

B

A

2

3

4

5

6

0 100 200 300 400 500

BTP treated (1µM)F340

/F38

0

time (s)

ionomycin

vehicle

1mM CaCl2

EGTA

ionomycin

1.51.71.92.12.32.52.72.93.13.33.5

0

28.5 57

85.5

114

143

171

200

228

257

285

314

342

371

399

428

456

485

time (s)

F340

/F38

0

1mM CaCl2

47

Figure 3.5 BTP inhibits entry of extracellular calcium. Jurkat T cells were pre-treated

with ionomycin, loaded with fura-2AM and then stimulated with ionomycin in Ringer’s

solution without extracellular calcium. 1 mM CaCl2 was later added to the buffer and

calcium mobilization through SOCs was followed.

1.5

2

2.5

3

3.5

4

4.5

0 100 200 300 400 500

vehicle

BTP treated (1µM)

time (s)

F340

/F38

0

No Ca2+ 1mM CaCl2

CaC

l2

ionomycin

48

BTP inhibits dynamic cytoskeletal changes in response to calcium ionophore.

The actin cytoskeleton has been implicated in intracellular Ca2+ homeostasis.

Disruption of actin cytoskeletal changes is one possible mechanism that BTP might

utilize to inhibit store-operated Ca2+ entry. We chose to examine the effects of BTP

treatment on an adherent cell line, HEK293T, in order to determine if BTP could affect

cytoskeletal rearrangements. Under resting conditions adherent cells, but not suspension

cells such as Jurkat T cells, develop focal adhesion plaques which serve to anchor the

cell. These plaques are rich in actin accessory proteins as well as F-actin (Fig. 3.6, top

left). Treatment of cells with a calcium ionophore such as ionomycin stimulates the cell

to break up most of its focal adhesions, presumably to facilitate greater mobility (Fig. 3.6,

top right). We treated adherent HEK293T cells with BTP prior to treatment with

ionomycin to determine if BTP could inhibit actin reorganization induced by calcium

signaling. When we treated HEK293T cells with BTP we observed a slight increase in F-

actin plaques that formed at the cell-coverslip interface, indicative of focal adhesions

(Fig. 3.6, bottom right). Interestingly, these plaques were not disrupted in BTP treated

cells following ionomycin treatment, as they were in the vehicle treated cells (Fig 3.6,

bottom left). In light of its interaction with a cytoskeletal reorganizing protein, this data

further suggest that the effects of BTP may be exerted through inhibition of cytoskeletal

changes.

49

Non-stimulated Ionomycin 2 min.

vehicle

BTP

Figure 3.6 BTP inhibits actin rearrangement following ionomycin treatment. HEK293T cells were treated with DMSO

(top panels) or BTP (bottom panels) and either left unstimulated (left panels) or stimulated for 2 min. with ionomycin. Cells

were then fixed and with Alexa-568-phalloidin (red) to detect F-actin. Note that in unstimulated cells, there are a large

number of actin punctae (white arrows) in the center of the cells. When control cells are stimulated, most of the central F-

actin punctae disappear. BTP treated cells have a large number of F-actin punctae remaining after stimulation.

50

BTP does not inhibit tyrosine kinase activation.

Stimulation of Jurkat T cells with antibodies against the CD3 chains of the TcR

results in activation of a number of non-receptor tyrosine kinases which couple to the

TcR and transmit the activation signal to activate a number of signaling pathways. These

kinases represent the earliest stage of T cell activation and as a result when they are

inhibited the T cell cannot be fully activated. In particular, activation of the Tec family

kinase ITK is crucial for activation of phospholipase-Cγ1 (PLC-γ1) which is responsible

for cleavage of PIP2 to generate the second messenger IP3 in order to induce release of

intracellular Ca2+ stores. When we examined tyrosine phosphorylation following

stimulation with anti-CD3 antibodies by western blotting, there was no difference

between BTP treated cells and those that were treated with vehicle alone (Fig. 3.7 a).

Similarly, BTP had no effect on the tyrosine phosphorylation of either ITK or PLC-γ1,

indicating that BTP’s effects may lie downstream of these proteins (Fig. 3.7b & c).

51

Figure 3.7 BTP does not affect tyrosine phosphorylation of cellular proteins, PLCγ1,

or ITK following TCR stimulation. Jurkat T cells were coated with anti-CD3

antibodies and stimulated for 5 min at 37°C following treatment with either DMSO or 1

µM BTP for 1 hr. A) Cells were lysed and a portion of the lysate was run on SDS-

PAGE and western blotted using anti-phosphotyrosine antibody. B) PLCγ1 was

immunoprecipitated from the lysate and blots were probed using anti-phosphotyrosine

antibody to detect PLCγ1 phosphorylation. C) ITK was immunoprecipitated from the

lysate and blots were probed using anti-phosphotyrosine to detect phosphorylated ITK.

52

BTP blunts MAP kinase signaling.

The immunosuppressants FK506 and CsA have been shown to inhibit

phosphorylation of the MAP kinases JNK and p38, but neither appears to affect

phosphorylation of the MAP kinase ERK (92). Based on their similar effects on NFAT

activation, we wanted to determine if BTP also affected phosphorylation of the MAP

kinases. Indeed, when we pretreated DT40 chicken B cells or Jurkat T cells with BTP

prior to stimulation with PMA and ionomycin, phosphorylation of p38 or JNK was

drastically reduced and lasted for a shorter amount of time than with the untreated control

(Fig. 3.8 a & b). Similar to reports on FK506 and CsA, ERK phosphorylation was

unaffected. However, phosphorylation of ERK was reduced after prolonged stimulation

(60 min.) (Fig 3.8 c). As expected, expression of c-fos, which is dependent on activation

of ERK, was decreased in cells treated with BTP (Fig. 3.8 d). Similarly phosphorylation

of c-jun, a target of JNK, was also decreased following BTP treatment (Fig. 3.8 e). It has

been demonstrated that extracellular calcium is important for nuclear localization of ERK

as well as production of c-fos (90). Since NFAT is inhibited by BTP and is also

regulated by entry of extracellular calcium, inhibition of the MAP kinases and NFAT by

BTP may both be mediated through a calcium dependent pathway.

53

P-p38

vehicle 1 µM BTP0 5 10 30 60 0 5 10 30 60

p38

B)

C)

Min.

P-JNK

JNK

0 5 10 30 60 0 5 10 30 60vehicle 1 µM BTP

A)Min.

0 2 5 10 30 60 0 2 5 10 30 60vehicle 1 µM BTP

P-ERK

ERK 1/2

Min.

c-fos

vehicle 1 µM BTP0 5 10 30 60 0 5 10 30 60Min.

vehicle 1 µM BTP

phospho c-jun0 5 10 30 60 0 5 10 30 60Min.

D)

E)

54

Figure 3.8 BTP inhibits MAP kinase activation. Jurkat T cells were pre-treated with

BTP for 1 hr prior to stimulation with PMA and ionomycin for the indicated times (min.).

Lysates were run on SDS-PAGE and blots were probed for the presence of phopho-p38

A), phospho-JNK B), phospho-ERK C), c-fos D), or phospho-c-jun E).

55

Discussion

Initial studies reported that BTP inhibits cytokine production by inhibiting

activation of the transcription factor NFAT, which is essential for transcription of many

cytokine genes (84-86, 93). Interestingly, BTP did not appear to inhibit NFAT

activation in a similar manner as other drugs which are known to inhibit NFAT

activation. In these initial reports it was unclear what the target of BTP was. We

therefore sought to characterize the effects of BTP on cells in order to gain insight into

possible mechanisms of action. We verified that BTP inhibits NFAT activation by

transfecting Jurkat T cells with an NFAT-luciferase reporter plasmid and stimulating the

cells in the presence or absence of BTP. As expected, in the presence of BTP NFAT

activation was blocked. We also observed similar results in primary splenocytes and

thymocytes from mice carrying the NFAT-luciferase reporter as a transgene. Next, we

transfected HEK293T cells, which do not normally express NFAT, with a plasmid

encoding NFAT4-GFP along with the NFAT-luciferase reporter. When transfected with

NFAT, HEK293T cells are capable of activating NFAT following stimulation with PMA

and ionomycin. Using this system we determined that BTP acts on a target that is more

widely expressed than NFAT as BTP was able to inhibit NFAT nuclear translocation and

transcriptional activation in these cells.

BTP was shown not to inhibit calcineurin phosphatase activity (84). However, it

is possible that BTP disrupts the interaction of calcineurin with NFAT or affects some

other process downstream of calcineurin activation. We found that this was not the case,

as BTP was unable to inhibit NFAT activation in the presence of a constitutively active

mutant of calcineurin.

56

Calcineurin is activated by increases in [Ca2+]i. Since it was determined that BTP

did not directly inhibit calcineurin phosphatase activity in vitro or disrupt processes

downstream of calcineurin activation it was possible that BTP might affect Ca2+

mobilization following cell stimulation. We loaded BTP treated cells with the Ca2+

sensitive dye Fura-2AM we observed a drastic defect in their ability to mobilize Ca2+ in

response to treatment with ionomycin. Specifically BTP inhibited entry of Ca2+ from the

extracellular space, a process carried out by unidentified plasma membrane ion-channels

known as store-operated channels (SOCs). The best electrophysiologically characterized

form of SOC are known as calcium release activated channels (CRAC). CRAC channels

have been found in many hematopoetic cells and account for most of the Ca2+ entry

mechanism in T cells. It appears as though different cell types may express different

forms of SOC, possibly by differential expression of channel subunits. The mechanism

by which the SOCs detect depletion of intracellular calcium stores remains elusive.

BTPs effects are not caused by inhibition of signals immediately downstream of

the T cell receptor as tyrosine phosphorylation in response to T cell receptor cross-linking

was unaffected. Specifically, we observed no differences in either Itk or PLC-γ1

phosphorylation following T cell receptor cross-linking. Activation of these proteins is

important for generation of IP3 which causes release of the intracellular stores. As

mentioned before, we observed no differences in intracellular store release with BTP

treatment.

BTP inhibited F-actin rearrangement following ionomycin treatment in HEK293T

cells. This demonstrates that BTP treatment can have effects on the cytoskeleton.

However, this effect could be compounded by BTPs effects on Ca2+ mobilization. It is

57

difficult to determine whether the effects observed are a result of BTP actin on

cytoskeletal proteins or a secondary result of BTP inhibiting Ca2+ mobilization, or a

combination of both.

The drugs FK506 and CsA have been shown to inhibit activation of JNK and p38

MAP kinases but not ERK MAP kinase (92). This effect appears to be dependent on the

method of activation as it was only observed following activation by treatment with a

PKC activator plus ionomycin. FK506 and CsA did not inhibit JNK activation when it

was induced by Fas cross-linking or high osmotic pressure (92). It was suggested that

these drugs inhibit the JNK and p38 and not ERK, by inhibiting activation of the

upstream kinase MEKK1 but not Raf1, which is upstream of ERK. However, these drugs

were not shown to directly inhibit MEKK1 and it is thought that they most likely work

upstream of this kinase. We observed similar effects of BTP on phosphorylation of these

proteins. All three of these compounds may inhibit activation of an upstream signaling

intermediate that is dependent on calcineurin, however a role for calcineurin has not been

established for activation of the MAP kinases.

58

CHAPTER 4

Purification and characterization of potential targets of

BTP

59

Rationale

The target of BTP is unknown at this time. Knowing the target of BTP will help

us understand its mechanism of action. Most signal transduction pathways utilize

protein-protein interactions to transduce signals from the plasma membrane to activate

transcription factors in order to activate transcription of appropriate genes. Affinity

purification is a widely used technique to identify proteins that interact with a specific

probe, usually an antibody or other protein. We made a derivative of BTP which retained

the majority of the BTP coupled to a linker and biotin. Biotin binds to the protein

streptavidin with the strongest affinity of any known natural interaction. Thus we

reasoned that the BTP-biotin compound could be immobilized onto streptavidin beads

and used to purify proteins that bind to BTP. These proteins could be targets of BTP and

further characterization of the interaction could then be used to confirm this possibility.

Affinity purification and mass spec identification of BTP-binding proteins.

In order to better understand the mechanism by which BTP inhibits SOCs, we set

up an affinity system to purify and identify BTP binding proteins. A derivative of BTP

was synthesized coupled to biotin as described in (Fig. 4.1). This biotinylated BTP

compound was then tested for inhibition of NFAT activation in primary thymocytes

carrying a transgenic NFAT luciferase reporter. Addition of the large linker and biotin

groups reduced the potency of this compound compared with BTP, however, it still had

significant activity in these cells (IC50 ~600 nM compared to ~15 nM for parent

compound) (Fig. 4.2a). BTP-biotin’s ability to inhibit Ca2+ mobilization was assayed by

treating Jurkat T cells with 10µM BTP-biotin prior to Fura-2AM calcium monitoring

60

following ionomycin treatment. As expected, BTP-biotin inhibited Ca2+ mobilization at

this concentration (Fig. 2b). Additional confirmation that the position used for the linker

addition was insensitive to modification was performed using a derivative of BTP that

was coupled to estrone instead of BTP (E-BTP). Mouse primary thymocytes were treated

with this compound and stimulated with PMA/ionomycin in culture medium. The

medium was collected and ELISA was performed to detect IL-2 production as an

indication of NFAT activity. Similar to BTP-biotin, E-BTP’s activity was only mildly

affected by addition at this position (Fig 4.3).

61

Figure 4.1 Synthesis of BTP-Biotin (1). Reagents and conditions: (a)

1,1,1,5,5,5-hexafluoro-2,4-pentanedione, HCl, EtOH, 100 °C. (b) H2, Pd(C), EtOH. (c) p-

iodobenzoyl chloride, DIEA, CH2Cl2. (d) t-Butyl N-propargyl carbamate, Pd(PPh3)2Cl2,

CuI, TEA. (e) TFA, CH2Cl2. (f) Biotin-PEG4-NHS ester, DIEA, CH2Cl2. (Synthesis

scheme courtesy of Laurie Mottram).

62

Figure 4.2. BTP-biotin retains inhibitory activity. A) NFAT-luciferase transgenic

mouse thymocytes were stimulated in the presence of the indicated concentration of BTP-

biotin with PMA/ionomycin for 24 hrs prior to assaying for luciferase activity. B) Jurkat

T cells were pre-treated with 10 µM BTP-biotin and loaded with Fura-2 prior to

stimulation with ionomycin in the presence of extracellular calcium.

10-9

10-8

10-7

10-6

10-5

10-4

0

25

50

75

100 BTP-biotin

concentration [M]

% in

hibi

tion

ionomycin

Time (s)

2.22.73.23.74.24.75.25.76.2

0 100 200 300 400 500

F340

/F38

0

BTP-biotin(10µM)

vehicle

A)

B)

63

Figure 4.3 Structure of estrone-BTP (E-BTP). BTP1 derivative coupled to estrone.

This compound retains inhibitory potency towards IL-2 production (IC50~5.75 nM)

(courtesy of Laurie Mottram).

Estrone-BTP

NN CF3

F3C

HN

O

HO

N O

O

NN

O

64

Figure 4.4 Estrone-BTP retains inhibitory activity towards IL-2 production. Mouse

primary thymocytes were treated with E-BTP and stimulated with PMA/ionomycin for

48 hours prior to collecting cell supernatants and assaying for IL-2 production by ELISA.

Stimulated, vehicle treated cells were set to 0% inhibition and unstimulated, vehicle

treated cells were used as 100% inhibition.

IL-2

10-1310-1210-1110-10 10-9 10-8 10-7 10-6 10-5

-25

0

25

50

75

100

125

IC50=5.75 nM

concentration [M]

% in

hibi

tion

65

The BTP-biotin compound was immobilized onto streptavidin coated agarose

beads and used to purify BTP binding proteins from cell lysates using the scheme

described in figure 4.5. Briefly, 1010 Jurkat T cells were lysed in 50 ml cell lysis buffer

for 30 minutes on ice with vigorous vortexing every 10 minutes. Lysates were then

cleared by centrifugation for 1 hour at 4°C at 10,733 x g. The protein concentration of

the lysate was determined to be 12.38 mg/ml by Bradford assay. Lysates were collected

in 25 ml aliquots and precleared 3 times by rocking overnight at 4°C with 500µl of

streptavidin-agarose beads. Following each pre-clear, beads were collected by

centrifugation and washed 3 times in lysis buffer prior to freezing at -20°C. After the

final wash lysates were incubated overnight with 100µl BTP-biotin coated streptavidin-

agarose beads. These beads were prepared by incubating the streptavidin-agarose beads

in 10ml lysis buffer containing 10µM BTP-biotin overnight, collecting the beads by

centrifugation, and then incubating the beads for an additional 2 hrs in lysis buffer

containing 10µM BTP-biotin prior to the final collection step. Following incubation of

the cell lysate with the BTP-biotin beads, beads were collected by centrifugation and

washed 5 times in 1ml of lysis buffer prior to boiling in SDS-PAGE reducing buffer to

release bound proteins from the beads. Preclear beads were treated in the same manner.

The reducing buffer containing the released proteins was then run on SDS-PAGE to

separate proteins by size. The gel was stained using colloidal Coomasie stain to detect

proteins. Unique bands were excised from the gel and subjected to in-gel tryptic

digestion and MALDI/TOF mass spectrometry based protein identification (Fig. 4.6).

This procedure was performed twice and the samples were sent to two different facilities

for mass spectrometry analysis, and both identified the 120 kDa protein as the actin

66

binding protein drebrin (Z score 2.32, 99.0 percentile, probability of a match = 1.0e+000

using ProFound in experiment 1, and p<0.05 using Mascot in experiment 2, Tables 4.1 &

4.2). The identity of the 75 kDa protein and the 4 kDa protein were determined to be

17β-hydroxysteroid-dehydrogenase 4 (17β-HSD4) and actin respectively after a single

identification (ProFound probability of match = 1.0e+000 for both, Z score = 2.22 and

2.28 respectively) (Tables 4.3 & 4.4).

67

Figure 4.5 Schematic representation of BTPBP affinity purification. Jurkat T cell

lysates were first pre-cleared of proteins that bound to the streptavidin coated beads.

BTP-biotin was then adsorbed onto streptavidin-agarose beads and incubated with the

pre-cleared lysate. Following wash steps to remove non-specifically bound proteins,

beads were boiled in SDS-PAGE reducing buffer to release proteins for SDS-PAGE.

1 X 1010 Jurkat T cells

+Streptavidin-beads

X 3

Initial lysate Pre-cleared lysate

+

Lyse

BTP-biotin coated beads

Boil beads in reducing buffer and run on gel

Wash 3X

Wash 3X

Boil beads in reducing buffer and run on gel

68

Figure 4.6 Purification of BTP-binding proteins. Jurkat T cell lysates were pre-cleared

3 times with streptavidin-agarose beads and then incubated with BTP-biotin coated

beads. Following incubation, beads were washed 5X in lysis buffer and then boiled in

SDS-PAGE reducing buffer to release protein from the beads. Proteins were separated

by SDS-PAGE and visualized using colloidal Coomasie staining. Highlighted bands

indicate the major unique bands found only on the BTP-biotin beads.

BTP-biotin

250

160

105

75

50

35

Mr (kDa)

Pre-clear #1

Pre-clear #2

Pre-clear #3 p120

p75

p40

69

23-42EESAADWALYTYEDGSDDLK

272-291SESEVEEAAAIIAQRPDNPR

43-62LAASGEGGLQELSGHFENQK

337-354SPSDSSTASTPVAEQIER

150-165LREDENAEPVGTTYQK

80-94YVLINWVGEDVPDAR

152-165EDENAEPVGTTYQK

227-236EREQQIEEHR

239-249QQTLEAEEAKR

238-248KQQTLEAEEAK

229-237EQQIEEHRR

178-186EQFWEQAKK

253-261EQSIFGDHR

229-236EQQIEEHR

178-185EQFWEQAK

328-336MAPTPIPTR

140-147LSSPVLHR

216-221QEQEER

186-191KEEELR

187-192EEELRK

Location in human DrebrinPeptide Sequence

Table 4.1 Peptide sequences obtained for p120 (Yale)

70

271-291KSESEVEEAAAIIAQRPDNPR

272-291SESEVEEAAAIIAQRPDNPR

337-354SPSDSSTASTPVAEQIER

150-165LREDENAEPVGTTYQK

80-94YVLINWVGEDVPDAR

63-71VMYGFCSVK

2-10AGVSFSGHR

Location in human DrebrinPeptide Sequence

Table 4.2 Peptide sequences obtained for p120 (Penn State)

71

480-506VAVAIPNRPPDAVLTDTTSLNQAALYR

146-168IIMTSSASGIYGNFGQANYSAAK

563-579FAKPVYPGQTLQTEMWK

404-419VLHGEQYLELYKPLPR

385-403SMMGGGLAEIPGLSINFAK

436-451GSGVVIIMDVYSYSEK

185-199SNIHCNTIAPNAGSR

169-183LGLLGLANSLAIEGR

316-331ATSTATSGFAGAIGQK

302-315IDSEGGVSANHTSR

622-633LQSTFVFEEIGR

69-81AVANYDSVEEGEK

111-121IDVVVNNAGILR

11-23VVLVTGAGAGLGR

425-435CEAVVADVLDK

261-270NHPMTPEAVK

24-32AYALAFAER

58-64VVEIRR

133-139AAWEHMK

85-92TALDAFGR

Location in 17β-HSD4Peptide sequence

Table 4.3 Peptides identified from p75

72

85-95IWHHTFYNELR

291-312KDYLANTVLSGGTTMYPGIADR

Location in actinPeptide sequence

257-284CPEALFQPSFLGMESCGIHETTFNSIMK

1-28MEEEIAALVIDNGSGMCKAGFAGDDAPR

292-312DYLANTVLSGGTTMYPGIADR

96-113VAPEEHPVLLTEAPLNPK

239-254SYELPDGQVITIGNER

313-326MQKEITALAPSTMK

360-372QEYDESGPSIVHR

29-39AVFPSIVGRPR

40-50HQGVMVGMGQK

197-206GYSFTTTAER

19-28AGFAGDDAPR

329-335IIAPPER

Table 4.4 Peptides sequences obtained for p40

73

We initially chose to confirm and further characterize the interaction of BTP with

drebrin for several reasons: 1) Drebrin plays a role in cytoskeletal rearrangements, which

have been shown to be important for intracellular calcium signaling. 2) The presence of

actin could be a secondary interaction through drebrin. 3) Reagents to confirm the

interaction (i.e. antibodies) as well as plasmids to further characterize the BTP/drebrin

interaction were readily available either commercially or through collaboration, whereas

reagents to study 17β-HSD4 were not readily available. Drebrin has been well studied in

neuronal cells and appears to be important for actin rearrangements such as those driving

neuronal dendritic spine outgrowth (73, 74, 94, 95). Binding of drebrin to BTP was

confirmed by performing pull-down assays using BTP coupled to a solid matrix,

followed by probing for endogenous drebrin, and further confirmed using a GFP tagged

drebrin protein (Fig. 4.7 b & c). When we expressed drebrin tagged with GST in E. coli.,

we still observed binding to BTP (Fig. 4.8). Bacteria lack most post-translational

modifications that animal cells carry out, such as serine/threonine or tyrosine

phosphorylation so the role of these kinds of modifications in the BTP-drebrin interaction

appears to be unnecessary. Also, because of the high level of divergence between

mammals and bacteria it is unlikely that drebrin is able to participate in multi-protein

complexes in the context of a bacterial cell. Thus, it is more likely that BTP binds

directly to drebrin rather than indirectly through another protein.

74

Ant

i-dre

brin

Ju

rkat

cel

ls

streptavidin

BTP-biotin

5% lysate

Dre

brin

-GFP

streptavidin

linker

BTP-biotin

Ant

i-GFP

5% lysate

B)

C) A)

75

Figure 4.7 BTP binds drebrin. A) Structure of BTP-biotin (1) and biotin-linker (2). B)

Jurkat T cell lysate was incubated with either BTP-biotin coated streptavidin-agarose

beads or streptavidin-agarose beads alone. Beads were boiled in SDS-PAGE reducing

buffer prior to SDS-PAGE and western blotting. Lysate representing 5% of the input was

also run to confirm protein expression. Blot was probed using anti-drebrin antibody. C)

HEK293T cells were transfected with GFP-drebrin plasmid. Cells were then lysed and

treated as in B) with the addition of biotin-linker coated beads as an additional specificity

control. Blot was probed using anti-GFP antibody.

76

Figure 4.8 BTP interacts with bacterially expressed drebrin. E. coli were

transformed with GST-drebrin plasmid and induced to express protein with IPTG for 2

hrs. Bacteria were lysed and lysates incubated with streptavidin-agarose beads, biotin-

linker beads, or BTP-biotin beads prior to SDS-PAGE and western blotting with anti-

drebrin antibody. A portion of the lysate was also run for confirmation of protein

expression.

BTP

-bio

tin

Stre

ptav

idin

Bio

tin-li

nker

Lysa

te

77

BTP binds to the N-terminal portion of drebrin.

In order to further verify the interaction with of BTP with drebrin, we obtained

plasmids which contained fragments of the drebrin cDNA fused to GFP. We wanted to

identify the region(s) of drebrin that BTP binds to in order to understand what effect BTP

binding might have on drebrin’s function. We performed the BTP-biotin pull-down

assay as before using lysates from HEK293T cells that had been transfected with the

different drebrin fragment plasmids. Analysis of fragments of drebrin for binding to the

BTP-biotin beads indicated that the full-length protein had the highest binding capacity,

and the N-terminal 1-366 amino acids retained strong binding. This region includes the

ADF-H and actin-binding domain, but lacks the proline rich region of drebrin. Further

analysis indicated that amino acids 233-366 of drebrin maintained minimal binding,

however this binding was much below that observed in the full length or N-terminal 366

amino acids (Fig. 4.9 a & b). This latter region includes the full actin binding domain

and a small region C-terminal to this region (compare to fragment containing amino acids

233-317, which contains just the actin binding domain). Thus the interaction between

BTP and drebrin includes the actin-binding domain, but requires residues that flank this

region at the N- and C-termini of this domain for optimal binding.

78

Figure 4.9 Mapping of BTP/drebrin interaction. HEK293T cells were transfected

with plasmids encoding various fragments of drebrin fused to GFP. Cells were lysed and

lysates were incubated with A) streptavidin-agarose beads, B) biotin-linker beads, or C)

BTP-biotin beads. Beads were boiled in SDS-PAGE reducing buffer and this was then

run on SDS-page and transferred to PVDF for western blotting with anti-GFP antibody.

D) Lysate corresponding to 5% of the input was also run to confirm expression of each

protein and give relative expression levels. Arrow indicates full-length drebrin. E)

Representation of relative binding of each drebrin fragment to BTP-biotin.

A)

C)

B)

E)

D)

Streptavidin beads Linker beads

BTP-biotin beads input

GFP

Drebrin1-366

233-300233-317

273-366

233-366

319-707

GFP

Drebrin1-366

233-300233-317

273-366

233-366

319-707

GFP

Drebrin1-366

233-300

233-317

273-366

233-366

319-707

GFP

Drebrin1-366

233-300

233-317

273-366

233-366

319-707

79

BTP blocks drebrin function.

When over-expressed in fibroblasts, drebrin causes the formation of long,

branched extensions and curved, thick actin bundles (67). We reasoned that if BTP

inhibits drebrin function in cells that BTP treatment of cells overexpressing drebrin may

alter the phenotype observed.

In order to assess drebrin as a target of BTP, GFP-tagged drebrin was over-

expressed in CHO cells, which caused cells to develop long, highly branched membrane

extensions (Fig. 4.10). We termed these extensions filopodia-like extensions (FLE)

because they resemble filopodia in that they are long membrane protrusions, but they are

much too large and branched to actually be filopodia. When drebrin over-expressing

cells were treated with BTP, there was a drastic reduction in filopodia-like extensions

(Fig 4.10). However, drebrin co-localization with actin was not affected, suggesting that

although the actin-binding site within drebrin forms part of the BTP binding domain, this

did not affect actin co-localization with drebrin. We counted the number of FLE per cell

and found that with BTP treatment there was an approximately 50% reduction in the

average number of FLE per cell as compared with vehicle treated cells (4.11 a)

Consistent with this, the number of cells containing only 1-5 FLE per cell was much

greater in the BTP treated group compared with the vehicle treated group (4.11 b). When

we counted the number of branch points on each FLE, we found that the drebrin treated

group had much fewer branches per FLE than did the vehicle treated group (4.11 c).

Interestingly, the average length of each FLE was unaffected by BTP treatment (Fig 4.11

d).

80

Figure 4.10 BTP inhibits drebrin function. CHO cells transfected with either GFP

(top) or GFP-drebrin (middle and bottom) and treated with either DMSO (vehicle, middle

panel) or BTP (bottom panel) prior to staining for F-actin (red) and visualization using

confocal laser scanning microscopy.

A)

vehicle

BTP treated

GFP alone

GFP Phalloidin Merge

81

4.11 Quantification of filopodia-like extensions. Branched cell extensions caused by

drebrin overexpression (filopodia-like extensions, FLE) (see fig. 4.9) were counted on

each cell in DMSO and BTP treated cells and expressed as average FLE per cell A) or as

the number of cells having a given range of FLEs per cell B). Additionally, the average

number of branch points per FLE C) and average length of each FLE D) were

determined. For all measurements n=25 cells.

FLE/cell

vehicle BTP0.0

2.5

5.0

7.5

10.0

12.5

outg

row

th #

A)FLE/cell

0 1-5 6-1011-1516-2002

4

6

8

10

12

# of outgrowths

# of

cel

ls

vehicleBTP

B)

C)average length

vehicle BTP0.0

2.5

5.0

7.5

10.0

arbi

trar

y un

its

D)branch points/FLE

vehicle BTP0

1

2

bran

ch p

oint

s

3

82

BTP does not affect drebrin protein expression.

One possible way that BTP might inhibit the ability of drebrin to induce

cytoskeletal changes is to decrease its expression level. To examine this possibility,

Jurkat T cells were treated for 0, 0.5, 1.0, 2.0, 4.0, and 6.0 hours with 1µM BTP. We

then lysed the cells and analyzed drebrin protein expression by western blotting. Over

the 6 hour time-course drebrin protein expression did not change in the presence of BTP,

indicating that BTP does not work by increasing turn-over of drebrin protein (Fig 4.12).

83

Figure 4.12 BTP does not affect drebrin protein expression. Jurkat T cells were

treated with BTP for the indicated amount of time and run on SDS-PAGE followed by

transfer to PVDF and western blotting with anti-drebrin antibody. Top panel shows

drebrin protein expression. Bottom panel shows actin protein levels to demonstrate equal

loading between lanes.

Anti-drebrin

Anti-actin

BTP treatment0 0.5 1 2 4 6hrs

84

Drebrin expression is required for SOC operation.

Drebrin has been studied in the context of neurite extension and subcellular

localization. Several protein-protein interactions have been suggested, but the function of

drebrin is still unclear. To date there have been no reports of drebrin being involved in

intracellular calcium regulation. Therefore, we wanted to determine if drebrin is involved

in this process. Particularly, we wanted to determine if loss of drebrin protein expression

altered the cells ability to mobilize calcium following intracellular store depletion.

Since actin rearrangement has previously been linked to SOC regulation we were

interested in determining if the actin reorganizing protein, drebrin, was important for

SOC operation. To assess the need for drebrin in Ca2+ signaling we utilized RNA

interference (RNAi). RNAi works by using small sequences of RNA (siRNA) that are

homologous to unique sequences within the mRNA of the protein of interest. When the

siRNA are present they activate a poorly understood mechanism which specifically

targets and destroys mRNA containing the specific sequence (96). In most cases even a

single base-pair difference between the siRNA and the target mRNA is enough to prevent

degradation of the mRNA. We used a pool of siRNA that were specific to the human

drebrin mRNA to abolish drebrin protein expression in Jurkat T cells. By 48 hours

following transfection of the siRNA into these cells there was a drastic reduction in

drebrin protein expression which was sustained until at least 96 hours post transfection

(Fig. 4.13). We observed varying degrees of protein expression following siRNA

transfection, however the expression level was consistently less than 50% of the original

expression level.

85

Figure 4.13 Time-course of drebrin knock-down by siRNA. Jurkat T cells were

transfected with either 100 nM or 200 nM drebrin-specific siRNAs by electroporation.

Cells were harvested at the indicated time following transfection and western blotting

was performed using anti-drebrin antibody to determine drebrin protein expression. The

drebrin siRNA decreases drebrin protein expression by 48 hours when used at 100 nM.

Significant loss of protein expression was seen by 24 hours when 200 nM was used, and

by 48 hours drebrin protein expression was below the detection level (top panel).

Western blotting for actin was performed as a loading control (bottom panel).

0 24 48 72 24 48 72

Anti-Drebrin

Anti-Actin

100 nM 200 nMTime (hr)siRNA conc.

86

Loss of drebrin protein expression should cause similar effects as inhibition of its

function. Since we had determined that BTP inhibits Ca2+ mobilization following

ionomycin treatment, we wanted to know if loss of drebrin protein expression would

cause a similar defect. To address this possibility, we used drebrin specific siRNA to

reduce drebrin protein expression in Jurkat T cells. We observed a rapid decline in

[Ca2+]i following ionomycin treatment in cells that had been transfected with drebrin-

specific siRNA, but not in cells transfected with control siRNA (Fig. 4.14 a). Drebrin

protein expression was reduced significantly in this assay by drebrin specific siRNA, but

not affected by control siRNA (Fig. 4.14 b).

BTP specifically inhibits entry of Ca2+ through SOCs. In order to determine is the

absence of drebrin affected Ca2+ mobilization in a similar manner, we utilized the Ca2+

add-back assay described previously. When drebrin-specific siRNA transfected cells

were stimulated with ionomycin in the absence of extracellular Ca2+, a spike in [Ca2+]i

corresponding to release of intracellular stores was observed which was similar in

kinetics and magnitude to control siRNA transfected cells. In contrast, when we added

extracellular calcium we observed only a slight increase in [Ca2+]i in cells transfected

with the drebrin-specific siRNA compared with control siRNA transfected cells (Fig.

4.15). Addition of BTP to control siRNA transfected cells reduced SOC activity as

expected. However, addition of BTP to the drebrin-specific siRNA transfected cells prior

to stimulation was unable to further inhibit Ca2+ mobilization suggesting that BTP

inhibits SOCs through the same pathway that drebrin is involved in (Fig. 4.15)

87

Figure 4.14 Loss of drebrin expression prevents calcium flux. A) Jurkat T cells were

transfected with either control siRNAs or drebrin-specific siRNAs and grown for 48

hours following transfection. Cells were then loaded with fura-2AM and intracellular

calcium concentration was monitored in the presence of 1 mM extracellular Ca2+ before

and after ionomycin treatment. B) Representative drebrin knock-down 48 hours post-

transfection. Jurkat T cells were either left untransfected, or transfected with 200 nM

control or drebrin-specific siRNA and allowed to grow for 48 hours before drebrin

expression was analyzed by western blotting with anti-drebrin antibody. Western

blotting with anti-actin antibody served as a loading control.

Time (s)

Drebrin siRNA

B)

A)

00.511.52

2.53

3.54

4.5

0 100 200 300 400 500

F340

/F38

0 ionomycin

Untreated cells

Control siR

NA

Drebrin siR

NA

Anti-drebrin

Anti-actin

Control siRNA

88

Figure 4.15 Drebrin is essential for store-operated channel function Jurkat T cells

were transfected with either control or drebrin-specific siRNA. 48 hours post-

transfection, cells were either left untreated, or treated with 1 µM BTP and then loaded

with fura-2AM. Intracellular calcium concentration was monitored following initial

stimulation with ionomycin in the absence of extracellular calcium. After [Ca2+]i

returned to baseline levels, 1mM CaCl2 was added and SOC activity was determined by

monitoring the change in fura-2AM fluorescence.

Time (s)

2

3

4

5

6

7

8

9

0 100 200 300 400 500

F340

/F38

0

No Ca2+ 1mM CaCl2

ionomycin

CaC

l2

Control siRNA

Control siRNA + BTP (1µM)

Drebrin siRNADrebrin siRNA + BTP (1µM)

89

Drebrin expression is necessary for NFAT activation.

Loss of drebrin protein expression has similar effects on Ca2+ mobilization as

treatment with BTP. Since Ca2+ mobilization is essential for NFAT activation and

treatment of cells with BTP blocks NFAT activation by inhibiting this process, we

expected to observe similar effects when drebrin protein expression was reduced with

drebrin specific siRNA.

When we co-transfected Jurkat T cells with drebrin siRNAs and NFAT luciferase

reporter we observed decreased NFAT activation following stimulation with

PMA/ionomycin (Fig. 4.16 a). Again, NFAT inhibition was not complete but this is

most likely a result of incomplete drebrin knockdown in the system (Fig. 4.16 b). This

data coupled with data showing that reduction in drebrin expression resulted in a block in

calcium activated SOC operation indicates that drebrin is essential for calcium signaling

and NFAT activation.

90

Anti-Drebrin

Anti-actin

Control siR

NA

Drebrin siR

NA

**P=0.0015

NFAT-luc activation

non P/I0.0

2.5

5.0

7.5

10.0

12.5

control

Drebrin siRNA

**

Treatment

Fold

act

ivat

ion

A)

B)

Figure 4.16 Drebrin is essential for NFAT activation. A) Jurkat T cells were

transfected with NFAT-luciferase plasmid plus either control siRNA or drebrin-specific

siRNA. 48 hours later cells were stimulated for 6 hrs with P/I and then assayed for

luciferase activity. B) Representative drebrin knock-down when RNAs are co-

transfected with NFAT-luciferase plasmid. Jurkat T cells were transfected with 100 nM

siRNA and 5 µg NFAT-luciferase plasmid. Cells were grown for 48 hours prior to

western blot analysis of drebrin protein expression using anti-drebrin antibody (top

panel). Western blotting with anti-actin antibody served as a loading control (bottom

panel).

91

Discussion

In order to identify potential targets of BTP, and to shed light on the mechanism

by which BTP inhibits Ca2+ influx, we developed an affinity-purification scheme for

identification of proteins that bind to BTP. This system was based on a derivative of

BTP1 which was coupled to a PEG4 linker and biotin via the para position on the terminal

phenyl ring. BTP-biotin was tested for its ability to inhibit NFAT activation and calcium

mobilization. While, the potency of this compound was reduced, it still retained

inhibitory activity. This demonstrates that addition of such a large group at this site on

BTP does not abolish its ability to bind to its target. This was further demonstrated with

another derivative of BTP which was linked to estrone via a different linker than the

biotin group. Like BTP-biotin, the estrone-biotin retained inhibitory potency. In fact

substitution of the entire ring does not appear to abolish the compounds inhibitory

activity as is evident by the action of BTP1, BTP2, and BTP3 which differ in this ring

structure but are still active (Fig. 4.17) (85). Others have demonstrated that BTP2 or

closely related compounds inhibit SOC activation (97-99). These compounds contain the

parent 3,5-bistrifluoromethyl pyrazole ring moiety, and have been shown to inhibit

cytokine production and block NFAT activation (84-86). In SAR studies substitution at

the ring structure opposite the 3,5-bistrifluoromethyl pyrazole ring altered inhibition only

slightly. In contrast, the trifluoromethyl groups on the pyrazole ring appears to be

essential for the compounds’ inhibitory activity as replacing them with less bulky groups,

such as methyl groups, greatly decreased the compounds’ potency (86).

92

Figure 4.17 Structure of BTPs. 3,5-bis(trifluoromethyl)pyrazole class of compounds.

share the core structure outlined including the 3,5-bis(trifluoromethyl)pyrazole ring, but

differ in the excluded ring structure.

93

The BTP-biotin compound was immobilized onto streptavidin coated agarose

beads via the strong interaction between biotin and streptavidin. These BTP-biotin

coated beads were then used to purify BTP binding proteins from crude cell lysates.

Three major protein bands were unique to the BTP-biotin compound as compared with

streptavidin-sepharose beads alone. These bands were identified as 17β-hydroxysteroid

dehydrogenase 4 (17β-HSD4), drebrin, and actin. We have not been able to verify the

interaction with 17β-HSD4 at this point due to a lack of reagents. However, it seems

unlikely that this enzyme, which inactivates estradiol and may participate in fatty acid

metabolism, would be involved in regulation of store operated channels. This of course

has not been confirmed so the possibility still exists.

Drebrin binds to actin with a very high affinity (Kd = 1.2 x 10-7 M) (77).

Therefore it is likely that association of actin with BTP is secondary to its association

with drebrin. This could be determined by performing binding studies of BTP with

purified actin, however this would be done in vitro and the interaction would still be

difficult to determine in vivo due to the presence of drebrin.

We further characterized the interaction between BTP and drebrin by performing

the affinity purification and transferring the proteins to PVDF membrane for western

blotting with anti-drebrin antibody. Additional verification was performed by performing

the above experiment with lysates from cells that had been transfected with a plasmid

encoding drebrin fused to GFP and then western blotting with anti-GFP antibody. This

served as both additional confirmation of the BTP-drebrin interaction and verified that

the drebrin antibody recognized drebrin. We used plasmid containing fragments of

drebrin fused to GFP in the affinity purification experiment and found that BTP binds to

94

the amino-terminal portion of drebrin near the actin binding domain. Specifically, BTP

strongly binds to the amino terminal 366 amino acids and quite weakly to a fragment

containing only amino acids 233-366, which contains the actin-binding domain plus a

short segment c-terminal to the actin-binding domain. Interestingly, while BTP inhibited

drebrin from causing extensive filopodia-like extensions it did not disrupt the association

between drebrin and actin. BTP most likely blocks drebrin from forming a protein-

protein interaction that is important for its effects on cells.

Consistent with the possibility that drebrin is a target of BTP, we found

expression of drebrin to be essential for SOC activation. When we reduced drebrin

protein expression with siRNAs specific for drebrin, the cells were no longer able to

activate SOCs when intracellular stores were depleted with ionomycin. The effects of

drebrin knock-down on Ca2+ mobilization were similar to those seen with BTP treatment.

In agreement with this, BTP treatment did not reduce Ca2+ influx further in cells treated

with drebrin siRNAs.

When we reduced drebrin protein expression in Jurkat T cells that had been

transfected with NFAT-luciferase reporter plasmid, we observed a decrease in NFAT

activation following stimulation with PMA and ionomycin. Anecdotally, using this

system we never observed full loss of drebrin expression. This is most likely a result of

the co-transfection protocol used. However, the reduction in NFAT activation seen was

proportional to the loss in drebrin protein expression.

95

CHAPTER 5

Discussion

96

In this study we identified the protein drebrin as a potential target of BTP. Little

is known about the function of drebrin in cells. It is clear that drebrin binds to

filamentous actin (F-actin) with high affinity (77). This binding appears to be mediated

through both the ADF-H and actin-binding domains (78). The ADF-H domain is a

widely used domain found in a number of other proteins. Cofilin is a small actin binding

protein that is essentially a single ADF-H domain alone. The biochemical function of

cofilin appears to be disruption of F-actin filaments however drebrin does not appear to

share this function. This is not entirely surprising since the ADF-H domain of drebrin

contains only 13-15% sequence homology with cofilin (100). The true activity of the

drebrin ADF-H domain is still unknown. Drebrin may use its ADF-H domain to increase

actin filament plasticity, allowing rapid reorganization of fiber structure without

completely breaking down the fiber. This would fit with drebrin’s ability to compete

with tropomyosin for actin binding, and with data that has shown association with other

actin depolymerizing factors such as gelsolin (76, 77).

TRP family ion-channels have been suggested to be involved in store-operated

Ca2+ entry. Considerable controversy revolves around the role of TRPC family

members in store-operated calcium entry. The evidence that intracellular-store depletion

initiates or modulates the activation of various TRP family members is overwhelming.

Evidence for this type of regulation exists for all TRPCs and also for TRP-vanilloid 6

(TRPV6), a member of one TRP subfamily (5, 101). It has been suggested that TRPC

channels are activated in a store independent manner by IP3 or DAG, rather than calcium

store depletion (99, 102). However, others have suggested that some TRPCs can respond

either to store depletion or to DAG depending on the level of TRPC protein expression

97

and possibly the cell type studied (102). Most TRPCs have a low Ca2+ selectivity with a

PCa/PNa between 0.1 and 10 and undoubtedly contribute to Ca2+ entry (1, 5, 27, 103). The

only Ca2+-impermeable TRPCs so far identified are members of another TRP subfamily,

TRP-melastatin 4 (TRPM4) and TRPM5 (104, 105). Very likely these family members

contribute to the regulation of Ca2+ entry through SOCs, but are not themselves store-

operated. TRPCs have exhibited considerable similarity with SOCs. Most of these

studies were done in heterologous expression systems. Results from cell systems in

which the signaling cascade between the ER and plasma membrane might be altered, the

correct stoichiometry of channels to regulatory proteins might be violated, or the correct

subunits might be missing, are suspect. In any overexpression systems, the effects of

local changes in Ca2+ concentration in a domain around the channel could be dramatic,

considering the high Ca2+ sensitivity of nearly all TRPCs this could be of considerable

consequence.

One criterion for identifying a channel protein as an SOC is that the SOC current

must disappear when expression of the candidate channel has been eliminated. To this

note, few studies have been done. TRPC4-/- mice exhibit an approximately 80-90%

inhibition in SOC activity in endothelial cells (31, 32). However, the

electrophysiological characteristics of heterogeneously expressed TRPC4 do not match

the missing currents from the TRPC4-/- mice (106, 107). Thus, it remains unclear

whether TRPC4 channels are SOCs or whether they merely regulate SOCs.

The most well characterized SOC current is the Ca2+ release-activated Current

CRAC. Unlike other SOCs described, CRAC appears to be highly selective for Ca2+.

The only highly Ca2+-selective channels in the TRP family described so far are TRPV5

98

and TRPV6, which have PCa/PNa > 100 (5, 27, 108). Several of their features are

identical with CRAC. However, single-channel conductance, open-pore block by

intracellular Mg2+, and permeability for Cs+, which reflect pore properties and

pharmacological properties, differ substantially between TRPV6 and CRAC (108).

Therefore, TRPV6 is very likely not CRAC. However, endogenous CRAC was markedly

depressed by expression of N-terminal TRPV6 fragments, indicating a possible

regulatory role of TRPV6 on CRAC (109).

Formation of hybrid TRPC channels composed of more than one TRPC is at

present the best explanation for the elusiveness of ICRAC properties of expressed TRPC

cDNAs and the relative paucity of data from normal cells that predict the

electrophysiological characteristics of the channels that appear upon transfection of TRPC

cDNAs. There is no doubt that multimerization occurs for many TRPs; these heteromers

include the complexes TRPC1-TRPC4-TRPC5, TRPC3-TRPC6-TRPC7, TRPV5-

TRPC6, TRPM4-TRPC5, and TRPM6-TRPC7. Further, it is clear that heteromer

formation changes the permeation and kinetic properties of these channels (29, 46, 110,

111).

Homer adapter proteins have also been shown to regulate store operated channels.

Homer proteins serve as adapters by binding to other proteins through the N-terminal

EVH1 domain via interactions with proline rich sequences, particularly PPxxF motif.

Homers facilitate multi-protein complex formation by homo-multimerization mediated by

EF-hand domains within the c-terminal coiled-coil domain (112). Pancreatic acinar cells

from Homer 1-/- mice exhibit spontaneous activation of store-operated channels. An

isoform of Homer 1 lacking the coiled-coil domain responsible for multimerization

99

(Homer 1a) causes spontaneous SOC activation (46). Similarly, TRPCs have been shown

to bind to Homer proteins, and this binding appears to regulate their activity (46).

TRPC1 mutants that are unable to bind Homers exhibit spontaneous activity as do wild-

type TRPC1 proteins co-expressed with Homer 1a (46).

A recent report demonstrated that BTP inhibits both TRPC3 and TRPC5 channel

activity (99). The TRPC/Homer complex has been suggested to bind to the IP3Rs. This

interaction is believed to regulate TRPC activity as prevention of this interaction induced

spontaneous TRPC activity and disassembly of this complex parallels TRPC activation

(46).

Our data suggests that BTP targets the actin binding protein drebrin.

Interestingly, drebrin has been found in complexes that also contain homer. Homer

binding sites are located in the C-terminal portion of the drebrin protein. One mechanism

by which drebrin may regulate TRPC channels is through reorganization of actin

associated with Homer/IP3R/TRPC complexes. Homer proteins also bind to members of

the Rho family small G-proteins (113). The Rho family proteins regulate actin

cytoskeletal structure by binding to cytoskeletal regulators and inducing their activation.

RhoA exerts its control on actin dynamics through a phosphatidylinositol 4-kinase (PI 4-

kinase) (114, 115). PI 4-kinase produces phosphatidylinositol 4-phosphate, a

phosphoinositide that can be subsequently phosphorylated by the PIP5-kinase to generate

PIP2. PIP2 interacts with and regulates numerous cytoskeletal proteins (116). Local

production of PIP2 on membranes has also been shown to initiate actin nucleation and

regulate membrane–cytoskeleton interactions (117, 118). The neural Wiskott–Aldrich

syndrome protein (N-WASP) links Cdc42 to actin polymerization though the actin-

100

related protein-2/3 (Arp2/3) complex, which promotes actin nucleation and

polymerization (119). Rac1 also activates actin nucleation by binding to the WASP

family member WAVE1 and disrupting an inhibitory complex of

WAVE1/PIR121/HSPC300 allowing active WAVE1 to activate the Arp2/3 complex

(120).

Drebrin could play a role in any of the three models for SOC activation presented

in the introduction (i.e. CIF, conformational coupling, or secretion-like coupling). In the

CIF model, drebrin may be directly activated by a small second-messenger. In turn, this

activation event might lead to rearrangement of the cortical actin layer, thus making the

SOCs more accessible to regulatory proteins or causing them to insert into the plasma

membrane (Fig. 5.1). Both the conformational coupling and secretion-like coupling

models could utilize drebrin’s ability to rearrange the actin cytoskeleton near sites of

SOCs at the plasma membrane. In the former, this could break down a physical barrier

separating the ER and plasma membrane or possibly induce an interaction between IP3R

and SOCs (Fig 5.2). In the latter case, this might allow access of vesicles to the plasma

membrane where they would fuse in order for the SOCs to gain access to extracellular

Ca2+ (Fig 5.3). Additionally, for the secretion-like coupling model, drebrin might play a

role in either release of the vesicles from within the cell or it might help to drive an actin-

based structure that moves the SOC vesicle from the ER to the plasma membrane. A role

for drebrin at this stage is attractive in the sense that it could interact with Homer proteins

associated with IP3Rs as well as the actin cytoskeleton, creating the possibility for multi-

protein complex formation.

101

Homer may recruit actin reorganizing proteins such as drebrin and Rho family

members to the homer/TRPC complex following activation. These proteins could then

facilitate either untethering of the TRPC from the IP3R complex and/or drive formation

of new actin structures that would act as a physical force to relocate TRPC complexes to

the plasma membrane (see fig. 5.4). BTP may prevent drebrin from binding to this

complex, or it may prevent drebrin from rearranging actin associated with the complex.

In either case, the net result of BTP treatment would be to prevent drebrin from changing

the actin structure associated with the complex when stores are full.

102

Figure 5.1. Possible role for Drebrin in the CIF model. In regards to the CIF model,

drebrin may be activated by CIF or one of its downstream effectors. This would most

likely induce drebrin-mediated cytoskeletal changes that would favor SOC activation.

TCR/BCRCa2+

SOC

PLCγPIP2

DAG

IP3PKC

Ras

ER

IP3R

Ca2+

Ca2+

Ca2+

CIF

drebrin

103

Figure 5.2. Possible role for drebrin in the conformational coupling model. In

regards to the conformational coupling model, drebrin may act to break down a layer of

cortical actin that acts as a physical barrier separating the ER and plasma membranes. Or

alternatively, drebrin-mediated cytoskeletal changes may cause the SOC and IP3r to

interact thus activating the SOC.

TCR/BCRCa2+

SOC

PLCγPIP2

DAG

PKC

Ras

IP3

ER

IP3R

Ca2+

Ca2+

Ca2+

drebrin

104

Figure 5.3. Possible role for drebrin in the secretion-like coupling model. Drebrin

may mediate changes at the plasma membrane that allow SOC containing vesicles to

dock with the plasma membrane. Or, drebrin may interact with Homer/IP3R complexes

at the ER to facilitate release or trafficking of the SOC vesicle to the plasma membrane.

TCR/BCRCa2+

SOC

PLCγPIP2

DAG

PKC

Ras

IP3

ER

IP3R

Ca2+

Ca2+

Ca2+

SOC

Rac

Actin

drebrin

Homer

TRPC?

105

Plasmamembrane

Corticalactinnetwork

ER

IP3R

Ca2+ Ca2+Ca2+

Ca2+

A) Stores full

Drebrin

Homer

Homer

Rac1GDP

Homer

HomerDrebrin

Homer

Homer

106

Figure 5.4 Model for Drebrin involvement in SOC activation. A) When intracellular

Ca2+ stores are full, channel proteins would be tethered to the IP3R via interaction with

Homer family members. This complex would likely include other Homer binding

partners such as drebrin and would be sequestered within the cell through interactions

between F-actin and both Homer and drebrin. B) Upon store depletion, a conformational

change in the IP3R would cause the complex to change, breaking the association of the

channel protein and the IP3R, but including other proteins such as active Rac1 in the

complex. The new complex would then rearrange actin associated with the complex and

drive the channel to the plasma membrane where it could insert and allow Ca2+ influx.

B) Stores depletedCa2+ Ca2+Ca2+

ER

IP3R

Ca2+

Ca2+

Drebrin

Homer

Homer

Rac1GTP

Drebrin HomerHomer

107

In our study, BTP did not affect the co-localization of drebrin with actin. It is

however possible that the interaction between drebrin and actin is affected in a more

subtle way than is apparent in these assays. Nevertheless, this data indicates that BTP

affects the function of drebrin. Using inhibitors of actin polymerization or

depolymerization, others have established a link between cytoskeletal rearrangement and

SOC regulation (51). However, no actin regulating proteins have been implicated in this

process at this point. Drebrin is involved in regulation of the actin cytoskeleton and has

profound effects on cell morphology. These effects appear to be mediated by drebrin’s

ability to induce branched and wavy actin filaments. The mechanism by which drebrin

accomplishes this is still unknown. Drebrin has been shown to compete for actin binding

with tropomyosin, fascin, and α-actinin (77, 121). It has also been shown to bind profilin

through drebrin’s proline-rich region. Recent evidence suggests that drebrin may form

complexes with other actin destabilizing proteins such as gelsolin (76). By competing

with actin stabilizing proteins for binding sites and by bringing other actin reorganizing

proteins into a complex, drebrin may create an environment where actin turnover is high

and the structure of surrounding fibers becomes more dynamic. Indeed, in dendritic

spines, a structure enriched in drebrin, actin within the spine has a very high turnover rate

and stabilizing proteins such as tropomyosin are excluded throughout the spine (75).

Given that inhibition of actin depolymerization by jasplakinolide prevents store-operated

channel operation, and that treatment with depolymerizing agents such as latrunculinB

enhance store-operated channel function, it appears as though SOCs are tightly regulated

by the plasticity of the actin structure within the cell (51, 56).

108

CHAPTER 6

Future Directions

109

Currently, studies are ongoing to determine the active portion of the BTP

molecule. Based on previous reports, we expect that the trifluoromethyl pyrazole ring is

important for the compound’s potency (86). To this end, we are testing a series of BTP

derivatives that have been altered by replacing the trifluoromethyl groups with less bulky

groups such as methyls, or deleting them entirely (Fig 6.1). These compounds are being

tested for their ability to inhibit NFAT activation in the luciferase system as well as their

ability to inhibit Ca2+ influx. Once this information is obtained, it may be useful in

optimizing BTPs for use as immunosuppressive drugs.

A number of possibilities exist for drebrin’s role in store-operated calcium entry.

Assuming a role for TRPCs in store-operated calcium entry, whether it is as the SOCs

themselves or as modulators of SOC function, drebrin may affect subcellular localization

or transport of TRPCs. Specifically, the possibility that a TRPC/Homer/drebrin complex

is present in cells is an attractive scenario, since homers have been shown to bind both

TRPCs and drebrin although it is unclear whether these interactions happen in the same

complex. Immunoprecipitation of TRPC and drebrin from cells overexpressing these

proteins should provide an initial idea of whether this interaction occurs. However, if

only a small portion of the drebrin within a cell is associated with TRPCs at any one time

the interaction may be difficult to detect. However, if the interaction is detected it will be

interesting to determine if coexpression of the non-dimerizing homer isoform, homer 1a,

can disrupt the interaction. This would indicate that the interaction is mediated through

homer as expected. Formation of this complex may also include Rho family GTPases,

which have been shown to associate with homer proteins (113). Formation of such a

complex could drive SOCs to the plasma membrane by activating Arp2/3 actin

110

Figure 6.1 Structure of BTP derivatives for determining active portion of BTP

molecule. Series of BTP1 derivatives that replace or delete trifluoromethyl constituents

on the pyrazole ring. (courtesy of Laurie Mottram)

NN

CF3

F3C

HN

O

Cl

NN

CH3

H3C

HN

O

Cl

bis-dimN

N

CF3

H3C

HN

O

Cl

3-trifluorN

N

CH3

F3C

HN

O

Cl

5-trifluor

NN

HN

O

Cl

NN

CF3

HN

O

Cl

3N

NF3C

HN

O

Cl

5

3,5-TFMBTP parent

3,5-DM LFM2-252

3TFM-5M LFM2-259

5TFM-3M LFM2-258

PyrazoleLFM2-253

3TFM LFM2-269

5TFM LFM2-267

111

nucleation, thus building new F-actin at the SOC site. Additionally, it will be important

to determine if BTP alters any interactions found between drebrin and other proteins in

order to more fully characterize BTP’s mechanism of action.

More sophisticated experiments such as fluorescence-resonance energy transfer

(FRET) between drebrin and TRPCs could also be used to determine if these proteins

interact. This approach has the advantage that it is more sensitive than

immunoprecipitation and can be used to determine what percentage of these proteins

interact within a cell given a specific set of circumstances, such as store filling or store

depletion.

Additionally, drebrin has been shown to bind to actin on Gogli membranes. This

raises the possibility that drebrin is involved in secretion or protein trafficking throughout

the cell. Functioning in this manner, drebrin could facilitate movement of vesicles

containing SOCs to the plasma membrane following Ca2+ store depletion. Along these

lines, blockade of drebrin function by BTP could prevent SOCs from accessing the

plasma membrane. Unfortunately, it is difficult to examine drebrin or BTP’s role in this

process because inducible secretion is regulated by increases in [Ca2+]i. Thus inhibition

of secretion by either drebrin knock-down or BTP treatment can’t be separated from

effects on Ca2+ mobilization. It may be possible to assess the role of drebrin in secretion,

or BTP’s ability to inhibit secretion, by examining constitutive secretion mechanisms that

do not rely on transient increases in [Ca2+]i. One system that may be useful in examining

this process was recently reported (122). This system utilizes a fluorescent protein fused

to a conditional aggregation domain (CAD) which can be turned off by addition of a

small molecule. In the absence of the small molecule, the proteins form aggregates that

112

are retained in the ER. When the small molecule is added, interactions of the CAD

domains are disrupted and the proteins are secreted through the cell’s constitutive

secretion process. This process can be followed by time-lapse fluorescent microscopy.

This system could be used to asses the effects of drebrin knock-down or BTP treatment

on secretion.

Loss of drebrin expression using siRNA specific for drebrin demonstrated that

proper Ca2+ mobilization requires drebrin protein expression. BTP binds to the N-

terminal portion of drebrin which contains the actin binding domain. However, we did

not observe differences in drebrin’s ability to bind actin in the presence of BTP. Most

likely, BTP disrupts the association of drebrin with other proteins that participate in Ca2+

influx. Unfortunately, with the exception of actin, homer, and profilin little is known

about proteins that bind to drebrin, or what the true biochemical activity of drebrin is.

Yeast 2-hybrid studies using different fragments of the drebrin cDNA may help to

identify other proteins that bind to drebrin. By using fragments spanning the N-terminal

366 amino acids and the c-terminal amino acids 317-707, binding sites can then be more

clearly identified by using more defined regions in mammalian immunoprecipitation

experiments. Identified proteins can then be screened using siRNA and overexpression

studies to determine if they have an effect on Ca2+ mobilization. Additionally, the

interaction between drebrin and the identified binding partners can be tested to see if BTP

treatment disrupts the interaction.

Long term it will be useful to develop mice deficient in drebrin. The drawback to

this approach is that these mice may have a lethal neuronal defect due to drebrin’s role in

dendrite outgrowth. If this is the case, development of T cell specific knock-out mice

113

using the CRE-Lox system may be a more useful system for determining if drebrin plays

a role in calcium signaling in vivo. These mice would be expected to have defects in T

cell development and activation due to inability to mobilize Ca2+ following stimulation

through the T cell receptor. Most obviously, NFAT dependent transcription would be

expected to be absent in these mice. Transgenic mice generated from the drebrin

deficient mice could carry different fragments of drebrin as transgenes in order to

determine a minimal portion of drebrin required for Ca2+ mobilization.

114

APPENDIX

Characterization of the Serine/Threonine Kinase,

Lymphocyte-Oriented Kinase (LOK)

115

Introduction

Lymphocyte oriented kinase (LOK) is a member of the Ste20 family of

serine/threonine kinases. Specifically, it belongs to the GCK sub-family of this family.

LOK, like all GCK sub-family members, contains an N-terminal Ste20 homology

serine/threonine kinase domain and a C-terminal coiled-coil region (Figs. A.1 & A.2)

(123). Little is known about the role of LOK in lymphocyte function. Knock-out mice

lacking the LOK gene appear normal. However, T cells taken from these animals exhibit

increased integrin clustering following stimulation with ConA (124). Thus it appears as

though LOK may play a modulatory role in lymphocyte activation.

LOK shares significant amino acid similarity with the Ste20 family kinase, Ste-

20-Like Kinase (SLK) (Table. A.1) (125). SLK has recently been implicated as an

upstream effector of the mitotic kinase, polo-like kinase (PLK) (126). PLK is involved in

transition from the G2 to M phase of mitosis. Thus, due to its significant sequence

similarity with SLK, LOK may also regulate G2/M transition during the cell cycle.

116

Figure A.1. Schematic representation of the Ste20 Group of serine/threonine

kinases. Ste20 family members fall into 2 sub-families. The Ste20/PAK family has a C-

terminal kinase domain and an N-terminal p21-binding domain, whereas members of the

GCK family contain an N-terminal kinase domain, no p21-binding domain and a C-

terminal unique or coiled-coil region.

p21-binding domain

GCK family

Structure of Ste20 Group of serine/threonine kinases

Serine/threonine kinase domain

Proline rich domain(s)

Unique or coiled-coiled domain

Ste20/PAK family

117

Figure A.2. Schematic representation of LOK. LOK has an N-terminal

serine/threonine kinase domain, a central proline rich region, and a C-terminal coiled-coil

domain.

Kinase domain

Pro-richregion

Coiled-coiled domain

Lymphocyte Oriented Kinase (LOK)

• GCK family serine/threonine kinase• Lymphocyte restricted expression• Does not activate any of the known kinase pathways• Knockout mice exhibit increased integrin aggregation in T

cells upon Con A stimulation

118

Table A.1. Amino acid homology between human LOK and other Ste20 family

members. LOK shares the most homology with the polo-like kinase kinases, xPlkk1 and

mSLK and very little homology with either GCK or PAK.

100%

36%

36%

37%

hGCK

100%hPAK1

10%hGCK

10%100%mSLK

11%72%100%xPlkk1

10%74%85%100%hLOK

hPAK1mSLKxPlkk1hLOKKinase

% Amino Acid Sequence Homology between hLOK and Other Ste20 Kinase Family Members

119

Effects on TCR signaling In order to asses the role of LOK in lymphocyte function, we generated Jurkat T

cells that stably express a constitutively active form of LOK which lacks the C-terminal

regulatory domain. When these cells were stimulated using Staphylococcal enterotoxin E

(SEE) coated Raji B cells, they produced very little IL-2 compared with control wild-type

Jurkat T cells (Fig A.3)(127). This deficiency was not due to inability to produce IL-2 as

stimulation with PMA and ionomycin, which bypass the TCR, was able to induce IL-2

production similar to that seen in wild-type Jurkat T cells (Fig A.3) (127). This data

indicates that constitutively active LOK blocks a signaling pathway upstream of IL-2

production, but between the TCR and activation of Ca2+-influx and PKC activation.

The IL-2 promoter contains binding sites for several inducible transcription

factors such as NFκB, AP-1, and NFAT. Additionally, the IL-2 promoter region contains

an NFκB/AP-1 composite binding site known as the CD28 responsive element

(CD28RE). Each of these binding sites are essential for IL-2 gene transcription.

120

Figure A.3. LOK kinase domain inhibits antigen induced IL-2 production in Jurkat

T cells. Wild-type Jurkat T cells (control) or Jurkat T cells stably expressing LOKK-

GFP were stimulated by incubation with Raji B cells (1:1) that had been coated with SEE

for 18 hours. Supernatants were collected and analyzed by IL-2 specific ELISA to

determine IL-2 production

LOK kinase domain inhibits antigen induced IL-2 production in Jurkat cells

Raji/SEE PMA/Ion0

2500

5000

7500

ControlLOKK-GFP

Stimulation

pg IL

-2

121

We sought to characterize which transcription factor(s) was inhibited by

expression of constitutively active LOK in order to gain insight into the upstream

signaling pathway that LOK modulates. Transcription driven by the CD28RE is

activated following activation of serine/threonine kinase MEKK1. In order to assess the

role of LOK in MEKK1 induced activation of the CD28RE, we co-transfected Jurkat T

cells with a constitutively active form of MEKK1 (CA-MEKK1) along with a luciferase

reporter plasmid that contained the luciferase gene driven by composite CD28RE sites

with or without full-length LOK. CA-MEKK1 strongly induced expression of the

luciferase gene by the CD28RE (Fig. A.4) (127). Remarkably, when we co-transfected

full-length LOK with CA-MEKK1, transcription of the luciferase gene was fully blocked

(Fig. A.4) (127).

122

Figure A.4. LOK downregulates MEKK1 induced activation of the CD28RE

transcriptional activity in Jurkat T cells. Jurkat cells were transfected with the

CD28RE/AP1-Luc reporter gene alone or with pFC-MEKK1 (2.5µg), LOKK (10µg) or

both MEKK1 plus LOKK or full length LOK. LOK alone had no effect on the

CD28RE/AP1 (results not shown). Luciferase activity was assayed 24h post-transfection.

Results are expressed as % MEKK1-induced activation.

Fold

Act

ivat

ion

Plasmid

MEKK1LOK

0

20

40

60

80

100

+ - - -- + - +- - + +

123

Transcriptional activation from the CD28RE is dependent upon activation of both

NFκB and AP-1. We wanted to know if LOK specifically inhibited activation of one of

these transcription factors downstream of MEKK1. Therefore, repeated the above

experiment except using either AP-1 luciferase reporter (Fig. A.5) or NFκB luciferase

reporter plasmids (Fig. A.6). In both cases expression of LOK blocked activation of the

reporter plasmid. However, inhibition of AP-1 activation appeared to be stronger than

inhibition of NFκB activation.

Since LOK appeared to inhibit both NFκB and AP-1 activation, it is possible that

LOK’s effects on transcription factors are more general. To test this, we transfected

Jurkat T cells that stably express the LOK kinase domain (LOKK) with an NFAT

luciferase reporter plasmid. This protein lacks the C-terminal regulatory region and is

therefore constitutively active. Compared with wild-type Jurkat T cells, Jurkat T cells

expressing LOKK, exhibited an inability to activate NFAT following either CD3 or CD3

and CD28 antibody crosslinking (Fig. A.7). As with IL-2 production, NFAT activation

in response to PMA/ionomycin stimulation was normal.

124

Figure A.5. LOK downregulates MEKK1 induced activation of AP-1

transcriptional activity in Jurkat T cells. Jurkat cells were transfected with the AP1-

Luc reporter gene alone or with pFC-MEKK1 (2.5µg) or both MEKK1 plus LOKK or

Luciferase activity was assayed 24h post-transfection.

.

Control MEKK1 MEKK1 + LOK0.0

2.5

5.0

7.5

Transfection

Fold

act

ivat

ion

125

Figure A.6. LOK downregulates MEKK1 induced activation of NFκB

transcriptional activation in Jurkat T cells. Jurkat cells were transfected with the

NFκB luciferase reporter gene alone or with pFC-MEKK1 (2.5µg)or both MEKK1 plus

LOKK. Luciferase activity was assayed 24h post-transfection

Control MEKK1 MEKK1 + LOK0

10

20

30

40

50

Transfection

Fold

act

ivat

ion

126

Figure A.7. LOK kinase domain inhibits NFAT activation. Wild-type Jurkat T cells

or Jurkat T cells stably expressing LOKK were transfected with the NFAT luciferase

reporter plasmid and then stimulated with anti-CD3, anti-CD3 plus anti-CD28, or

PMA/ionomycin for 6 hours prior to performing luciferase assay.

JurkatJurkat-LOKK

0

0.5

1

1.5

2

2.5

3

3.5R

elat

ive

light

uni

ts

non-stim CD3/CD28CD305

101520253035

non-stim PMA/Ion

127

Since LOK appeared to inhibit multiple signaling pathways, we sought to

determine if LOK inhibited activation of one of the non-receptor tyrosine kinases that are

activated following TCR stimulation. When wild-type Jurkat T cells are stimulated by

cross-linking the TCR associated CD3 chains with antibodies, tyrosine phosphorylation

of several proteins can be detected within minutes. When we performed this experiment

with Jurkat T cells stably expressing LOKK, there was a marked decrease in tyrosine

phosphorylation after stimulation. (Fig. A.8). This indicates that LOK acts very early in

TCR mediated T cell activation. One of the earliest events following TCR cross-linking

is phosphorylation of the CD3-ζ chains by the Src family kinase Lck. We examined the

phosphorylation status of the CD3-ζ chains following CD3 cross-linking and found that

Jurkat T cells expressing LOKK are unable to phosphorylate CD3-ζ chains (Fig A.9).

Therefore, LOK blocks one of the earliest events in T cell activation. Additionally,

cross-linking of the co-stimulatory molecule, CD28, in addition to CD3 was unable to

rescue the defect (Fig A.10).

128

Figure A.8. LOKK decreases tyrosine phosphorylation following TCR stimulation.

Wild-type or LOKK expressing Jurkat T cells were stimulated for 5 min with anti-CD3

antibody and tyrosine phosphorylation was assessed by western blotting with anti-

phosphotyrosine antibody. Western blot for actin confirmed equal loading between

samples.

LOKK decreases tyrosine phosphorylation upon CD3 stimulation

WT LOKK WT LOKK

α-actin blot

α-pY blot

αααα-CD3:

250160105

50

35

3025

75

1 2 3 4- - + +

actin

129

Figure A.9. LOKK inhibits TCR-ζ chain and ZAP-70 phosphorylation. Wild-type

or LOKK expressing Jurkat T cells were stimulated for 5 min. with anti-CD3 antibody

prior to performing anti-phosphotyrosine blot, followed by blotting for Zap-70 and ζ-

chain.

αααα-pY blot

αααα−−−−ζζζζ blot ζ-chain of TcR

1 2 3 4αααα-CD3:

WT LOKK- + - +

Ip: α-ζ

ζ-chain of TcR

Zap-70

LOKK inhibits TcR ζζζζ-chain phosphorylation and Zap-70 phosphorylation

130

Figure A.10. CD28 costimulation does not rescue tyrosine phosphorylation in

LOKK cells. Wild-type of LOKK expressing Jurkat T cells were stimulated for 5 min.

with both anti-CD3 and anti-CD28 antibodies prior to western blotting with anti-

phosphotyrosine antibody.

CD 28 Costimulation does not rescuetyrosine phosphorylation in LOKK cells

α-pY blot

1 2 3 4αααα-CD3/CD28:

WT LOKK- + - +

Ip: α-pY

131

One mechanism by which signaling intermediates are prevented from interacting

in resting cells is by preferential localization of proteins into membrane micro-domains

known as lipid rafts. Upon stimulation, many signaling intermediates are recruited to the

lipid rafts where they can act upon their substrates. Lck, the protein responsible for

phosphorylating the CD3-ζ chains is excluded from the lipid rafts in the resting state and

is then recruited to lipid rafts, which are enriched in TCR, upon stimulation. LOK might

prevent Lck from being recruited to the lipid rafts, and therefore prevent CD3-ζ chain

phosphorylation. Because the lipid rafts are composed of different lipid components than

the rest of the membrane, they are less dense than surrounding membrane and can be

separated, along with proteins found in them, by density gradient ultracentrifugation.

When we performed this type of experiment with Jurkat cells expressing LOKK, we saw

no difference in recruitment of Lck to the lipid rafts (Fig. A.11), despite the overall defect

in tyrosine phosphorylation (Fig. A.12). In agreement with LOK having no role in lipid

raft formation, the LOK kinase domain was not found to be associated with lipid rafts

(Fig. A.13)

132

Figure A.11. Jurkat-LOKK cells are deficient in lipid raft associated tyrosine

phosphorylation. Cells were stimulated for 5 min with anti-CD3 antibody, lysed in

hypotonic lysis buffer containing 1% Triton-X 100 at 4°C, and then loaded onto a

discontinuous sucrose density gradient and spun at 200,000 x g for 4 hours at 4°C. 10

fractions were taken from the top of the gradient and separated by SDS-PAGE prior to

western blotting with anti-phosphotyrosine antibody.

Lipid Raft-associated tyrosine-phosphorylationNon stim. αααα-CD3

Jurkat LOKK

αααα-pY blot

Jurkat WT

αααα-pY blot

Lipid rafts TX-100 soluble Lipid rafts TX-100 soluble

133

Figure A.12. Normal localization of Lck to lipid rafts in Jurkat LOKK cells. Wild-

type of LOKK expressing Jurkat T cells were stimulated with anti-CD3 and lipid rafts

were separated as previously described. Blots were then probed with anti-Lck to detect

the presence of Lck in each fraction.

Normal Localization of Lck to Lipid Rafts

αααα-Lck blot

Jurkat WT

Jurkat LOKK

Lipid rafts Lipid raftsTX-100 soluble TX-100 soluble

Non stim αααα-CD3 stim

Lck

Lipid rafts TX-100 soluble Lipid rafts TX-100 soluble

αααα-Lck blot Lck

134

Figure A.13. LOK kinase domain does not localize to lipid rafts. Lipid rafts were

isolated from non-stimulated or stimulated Jurkat T cells expressing LOKK. Blots were

then probed using anti-GFP antibody to detect the presence of LOKK.

LOK kinase domain is not associated with lipid rafts

Lipid rafts TX-100 soluble Lipid rafts TX-100 soluble

Non stim αααα-CD3 stimJurkat LOKK

135

Discussion (part I)

The serine/threonine kinase, LOK, inhibited production of IL-2 in Jurkat T cells

following stimulation with Raji B cells coated with the super-antigen SEE. We examined

the ability of LOK to inhibit activation of both NFκB and AP-1 downstream of MEKK1

and found that LOK inhibits activation of both of these transcription factors.

Additionally, we found that LOK is able to inhibit activation of the transcription factor

NFAT following CD3 or CD3/CD28 cross-linking. Thus LOK appears to inhibit

activation of multiple signaling pathways upstream of IL-2 gene transcription.

We followed these experiments by assessing the ability of Jurkat T cells

expressing constitutively active LOK to activate the tyrosine kinase cascade that follows

TCR cross-linking. Surprisingly, we found that LOK inhibits activation of the earliest

events in TCR mediated activation, specifically phosphorylation of the CD3-ζ chains.

Although the mechanism for LOK mediated inhibition of CD3-ζ chain phosphorylation is

unclear, it does not involve exclusion of Lck from lipid rafts.

136

Effects on cell-cycle

Because LOK shares significant amino acid sequence homology with kinases that

regulate the mitotic kinase polo-like kinase (PLK), we sought to determine if LOK had

effects on the cell-cycle.

Many cell-cycle regulators associate with microtubules. We created a plasmid

that encodes the C-terminal coiled-coil domain of LOK in order to determine subcellular

localization of this portion of LOK. When we transfected this construct into CHO cells,

we observed peri-nuclear localization of the protein to structures that resemble

microtubules (Fig. A.14). In agreement with this, the related kinase SLK has been shown

to localize to microtubules (128).

We sought to determine if expression of constitutively active LOK would affect

progression through the cell-cycle. To determine this, we transfected NIH3T3 fibroblasts

with LOKK and then serum-starved for 48 hours in order to synchronize the cells in S-

phase of the cell-cycle. We then loaded the cells with propidium iodide to determine

their DNA content and indirectly to determine the phase of the cell-cycle they were in. In

non-transfected cells, most of the cells were in S-phase after 48 hours of serum

starvation, as expected. However, in the transfected cell population (detected by the

presence of GFP) there was a buildup of cells in the G2/M phase (Fig A.15).

Interestingly, when the cells were released to re-enter the cell-cycle by addition of serum

to the culture medium, this buildup at the G2/M phase gradually disappeared so that there

was no detectable difference between the transfected and non-transfected populations by

24 hours following reintroduction of serum (Figs. A.16-19).

137

Figure A.14. LOK coiled-coil region exhibits a unique sub-cellular localization

pattern. CHO cells were transfected with a plasmid encoding the LOK coiled-coil

domain fused to GFP and visualized by fluorescent microscopy using a 40X objective.

LOK coiled-coil region exhibits a unique sub-cellular localization pattern

LOKcc-GFP

138

Figure A.15. LOKK causes cells to arrest in G2/M phase following serum

starvation. NIH3T3 cells were transfected with LOKK-GFP and serum starved for 48

hrs prior to staining with propidium iodide and analysis by flow cytometry. LOKK-GFP

positive cells were separated from non-transfected cells on the basis of GFP expression

and each group was analyzed for DNA content as an indication of their progression

through the cell-cycle.

LOKK 0 hrs post serum-starve

05

10152025303540

G0/G1 S G2/M Apoptosis

GFP-

GFP+

% o

f cel

ls

139

Figure A.16. Serum allows LOKK expressing cells to overcome G2/M phase arrest.

NIH3T3 cells were transfected with LOKK-GFP and serum starved for 48 hrs and then

released to progress through the cell-cycle by re-addition of 10% serum for 16 hours prior

to staining with propidium iodide and analysis by flow cytometry. LOKK-GFP positive

cells were separated from non-transfected cells on the basis of GFP expression and each

group was analyzed for DNA content as an indication of their progression through the

cell-cycle.

LOKK 16 hrs post serum-starve

051015202530354045

G0/G1 S G2/M Apoptosis

GFP-

GFP+

% o

f cel

ls

140

Figure A.17. Serum allows LOKK expressing cells to overcome G2/M phase arrest

(20 hrs). NIH3T3 cells were transfected with LOKK-GFP and serum starved for 48 hrs

and then released to progress through the cell-cycle by re-addition of 10% serum for 20

hours prior to staining with propidium iodide and analysis by flow cytometry. LOKK-

GFP positive cells were separated from non-transfected cells on the basis of GFP

expression and each group was analyzed for DNA content as an indication of their

progression through the cell-cycle.

LOKK 20 hours post serum-starve

0

10

20

30

40

50

60

G0/G1 S G2/M Apoptosis

GFP-

GFP+

% o

f cel

ls

141

Figure A.18. Serum allows LOKK expressing cells to overcome G2/M phase arrest.

NIH3T3 cells were transfected with LOKK-GFP and serum starved for 48 hrs and then

released to progress through the cell-cycle by re-addition of 10% serum for 24 hours prior

to staining with propidium iodide and analysis by flow cytometry. LOKK-GFP positive

cells were separated from non-transfected cells on the basis of GFP expression and each

group was analyzed for DNA content as an indication of their progression through the

cell-cycle.

LOKK 24 hours post serum-starve

05

10

15

2025

3035

G0/G1 S G2/M Apoptosis

GFP-

GFP+

142

Discussion (part II)

The substrate of LOK remains unknown. However, due to its similarity with

kinases that phosphorylate PLK, namely Xenopus polo-like kinase 1 (xPLKK1) and

mammalian SLK, LOK is a possible PLK kinase. In this role, constitutive LOK kinase

activity would be expected to have some effect on the cell-cycle. Indeed this is the case.

Expression of constitutively active LOK was able to cause an increase in the percentage

of cells that remained in G2/M phase following 48 hours of serum starvation, which

normally arrests cells in S phase. This is likely an effect of persistent phosphorylation of

PLK. In fact, it was recently demonstrated that LOK can phosphorylate PLK, making

this a likely scenario (129). However, when cells were released to re-enter the cell-cycle,

the block at G2/M was overcome. Presumably, some factor in serum activates another

factor that is able to overcome the persistent PLK phosphorylation, perhaps a

phosphatase that opposes LOK. Another possibility is that the cells re-enter S phase and

become multi-ploidal and thus are out of range for the measurements used and

consequently go undetected. This was reported to be one effect of LOK expression by

another group, however we have not confirmed this possibility in our system (129).

143

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Vita

Name Jason C. Mercer Education 2000-2005 Doctor of Philosophy in Biochemistry, Microbiology, and

Molecular Biology The Pennsylvania State University University Park, PA 1996-2000 Bachelor of Science in Biology The University of Texas at Dallas Richardson, TX Research Experience 2000-2005 Department of Biochemistry and Molecular Biology The Pennsylvania State University Advisor: Dr. Avery August Thesis Project: 3,5-bistrifluoromethyl pyrazole (BTP) compounds

and regulation of store-operated calcium channels by the actin binding protein Drebrin

Teaching Experience Spring 2001 Teaching Assistant – Microbiology: Introduction to Microbiology

(Micro 202) Fall 2001 Teaching Assistant – Biochemistry and Molecular Biology:

Protein and Molecular Cloning lab (BMB 342) Publications Tao L, Wadsworth S, Mercer J, Mueller C, Lynn K, Siekierka J, and August A. Opposing roles of serine/threonine kinases MEKK1 and LOK in regulating the CD28 responsive element in T-cells. Biochem J. 2002, 363; 175 Fisher A, Mercer J, Iyer A, Ragin M, and August A. Regulation of CXCR4 mediated migration by the Tec family tyrosine kinase Itk. J Biol Chem. 2004, 279; 29816 Mercer JC, Ragin MJ, and August A. Natural Killer T cells: Rapid responders controlling immunity and disease. Int. J. of Biochem & Cell Biol. In Press.