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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.