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Dual functions of Vav in Ras-related small GTPases signaling regulation

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Dual functions of Vav in Ras-related small GTPases signaling regulation

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DUAL FUNCTIONS OF VAV IN RAS-RELATED SMALL
GTPASES SIGNALING REGULATION
by
Yi Xia
A Thesis Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(Biochemistry & Molecular Biology)
December 1999
Copyright 1999 Yi Xia
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UMI Number: 1409666
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UNIVERSITY O F SOUTHERN CALIFORNIA
THE GRADUATE SCHOOL
UNIVERSITY PARK
LOS ANGELES. CALIFORNIA MOOT
This thesis, written by
tstu , _________
under the direction of A.x.£.—Thesis Committee,
and approved by all its members, has been pre
sented to and accepted by the Dean of The
Graduate School, in partial fulfillment of the
requirements for the degree of
M a s te r o f S c ie n c e
Date vJ ® mber 2 9 , 1999
TH£SIS COMMITTEE
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ACKNOWLEDGEMENTS
First of all, I would like to thank Dr. Daniel Broek, who provided me the great
opportunity to study and research in his laboratory with his team. I appreciate his
encouragement and guidance.
I also thank all the people, who gave me their technical assistance and valuable
suggestions.
Finally, I really thank my wife Han Ma, whose love and support are always inside
my heart, and my baby girl Jessica Xia, who brings me happiness.
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TABLE OF CONTENTS
Acknowledgements...............................................................................................................ii
List o f tables and figures......................................................................................................v
Abstract..................................................................................................................................vi
Chapter I. Introduction
1.1 Role of vav in hematopoiesis, lymphocyte development and activation...................... 1
1.2 Signaling motifs and possible function of vav in intracellular signaling.......................2
1.3 Function and regulation of Ras and Ras-related small GTPases in intracellular
signal transduction...........................................................................................................5
1.4 Purpose of our research: Studying the role of vav in regulation o f Ras-related
GTPase signaling.............................................................................................................6
Chapter 2. Vav/Y-DH alone is sufficient for its GEF activity and its binding
affinity for Rac is influenced by Lck phosphorylation
2.1 Introduction......................................................................................................................16
2.2 Recombinant Lck is able to phosphorylated vav/Y-DH in vitro, thereby to
influence its binding affinity to Rac-GTPase.................................................................18
2.3 Vav/Y-DH alone is sufficient to exert its GEF activity for Rac-GTPase.....................20
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Chapter 3. Vav acts as a GDI for Ras and is regulated by Lck Phosphorylation
3.1 Introduction.....................................................................................................................29
3.2 Vav/Y-DH is able to bind Ras-GTPase in vitro............................................................ 30
3.3 Vav/Y-DH serves as a GDI for Ras-GTPase................................................................ 31
Chapter 4. Conclusion....................................................................................................... 41
Chapter 5. Materials and Methods................................................................................... 47
Chapter 6. References.........................................................................................................58
iv
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LIST OF TABLES AND FIGURES
Figure 1.1 Schematic structure of proto-oncogene vav......................................................8
Figure 1.2 Interaction of the principal domains of vav with other signaling
molecules............................................................................................................ 10
Figure 1.3 Structure of Dbl protein families........................................................................ 12
Figure 1.4 Activation/inactivation cycle o f Ras superfamily of small GTPases.............. 14
Table 2.1 Dbl-related GEF family of proteins....................................................................22
Figure 2.2. Vav/Y-DH protein can be phosphorylated by Lck in vitro............................. 23
Figure 2.3. The results of in vitro vav A"-DH and Rac-GTPase binding assay..................25
Figure 2.4. The results of in vitro 3 H-GDP-releasing assay...............................................27
Figure 3.1. In vitro vav/Y-DH and Ras-GTPase binding essay..........................................35
Figure 3.2. The results of in vitro 3 H-GDP-releasing assay...............................................37
Figure 3.3. The results of 3 H-GTP-releasing assay............................................................. 39
Figure 4.1. Schematic mode of modulation of vav catalytic activity by both Lck
and phosphoinositides.........................................................................................43
Figure 4.2. The effects of Phosphoinositides on phosphorylation of Tiam by PKC
in vitro.................................................................................................................45
Figure 5.1. Construction of vav Dbl-homology domain (vav/Y-DH)............................... 56
v
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ABSTRACT
Vav, a 95 IcDa protein which was originally identified as a transforming oncogene
during gene transfer studies, is expressed solely in cells o f hematopoietic origin
regardless o f their differentiation lineage including lymphoid, myeloid, and erythroid
lineage. It is well established that vav plays a critical role in the activation and
modulation o f Ras-related small GTPases, which are involved in distinct intracellular
signaling cascades such as regulation o f cell growth and differentiation and organization
o f cytoskeleton. Our previous studies have reported that vav acts as a guanine nucleotide
exchange factor (GEF) for Rac/Rho family o f GTPases and observed that vav was able to
bind Ras-GTPase and influenced its guanine nucleotides exchange. In this study, we
further study the role o f vav for Rac and Ras-GTPases. We found that the Dbl-homology
domain o f vav (vav/Y-DH) alone was sufficient to exert its GEF activity towards Rac-
GTPase, and Lck phosphorylation o f vav/Y-DH influenced its binding affinity for Rac-
GTPase, thereby to further modulate its catalytic activity for Rac-GTPase. Also we
observed that vav/Y-DH protein inhibited the release o f 3 H-GDP from GDP/Ras-GTPase
under unphosphorylated state. Further in vivo studies are needed to examine the role o f
vav under phosphorylation condition. Thus these findings support our conclusions that
vav possesses dual functions in Ras-related small GTPase signaling regulation, i.e., vav
acts as a GEF for Rac-related family o f small GTPases and serves as a guanine nucleotide
dissociation inhibitor (GDI) for Ras-GTPase.
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CHAPTER 1: INTRODUCTION
1.1 Role of vav in hematopoiesis, lymphocyte development and activation
Vav was originally identified as a transforming oncogene during gene transfer
studies that involved transfection of murine NIH 3T3 fibroblasts with DNA from human
esophageal carcinomas and tumorigenicity assays in athymic nude mice (I, 2). Analysis
of cDNA clones corresponding to the vav oncogene, and subsequent cloning of the
complete human and murine proto-oncogene vav cDNA, indicated that the vav oncogene
was generated by a genetic rearrangement (2, 3, 4). Vav oncogene found in the
transfected NIH 3T3 cells, revealed that its N-terminal amino acid sequence was replaced
by the Tn5 transposase, which is derived from the pSV2neo plasmid co-transfected as a
selectable marker in gene transfer assays (3). Therefore, deletion of N-terminal sequence
of proto-oncogene vav causes it to become oncogenic. This hypothesis was supported by
Katzav S et al, who observed that the full-length proto-vav cDNA clone failed to
transform NIH 3T3 cells, whereas truncation of amino-end HLH domain of proto-vav
resulted in oncogenic activity (3, 4).
Vav is expressed solely in cells of hematopoietic origin regardless of their
differentiation lineage including lymphoid, myeloid, and erythroid lineage (2). It is likely
to play a critical role in hematopoiesis. This notion is supported by the finding that vav
expression is first detected in in vitro differentiating murine embryotic stem (ES) cells
just prior to the early onset of hematopoietic differentiation (5-7). Stable expression of
l
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vav antisense RNA in ES ceUs inhibited their spontaneous or cytokine-induced
differentiation into erythroid and non-erythroid hematopoietic cells, without interfering
with the development of embryoid body (5). Moreover, studies with vav knockout mice
have revealed that ablation of vav gene led to block in the development of positive and
negative T-cell selections, a severe reduction in the number of thymic double-positive
and double-negative thymocytes as well as mature peripheral CD4" or CD8" T-cells (8,
9). Similarly, absence of vav also results in defects in B-cell development and B-cell
receptor (BCR) signaling pathway (8, 10). The total numbers of peripheral B cells were
reduced and peritoneal CDS" B cells were absent. Furthermore, overexpression of vav
was found to promote antigen mediated transcriptional activation of nuclear factor of
activated T cells (NFAT), which controls antigen receptor-induced activation of genes for
cytokines such as D L-2, EL-4, and TNF-a (11-13). Thus vav is implicated in
hematopoiesis, lymphocyte development and activation.
i.2 Signaling motifs and possible function of vav in intracellular signal transduction
Analysis of the putative sequence of the vav oncogene and proto-oncogene
products revealed that vav gene codes for a 95 kDa protein, which contains several highly
conserved signaling motifs (2, 3). The abundance of such motifs suggests that vav could
be involved in distinct intracellular signaling pathways and possibly mediate crosstalk
among such signaling pathways. A schematic view o f the multiple modular domains of
vav is shown in Figure 1.1. The following description of these structural domains
corresponds to their order in the amino acid sequence o f proto-oncogene vav protein.
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1) The N-terminal leucine-rich region. The N-terminal leucine-rich region of vav
was predicted to be an amphipathic helix-loop-helix (HLH) followed by a leucine zipper-
like (LZ-like) domain, which resemble the C-terminal of Myc protein and the steroid
binding domain of nuclear receptors (2, 3). Truncation of this region from the proto
oncogene vav product confers its transforming activity (3).
2) The Acidic residue-rich domain. A region downstream of the leucine zipper
like domain contains 49% glutamic or aspartic acid and corresponds to residues 132-176
in proto-oncogene vav product. The 174-tyrosine residue in this region can interact with
SH2 domains of Lck, Fyn, and p85 (PI3-K) upon phosphorylation (Bonnefoy-Berard N.
et al., unpublished) (Fig. 1.2).
3) The Dbl-homology (DH) domain. The Dbl oncoprotein was first designated as
a guanine nucleotide exchange factor (GEF) for Rho-related family (14). Thereafter, a
number of Dbl-related proteins were discovered to possess GEF enzyme activity for Rho
family (Fig. 1.3), such as Tiam, Lfc, and Lsc (IS, 16). These guanine nucleotide
exchange factors (GEFs) activate small GTPases by exchanging of bound GDP for GTP,
therefore, converting the small GTPases from an inactivated state (GDP-bound form) to
an activated state (GTP-bound form). This GDP/GTP exchange in GTPases is a critical
step in the stimulation of downstream effector signaling cascades (17-20). Vav also
contains an approximately 250 amino acid motif called Dbl-homology (DH) domain and
it has been reported to possess GEF enzyme activity towards the Rho-related family of
GTPase (21,22).
3
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4) The Pleckstrin-homology (PH) domain. The PH domain was first defined as
two 100-amino acid repeats in pleckstrin, the major PKC substrate in platelet. This PH
domain is present in all proteins that contain a DH domain. It is usually located
downstream of the DH domain and exerts its function association with the adjacent DH
domain. Ligands that were found to bind in vitro to PH domains include the Py subunits
of trimeric G proteins, several protein kinase C (PKC) isoforms and a lipid second
messenger, phosphatidylinositol 4, 5-bis-phosphate (23-26). A recent study has repoited
that the PH domain of vav can bind phosphoinositides (22). The PH domain of vav binds
in vitro to PI-3,4,5 Pj.a product of the reaction catalyzed by phosphatidylinositol 3-kinase
(PI-3-K), and this interaction promotes phosphorylation of vav by Lck thereby activating
vav’s GEF catalytic function for Rac/Rho family of GTPases. Whereas vav binding PI-
4,5 P2. a substrate of PI-3 K, exhibits inhibitory modulation of its catalytic function. Thus
the PH domain might possess a regulatory function to allosterically modify DH domain’s
catalytic activity (22).
5) The Cysteine-rich domain. Proto-vav has a cysteine-rich, zinc finger-like
domain corresponding to amino acid 516-563 residues, which displays strong similarity
to the phorbol ester binding domain of protein kinase C. A study has revealed that Cys-
528 in this region was essential for phorbol ester binding in vivo (27).
6) The C-terminal Src-homology 2 and 3 (SH2/SH3) domain. SH2 and SH3 domains
are frequently present together and serve as non-catalytic modules that regulate cellular
process through protein-protein interactions (28, 29). SH2 domains associate with
4
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specific proteins via sequences containing phosphotyrosine, whereas SH3 domains
appear to bind preferentially to proline-rich motif. The C-terminal region of vav
constitutes two SH3 domains, which are separated by a single SH2 domain. A short
proline-rich sequence, which lies just upstream of the proximal SH3 domain of vav,
could potentially interact with SH3 domains of other signaling molecules. So far no such
molecule has been identified to be able to bind to this proline-rich sequence of vav.
1.3 Function and regulation of Has and Ras-related small GTPases in intracellular
signal transduction
Ras superfamily comprises more than S O closely related proteins which have the
ability to bind and hydrolyze guanosine triphosphate (GTP). They can be further divided
into at least six subfamilies based upon their primary sequence, i.e., Ras, Rho, Rab, Ran,
Rad, and Arf (17, 18). The members of these small GTPases subfamilies have been
shown to possess diverse biochemical and biological functions in intracellular signal
transduction. For examples, Ras is involved in regulation of cell proliferation and
differentiation, Rab and Arf modulate the vesicular transport and membrane traffic, Rho
participate in organization of cytoskeleton and generation of free radicals, and Ran
regulates the nuclear transport and cell cycle (30-33). Ras-related small GTPases function
as regulated molecular switchers that cycle between an activated GTP-bound form (on
state) and an inactive GDP-bound form (off state). These GTP/GDP binding states are
tightly controlled by three classes of regulatory molecules. The guanine nucleotide
exchange factors (GEFs), promote release of GDP from the small GTPases and
5
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consequently facilitate GTP binding and the GTPase activation, whereas GTPase-
activating proteins (GAPs) hydrolyze GTP and cycle these GTPases back to the inactive
GDP-bound form. A third class of the regulatory molecules is guanine nucleotide
dissociation inhibitors (GDIs), which associates with GTPases to maintain the existing
nucleotide-bound state (Fig. 1.4).
1.4 Purpose o f our research: Studying the role of vav in regulation of Ras and Ras-
related GTPases signaling
The highly specific expression of vav in hematopoietic cells indicates that it might
play a critical role in signaling pathways unique to these cells. Vav is coupled to distinct
hematopoietic receptors. These receptors include antigen receptors on T and B
lymphocytes (34-36); accessory receptors, i.e., CD28 on T cells (37) and CD19 on B cells
(38); several protein tyrosine kinase (PTK) receptors such as c-kit (39, 40); and platelet
derived growth factor (PDGF) receptor in vav-transfected NIH 3T3 fibroblasts (41).
Activation of these receptors upon ligand binding leads to a rapid increase in the tyrosine
phosphorylation of vav. The phosphorylated vav is able to modulate the activity of Ras-
GTPase or/and Ras-related small GTPases such as Rac and Rho via direct protein-protein
interaction, thereby to further trigger the subsequent signaling cascades in diverse
intracellular signaling pathways (22, 34).
Our previous studies have revealed that phosphorylated vav stimulated the release
of 3 H-GDP from ^H-GDP/Rac-GTPase complex and the products of PI-3 K (PI-3,4 P2
6
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and PI-3,4,5 P3) also exhibited two-fold stimulation of vav GEF activity. In contrast, in
the presence of the substrates of PI-3 K (PI-4,5 P2) observed a 90% inhibition of vav GEF
activity (22). Thus these findings suggest that vav acts as a GEF for Rac/Rho family of
small GTPases and is regulated by both Lck phosphorylation and phosphoinositides (22).
Furthermore, our preliminary data have revealed that vav is able to bind Ras-GTPase in
vitro and this interaction can influence the guanine nucleotide-binding status of Ras-
GTPase (Han JW and Broek D unpublished data). So the goals of this thesis project are
1) To further study the role of vav as a GEF for Rac-related GTPases. We will examine
whether the individual Dbl-homology (DH) alone is sufficient to exert vav’s GEF activity
for Rac-GTPase and whether Lck phosphorylation of vav influence its binding affinity
for Rac-GTPase, thereby to further modulate its GEF activity for Rac-GTPase. This part
of study will be demonstrated in chapter2. 2) To examine the role of vav for Ras-GTPase.
We will test whether vav is an upstream regulatory molecule for Ras-GTPase and what is
its functional role in Ras-GTPase signaling regulation? Is it a guanine nucleotide
dissociation inhibitor (GDI) for Ras-GTPase? Can its catalytic activity be regulated by
Lck phosphorylation or/and phosphoinositides? This part of study will be discussed in
chapter 3.
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Figure 1.1 Schematic structure of proto-oncogene vav. LR: leucine-rich domain, Ac:
acidic region, YEDL motif is the phosphorylation site of tyrosine residue, DH: Dbl-
homology domain, PH: pleckstrin-homology domain, CR: cysteine-rich domain, Cys-
528: phorbol ester binding site, PPPP: proline-rich region, SH2: Src-homology 2 domain,
SH3: Src-homology 3 domain.
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LR Ac DH PH CR SH3 SH2 SH3
PPPP
YEDL
Oncogenic
deletion
9
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Figure 1.2 Interaction of the principal domains o f vav with other signaling
molecules. LR: leucine-rich domain, Ac: acidic region, DH: Dbl-homology domain, PH:
pleckstrin-homology domain, CR: cysteine-rich region, SH2: Src-homology 2 domain,
SH3: Src-homology 3 domain, Y: Tyr residue interacts with SH2 domains of other
signaling molecules upon phosphorylation, P: proline-rich region possible binds SH3
domains of other protein.
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Syk, ZAP-70, JAK
SLP-76, SHP-1
Nek, Crk,
tubulin
Oncogenic ?
deletion
E-actin
Grb-2
i
i
*
hnRNP K ,
Ku-70, zyxin
LR DH PH CR SH3 SH2 SH3
( R
Ptyr residue
t
SH2 domains of lck,syn,src,
PLCyl ,GAP,p85(PI-3K)
Raf, PI-3K
?
u
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Figure 1.3 Structure o f Dbl protein families. The domains are as indicated. PH:
pleckstrin-homology domain, DH: Dbl-homology domain, Ras GEF: GEF domain for
Ras-GTPase, DAG: phorbol ester binding domain, SH2/3: Src-homology domains.
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Proto-Dbl
Tim
Cdc24
Ost
Tiam-1
Ect-2
Lbc
Dbs
Vav
Abr
RasGRF
MSos
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Figure 1.4 Activation/inactivation cycle of Ras superfamily of small GTPases. GEF:
guanine nucleotide exchange factor, GDI: guanine nucleotide dissociation inhibitor,
GAP: GTPase-activating protein.
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Extracellular stimulation
GDP
GEF GTP
GDI
OFF
GTPase
GDP
GTPase
G T P
ON
GDI
GAP
Effectors
signaling
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CHAPTER 2: VAV/Y-DH ALONE IS SUFFICIENT FOR ITS GEF ACTIVITY
AND ITS BINDING AFFINITY FOR Rac IS INFLUENCED BY Lck
PHOSPHORYLATION
2.1 Introduction
Rho subfamily of small GTPases in the mammlian cells comprises more than ten
members including Rho, Rac, Cdc42, TC10, RhoG, and RhoE (42). Members of the Rho
subfamily of small GTPases control the adhesion, morphology, and motility of
mammalian cells. Rho, Rac, and Cdc42 share 50% identity at the amino acid sequence
level and have been shown to regulate the signal transduction pathways that link
extracellular signals to the formation of stress fiber, lamellipodia, and filopodia,
respectively, in experimental Swiss 3T3 cell line (43-45). Moreover, members of Rho
subfamily of small GTPases are capable of activating signaling pathways such as
activation of SAPK/JNK and p38 MAPK during stress (46-48) as well as activation of
transcriptional factors SRF and NF-kB (49, 50). Furthermore, members of Rho subfamily
of small GTPases are also involved in signaling regulation of cell growth control and
their activities are essential for Ras-induced cell transformation (42, 51). The activation
of Rho subfamily of small GTPases is determined by their GTP: GDP content, which is
regulated by guanine nucleotide exchange factors (GEFs), GTPase-activating proteins
(GAPs), and guanine nucleotide dissociation inhibitors (GDIs). (See figure 1.4)
16
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The guanine nucleotide exchange factors (GEFs) for Rho subfamily of small
GTPases are characterized by a common structural motif designated Dbl-homology (DH)
domain, which is immediately followed by a pleckstrin homology (PH) domain. (49).
These are the Dbl-related family o f proteins (Table 2.1).
Among these putative GEFs, Vav is the most controversial one. Some studies
demonstrated that vav is a GEF for Ras-GTPase (34, 52), whereas others speculated that
vav might be a GEF for Rho family of GTPases, but not for Ras-GTPase (53, 54). Vav
contains a DH domain in tandem with a PH domain, which is similar to the structure of
the GEFs for Rho family such as Dbl, but vav does not have CDC25 catalytic domain as
seen in the GEFs for Ras-GTPase such as RasGRF, mSos, and CDC25 (4). Recently,
some studies have reported that vav acts as a GEF for Rac/Rho family of GTPases and is
regulated by both Lck phosphorylation and phosphoinositides (21, 22).
The goal of this thesis project is to further study the role of vav as a GEF for Rac-
related GTPases. We will examine whether the individual Dbl-homology (DH) alone is
sufficient to exert vav’s GEF activity for Rac-GTPase, and whether Lck phosphorylation
of vav influences its binding affinity for Rac-GTPase, thereby further modulating its GEF
activity for Rac-GTPase.
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2.2 Recombinant Lck is able to phosphorylate vav/Y-DH in vitro, thereby to
influence its binding affinity to Rac-GTPase
Some studies have demonstrated that vav is constitutively phosphorylated on its
tyrosine and serine residues in resting T lymphocyte, B lymphocyte, macrophage, and
vav-transfected NIH 3T3 cells (41, SS, 56). Upon ligand stimulation of a number of
receptors including antigen receptors on T and B lymphocytes (34, 35),
lipopolysaccharide receptors on macrophages (57), and platelet derived growth factor
(PDGF) receptors in vav-transfected fibroblasts (41), phosphorylation of vav on the
tyrosine residue is rapidly increased. Then the phosphorylated vav is able to activate Ras
and Rho family GTPases and further triggers subsequent signaling cascade in distinct
intracellular signaling pathways (21, 22).
The identification of the protein tyrosine kinases (PTKs) that are responsible for
the phosphorylation of vav under physiological conditions is still unclear. Several
candidates of Src-related kinases such as Zap-70, Lck, and Syk were reported to have the
ability to phosphorylate vav in vitro and in vivo (58-60). In our experiments, we were
able to show that the vav/Y-DH protein, which is the DH domain of vav including 174
tyrosine residue, could be phosphorylated by recombinant Lck kinase, which was
expressed in insect sf9 cells. We first constructed the Dbl-homology domain of vav
including the 174-tyrosine residue into pRSET vector system to express vav/Y-DH fusion
protein and produced recombinant Lck kinase in baculoviral expression system (sf9
insect cell). After purification of vav/Y-DH protein and preparation of recombinant Lck
18
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kinase from sf9 insect cell lysate, the 100 pmoles o f vav/Y-DH protein were incubated
with 3 ui of recombinant Lck cell lysate at 37°C for 20 minutes during in vitro
phosphorylation assay.
As shown in figure 2.2, all recombinant Lck kinase prepared from three individual
batches of induced insect sf9 cells were able to phosphorylate the vav/Y-DH protein
compared to non-induced insect cell lysate in vitro. These data revealed that the
recombinant Lck kinase was expressed constantly and comparatively in the baculoviral
expression system, and its kinase activity was efficient. Further, we tested the binding
affinity of vav/Y-DH protein for Rac-GTPase under phosphoralated and
unphosphorylated states. During the vav/Y-DH protein and Rac-GTPase in vitro binding
assay, 100 pmoles of vav/Y-DH protein and 300 pmoles of soluble GST-Rac were used.
The phosphorylated vav/Y-DH protein by recombinant Lck kinase was prepared as
described in above in vitro phosphorylation assay. Following in vitro binding reaction,
the SDS PAGE and Western blotting were performed. The results of immunodetection of
bound GST/Rac in the binding assay by using anti-Rac antibody are present in the figure
2.3.
As shown in figure 2.3, the vav/Y-DH protein, which contains the DH domain
including Y-174 phosphorylation site was able to bind Rac-GTPase in vitro. The
phosphorylated vav/Ptyr-DH bound Rac-GTPase stronger than unphosphorylated vav/Y-
DH did. Our previous studies have provided direct evidence for the role of vav as a GEF
for Rho family members and this activity was regulated by both Lck phosphoryiaton and
19
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phosphoinositides modulation (21, 22). Thus the results of above in vitro binding assay
suggest that phosphoiylation of vav on tyrosine-174 residue might modulate vav’s GEF
catalytic activity by directly influencing its binding affinity for Rac-GTPase, thereby to
further trigger distinct downstream signaling pathways.
2.3 Vav/Y-DH alone is sufficient to exert its guanine nucleotide exchange factor
(GEF) activity for Rac-GTPase
We have demonstrated that vav is a guanine nucleotide exchange factor (GEF) for
Rac-GTPase (21, 22). Further, we need to determine whether the vav/Y-DH alone is
sufficient to exert vav’s GEF catalytic activity for Rac-GTPase. For this purpose, in vitro
3 H-GDP-releasing assay was performed. During in vitro 3 H-GDP-releasing assay, 10
pmoles of soluble 3 H-GDP coupled GST-Rac protein were incubated with IS pmoles of
his-tagged vav/Y-DH protein or Ni2"agarose-beads (as a negative control) in S00 ul of
buffer G (without DTT) at 37°C. Then 10 uM o f cold GTP and 500 ug of BSA were
added to the reaction. Thereafter, 60 ul of aliquots from each reaction sample was
removed at 0, IS, 30 minutes time points, and then mounted on the nitrocellulose
membrane filter for radioactivity detection (see chapter S methods and materials). The
results of3 H-GDP-releasing assay at IS minutes time point are present in figure-2.4.
As shown in figure 2.4, the vav/DH protein alone enhanced the 3 H-GDP releasing
from 3 H-GDP-Rac-GTPase complex by approximately two-fold by comparing with the
blank that indicated the spontaneous dissociation o f 3 H-GDP from ^-GDP/Rac-GTPase
20
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complex. These data suggest that the Dbl-homology domain of vav alone is sufficient to
exert its GEF activity for Rac-GTPase. Thus vav acts as a GEF for Rac-GTPase via direct
interaction between vav DH domain and Rac-GTPase, and phosphorylation of vav by
Lck kinase influences this interaction, thereby to further modulate vav’s catalytic activity
for Rac-GTPase.
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Tab. 2.1 Dbl-related GEF family of proteins
(Modified from Van Aelst et al, 1997)
Biochemical function Biological properties Tissue distribution
Dbl GEF for Rho/Cdc42 oncogenic brain, adrenal glands,
Lfc GEF for Rho oncogenic hematopoietic cells,
Lsc GEF for Rho oncogenic hematopoietic cells,
Dbs
?
oncogenic kidney, lung,
Lbc GEF for Rho oncogenic heart, lung,
Tiam GEF for Rac oncogenic, metastatic brain, testis
vav GEF for Rho/Rac ? oncogenic hematopoietic cells
FGD1 GEF for Cdc42 implicated in brain, heart, lung,
Ost GEF for Rho/Cdc42 oncogenic brain, lung, heart, liver
Bcr GEF for Rho/Cdc42/Rac implicated in leukemia predominantly brain
Abr GEF for Rho/Cdc42/Rac
?
predominantly brain
Ect-2 binds to Rho/Rac ? oncogenic testis, liver, kidney,
Trio GEF for Rho/Rac cell migration ? ubiquitous
NET1 GEF for Rho oncogenic ubiquitous
Tim
?
oncogenic kidney, liver, placenta,
Sos GEF for Ras/Rac
?
ubiquitious
RasGRF GEF for Ras/Rac
?
brain
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Figure 2.2 Vav/Y-DH protein can be phosphorylated by Lck in vitro. 100 pmoles of
vav/Y-dh protein were incubated with 3 ul of insect cell lysate at 37°C for 20 minutes and
with 3 ul of non-induced cell lysate as a negative control. Lane 1, 2, 3: Vav/Y-DH plus
recombinant Lck (different batches of insect cell lysate), Lane 4: unloaded, Lane 5:
Vav/Y-DH plus non-induced insect cell lysate.
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Lane 1 2 3 4 5
Vav/Y-DH
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Figure 2.3 The results of in vitro vav/Y-DH and Rac-GTPase binding assay. 100
pmoles of phosphorylated or unphosphorylated his-tagged vav/DH protein were
incubated with 300 pmoles Rac/GST fusion protein at 37°C for one hour. Anti-Rac
antibody was used to detect Rac/GST in western blotting. Lane I: unphosphorylated
vav/Y-DH and GST/Rac, Lane 2: phosphorylated vav/Ptyr-DH and GST/Rac, Lane 3:
GST/Rac only as positive control.
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Lane 1 2 3
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Figure 2.4 The results of in vitro 3H-GDP-releasing assay. 10 pmoles of soluble 3 H-
GDP coupled GST-Rac protein were incubated with IS pmoles of his-tagged and
phosphorylated vav/Y-DH protein or equal volume of Ni2~agarose-beads only at 37°C for
15 minutes. The column blank indicated 3 H-GDP/Rac-GTPase only as a blank control for
spontaneous dissociation of 3 H-GDP from 3 H-GDP/Rac-GTP ase complex, whereas
column beads for Ni2 ’ * ’ agarose beads plus 3 H-GDP/Rac-GTPase as a negative control.
The column vavDH represented that vavDH protein was incubated with 3 H-GDP/Rac-
GTPase.
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5 2 1 0
Blank GST V avD H
28
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CHAPTER 3: VAV ACTS AS A GUANINE NUCLEOTIDE DISSOCIATION
INHIBITOR (GDI) FOR Ras-GTPase AND IS REGULATED BY Lck
PHOSPHORYLATION
3.1 Introduction
Ras gene was first known as a transforming gene in human tumors in 1980, i.e., v-
H-Ras for Harvey, BALB, and Rasheed sarcoma viruses, and v-K-Ras for Kirsten
sarcoma viruses (61). Almost two decades have passed. Ras and Ras-related small
GTPases have become a large family, and they exert their critical biochemical and
biological functions in diverse intracellular signaling pathways.
Vav was found to have a GEF activity for Ras-GTPase by Gulbins et al, but other
researchers have different opinions (IS, 54). The reasons were 1) Vav is one of the Dbl-
related protein family members; it possesses a Dbl-homology domain, but does not have
a CDC2S like catalytic domain as seen in other Ras GEFs such as CDC2S, mSos, and
RasGRF (4). 2) Vav-transformed cells do not exhibit an increased cellular Ras-GTP
level, in contrast to Ras GEFs transformed cells (IS). 3) Vav-transformed and Dbl-
transformed cells exhibited the same properties as Rho-transformed cells, but did not
exhibit the characteristics as Ras-transformed cells (IS, 54). These morphological
differences suggest that vav transformation is not a consequence of activation of Ras-
GTPase, but might be a consequence of activation of Rho-related family of GTPases. 4)
29
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Some studies have demonstrated that vav is a GEF for Rho-related family o f small
GTPases (IS, 21), these results are consistent with the above observations and
hypotheses.
Thus we decided to examine the role of vav in Ras-GTPase activation. Is vav a
GEF or a GDI for Ras-GTPase? Can this catalytic activity be regulated by Lck
phosphorylation or/and phosphoinositides as seen in the Rho-related GTPase pathways?
3.2 Vav/Y-DH protein is able to bind Ras-GTPase in vitro
Our previous experiments have indicated that vav can directly bind to Ras-
GTPase, but which individual domain is responsible for this interaction between vav and
Ras-GTPase is still uncertain. We speculate that the Dbl-homology domain of vav is a
candidate for this interaction. Our hypothesis is confirmed by in vitro protein binding
assay.
During in vitro vav/Y-DH and Ras-GTPase binding assay, 100 pmol of Ni2 '
agarose bead bound his-tagged vav/Y-DH, vav/JW-DH, vav/PH, and vavL proteins were
incubated with 300 pmol of soluble GST/Ras respectively. 500 ug/ml BSA and 5 mM
Imidazole were added to the above reactions. The binding reaction mixtures were rotated
at 4°C for one hour. Followed in vitro binding reaction, the SDS PAGE and Western
blotting were performed (see chapter 5 methods for details). The results of
immunodetection of bound GST/Ras in the binding assay are present in the figure 3.1.
30
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As shown in figure 3.1, the results of in vitro binding assay reveal that vav/Y-DH
protein was able to strongly bind to Ras-GTPase as well as vav/JW-DH protein in vitro,
whereas vav/PH protein bound only weakly to Ras-GTPase. Thus vav might bind directly
to Ras-GTPase via its Dbl-homology domain, thereby to further modulate Ras-GTPase
activity in Ras and Ras-related signaling pathways.
3.3 Vav/Y-DH serves as a guanine nucleotide dissociation inhibitor (GDI) for Ras-
GTPase
Since Ras was discovered two decades ago, a number of GEFs and GAPs for Ras-
GTPase have emerged, but no GDI for Ras-GTPase is known so far. As previous chapters
described, the role of vav for Ras-GTPase was contradictory. We decided to examine the
functional role of vav for Ras-GTPase.
During in vitro 3 H-GDP(GTP) releasing assay, 10 pmoles of soluble 3 H-
GDP(GTP) coupled GST-Ras protein were incubated with 15 pmoles of his-tagged
vav/Y-DH protein or Ni2~agarose-beads (as a negative control) in 500 ul of buffer G
(without DTT) at 37°C. Then 10 uM unlabeled GTP and 500 ug of bovine serum albumin
(BSA) were added to the reaction. Thereafter, 60 ul aliquots from each reaction sample
was removed at 0, 15, 30 minutes time points, and then mounted on the nitrocellulose
membrane filter for radioactivity detection (see chapter 5 methods). The results of 3 H-
GDP-releasing assay are present in figure-3.2. As shown in the figure-3.2, the
unphosphorylated vav/DH protein showed about 40% inhibition of the releasing of 3 H-
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GDP from JH-GDP/Ras-GTPase complex, whereas the phosphorylated vav/DH protein
did not effect the releasing of 3 H-GDP from 3 H-GDP/Ras-GTPase complex.
Moreover, the results of GTP-releasing assay are present in figure 3.3. The
methods and column contents were the same as those in the 3 H-GDP-releasing assay,
except that 3 H-GTP/Ras-GTPase was in stead of 3 H-GDP/Ras-GTPase and the release of
3 H-GTP from 3 H-GTP/Ras-GTPase complex was measured. As shown in the figure-3.3,
the phosphorylated vav/DH protein enhanced about two-fold of 3 H-GTP from 3 H-
GTP/Ras-GTPase complex, whereas the unphosphorylated vav/DH protein did not effect
the releasing o f 3 H-GTP from3 H-GTP/Ras-GTPase complex.
These findings suggest that the unphosphorylated vav is able to stabilize the 3 H-
GDP/Ras-GTPase complex and serves as a guanine nucleotide dissociation inhibitor
(GDI) for GDP/Ras-GTPase. In contrast, the phosphorylated vav stimulates dissociation
of 3 H-GTP from 3 H-GTP/Ras-GTPase and functions as a guanine nucleotide dissociation
stimulator (GDS) for GTP/Ras-GTPase complex. Thus vav favors the GDP bound form
(inactivated state) of Ras-GTPase and possesses an inhibitory role for Ras-GTPase under
either unphosphorylated or phosphorylated condition.
However, previous studies in our laboratory have confirmed that
unphosphorylated vav functions as a GDI for GDP/Ras-GTPase, but their study with
phosphorylated vav indicated that the PTyr/vav possessed GDI activity for GTP/Ras-
GTPase (data not shown). If this is the case, we are able to explain the model of Ras
32
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activation by receptor PTK or receptor associated PTK. Upon agonists stimulation of
these receptors, vav becomes phosphorylated. The phosphorylated vav holds Ras-GTPase
to its GTP-bound form (activated state), thereby to further trigger the Ras-related
intracellular signaling cascades. In contrast, unphosphorylated vav is able to stabilize
GDP/Ras-GTPase complex to maintain its inactive state in resting cells. Thus vav might
override other GEFs and GAPs of Ras-GTPase to control the Ras-GTPase’s activities in
the intracellular signal transduction.
It is still unknown whether vav is a guanine nucleotide dissociation stimulator
(GDS) or guanine nucleotide dissociation inhibitor (GDI) for GTP/Ras-GTPase complex.
Some studies have reported that Cells overexpressing vav do not have increased levels of
GTP-bound Ras protein (11.7% in vav-transfected cells comparing to 13.8% in non
transfected NIH 3T3 cells) (14, 53, 62). Others have reported that Ras-GTP levels were
elevated in vav-transfected cells (35%) (63, 64) and observed that MAPKs were
constitutively activated in vav-transformed cells (14, 62). The basis for the differences
between above observations is not clear. Further in vivo studies are needed to answer the
remaining questions. For examples, 1) We can determine whether vav is a GDI for
GDP/Ras-GTPase under unstimulated condition. We can transfect vav and vav mutants
that are defective in Ras-binding into NIH 3T3 cell, and then measure the cellular levels
of Ras-bound GDP or/and GTP in these transfected cells. If vav is a GDI for GDP/Ras-
GTPase, an elevated level of GDP/Ras-GTP in vav-transfected cell is predicted by
comparing to the mutants of vav transfected cells; 2) We can also test whether the
phosphorylated vav is a GDI or a GDS for GTP/Ras-GTPase. We can still use above vav
33
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and vav mutants to transfect NIH 3T3 cell, and then measure the cellular levels of Ras-
bound GDP or/and GTP in the transfected cells under condition of bovine serum
stimulation. Since the phosphorylation of vav in the vav-transfected cell is enhanced
under bovine serum stimulation (Das b and Broek D unpublished data). If vav is a GDI
for GTP/Ras-GTPase, an elevated level of GTP/Ras-GTPase is predicted by comparing to
the mutants of vav transfected cells, whereas, if vav is a GDS for GTP/Ras-GTPase, a
decreased level of GTP/Ras-GTPase is expected. Alternatively, we can examine the role
of phosphorylated vav for Ras-GTPase by cotransfection of vav and Src-related tyrosine
kinase such as Lck and Syk. By comparing the levels of GTP/Ras-GTPase in vav/lck
cotransfected cells and non-transfected cells, we might be able to distinquish the role of
vav either as a GDI or a GDS for GTP/Ras-GTPase under Lck phosphorylation condition.
However, the fit vivo studies, especially which are involved in Ras-related
GTPases signaling pathways, are more complicated and more difficult. Vav plays a
central role in the Ras-related GTPases signaling network. So it is very important how to
interpret the observations during in vivo studies and many possibilities should be
concerned. During vav transfection studies, which signaling pathway is mainly involved
in such as Ras, Rac pathways, should be distinguished. Downstream events such as
mitogen-activated protein kinase (MAPK) can be used as an indicator for activation of
Ras-GTPase, whereas c-Jun N-terminal kinase (JNK) for activation of Rac/Rho family
GTPase. The morphological changes of the vav-transfected cells also can be used as an
another indicator.
34
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Figure 3.1 In vitro vav/Y-DH and Ras-GTPase binding essay. 100 pmol of Ni2'
agarose beads bound his-tagged vav/Y-DH, vav/JW-DH, vav/PH, and vavL proteins were
added to buffer G (without DTT) and reacted with 300 pmol of soluble GST/Ras
respectively at 4°C for one hour. Lane 1 : vav/JW-DH plus Ras/GST as a positive control,
Lane 2: vav/Y-DH plus GST only as a negative control, Lane 3: vav/Y-DH plus
Ras/GST, Lane 4: vav/PH plus Ras/GST, Lane 5: vavL plus Ras/GST.
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Lane
Ras/GST
36
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Figure 3.2 The results of in vitro 3H-GDP-releasing assay. 10 pmoles of soluble 3 H-
GDP coupled GST-Ras protein were incubated with IS pmoles of his-tagged vav/Y-DH
protein or Ni2~agarose-beads (as a negative control) respectively at 37°C for 30 minutes.
Blank: 3 H-GDP/Ras-GTPase only as a blank control for spontaneously dissociation of
GDP from GDP/Ras-GTPase complex, Beads: Ni2 + agarose beads plus ^-GDP/Ras-
GTPase as a negative control, PTyrDH: Lck phosphorylated vav/DH plus ^-GDP/Ras-
GTPase, and Y-DH: Unphosphorylated vav/DH plus 3 H-GDP/Ras-GTPase.
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03 20
Blank Beads PTyrDH Y-DH
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Figure 3.3 The results o f 3 H-GTP-releasing assay. 10 pmoles of soluble 3 H-GTP
coupled GST-Ras protein were incubated with IS pmoles of his-tagged vav/Y-DH
protein or Ni2'agarose-beads (as a negative control) respectively at 37°C for 30 minutes.
Blank: 3 H-GTP/Ras-GTPase only as a blank control for spontaneously dissociation of 3 H-
GTP from 3 H-GTP /Ras-GTPase complex, Beads: Ni2 + agarose beads plus JH-GTP/Ras-
GTPase as a negative control, PTyrDH: Lck phosphorylated vav/DH plus ^-GTP/Ras-
GTPase, and Y-DH: Unphosphorylated vav/DH plus 3 H-GTP/Ras-GTPase.
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C L 60
H
O
o>
c
<0
(0
0
© 20
c.
Blank B ead s PTyrDH Y-DH
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CHAPTER 4: CONCLUSION
We have determined the ability of vav to bind to Rac-GTPase or Ras-GTPase, and
to serve as a guanine nucleotide exchange factor (GEF) for Rac-related family of small
GTPases and a guanine nucleotide dissociation inhibitor (GDI) for Ras-GTPase. The
evidence of the dual functions of vav for Ras-related family of small GTPases was
obtained from several distinct biochemical and biological assays, i.e., in vitro protein
binding assay, in vitro GDP or GTP releasing assay, and so forth. The above data have
strongly supported our conclusions that vav possesses dual function in Ras-related
GTPase regulation, i.e., vav is a GEF for Rac-related family of small GTPases and serves
as a GDI for Ras-GDP complex under unphosphorylated physiological state. Further in
vivo studies are needed to determine whether vav is a GDI or GDS for Ras-GTP complex
under phosphorylation condition (see chapter 3).
We have established the model of modulation o f vav GEF activity by both Lck
and phosphoinositides (Fig. 4.1). In this model, PI-4,5 P2, the subatrate of PI-3 K could
inhibit the vav activity by shed the Ptyr-174 DH domain of vav that is essential for its
GEF activity, whereas PI-3,4,5 P3, the product of PI-3 K could enhance vav activity by
open its structure and make its Ptyr-174 DH domain more accessible for the other signali
molecules. This mode also raises new questions concerning signaling and other Dbl-
related family proteins (see below).
Recently, we have observed that phosphoinositides influenced the
phosphorylation of Tiam by protein kinase C (PKC) (Fig. 4.2). During in vitro
41
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phosphorylation assay, S O pmoles o f Tiam protein were incubated with 2units of
recombinant PKC (GIBCO BRL. Cat. #10830-016) at 37°C for 20 minutes. As shown in
figure 4.2, the PI-4,5 P2 enhanced phosphorylation of Tiam by PKC, whereas the PI-3, 4,
5 P3 inhibited this kinase reaction. Does the same regulation exist in the Tiam signaling
cascade? Does this modulation by phosphoinositides further effect on the GEF activity of
Tiam? As we known, Tiam consists of two PH domains separated by a DH domain and
possesses GEF activity for Rho-related family of small GTPases (15). So it is necessary
to determine which individual PH domain(s) of Tiam is involved in phosphoinositides
binding and its modulation. Is any difference of this phosphoinositides modulation
between vav and Tiam molecules? These questions will lead to understand whether this
phosphoinosotides modulation is specific for vav or is present in a similar manner in the
regulation of other DH-PH containing proteins. Since Ras oncogene discovered, the
biochemical and biological roles of Ras-GTPase in cell growth control and differentiation
have been well established (15, 21, 61). In terms of human cancer, any genetic defective
and misregulation in the Ras and Ras-related small GTPases signaling cascades is clearly
critical and important. Vav possesses a central role in the activation and modulation of
these Ras-related small GTPases and is implicated physiologically significant in these
intracellular signaling pathways. Our finding might lead to a better understanding of
tumorigenicity and to eventually protect people from tumors and other diseases.
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Figure 4.1 Schematic model of modulation of vav catalytic activity by both Lck and
phosphatidylinositides. PIP2 : phosphatidyl inositol 4, 5-bisphosphate, the substrate of PI-
3 K, PIP3: phosphatidylinositol 3,4,5-trisphosphate, the product of PI-3 K, PI-3 K:
phosphatidylinositol 3-kinase.
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Lck
rirw
I ______
( " w T H DH
DH
iY174
PIP2|^ P I P 3
a ^ P H )-PiP2
Y 174
DH 1 f
T PIP2
PIP3I I
PH -Pf>2
\
DH
■ W
44
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Figure 4.2 The effects of Phosphoinositides on phosphorylation of Tiam by PKC in
vitro. S O pmoles of Tiam protein were incubated with 2 units of recombinant protein
kinase C (PKC). Lane 1, 4: Tiam plus PKC only, Lane 2, 5: Tiam and PKC plus
phosphatidylinositol-4,5 bisphosphate (PIP2), Lane 3, 6: Tiam and PKC plus
phosphatidyl inositol-3,4,5 triphosphate (PIP3).
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Lane 1 2 3 4 5 6
Phosphorylated
Tiam
46
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CHAPTER S: METHODS AND MATERIALS
Plasmids
The vav Dbl-homology domain including phosphorylation site of Y-174 residue
(vav/Y-DH) was constructed into TA cloning vector system (Invitrogen. Cat. #K2000-01)
by means of PCR-based site-direct DNA mutagenesis (Table. 4.1 shown the primers used
in PCR). The desired insert was then subcloned into PRSET his-tagged expression
system (Invitrogen. Cat. #K880-01) at Hind III and Xhol restriction enzyme sites. The
subcloned vav/Y-DH sequence was confirmed by DNA sequencing. Then the constructed
vav/Y-DH/PRSET plasmid was transformed into E. Coli to express fusion protein (Fig.
5-1).
Expression of recombinant Lck in insect s!9 cell (baculoviral expression system)
The construction of recombinant Lck/baculoviral vector was obtained from Dr.
Michael White’s laboratory (Department of Pharmacology, University of North Carolina
at Chapel Hill.) and the recombinant protein was expressed in the insect sf9 cell
(Pharmingen Cat. #21300L) monolayer cultures as following. Approximately 2X 106 sf9
cells/per plate were seeded in several individual 10 cm tissue culture plates (Falcon Cat.
#3025), the fresh TNM-FH medium (Pharmingen Cat. #21227M) was added to make up
a total of 12 ml media per plate. The seeded cells were infected with 50 ul of high-titer
recombinant baculoviruses stock solution (1-2X 108 pfu/ml) and incubated at 27°C for 3
47
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days, then the signs of infection should be checked every 2-3 days until the cells were
harvested around 9-10 days after viral infection.
Sf9 cell lysate preparation
The above insect cells infected with recombinant viruses were collected by pipette
washing off cells from the wall of culture plates and spun down at 2,500X g for S
minutes. The cell pellet contained non-secreted recombinant lck protein and was
resuspended in ice-cold insect cell lysis buffer (Pharmingen Cat. #21425A) with
reconstituted protease inhibitor cocktail (Pharmingen Cat. # 21426Z). The cells were set
on ice for 4S minutes with 1 ml of lysis buffer/per 2X 107 cells. Clear lysate from cellular
debris was obtained by centrifuging at 40,000X g for 60 minutes. The clear supemant,
which should contain recombinant Lck protein, was collected and stored at -70°C for
activity testing and further experiments.
Phosphorylation o f vav L, vav L/mutants, and vav/Y-DH by Lck in in vitro kinase
reaction
20 ul (500 ng) of Ni2 " agarose beads bound his-Y-vav/DH protein was washed
three times with 500 ul of ice-cold kinase buffer (lOmM HEPES, 12 mM MgClj, pH 7.4).
The beads were resuspended in 30 ul of kinase buffer with 10 uM o f cold ATP for further
kinase reaction. Then 1 ul of y3 2 P-ATP (3000 mCi/mmole, 10 mCi/ml, NEN-Dupont) and
2 ul of the supemant containing recombinant Lck protein expressed in insect sf9 cells
48
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were added to above reaction solution. The reaction sample was incubated at 37oC for 20
minutes. The Ni2 " agarose beads were washed with 500 ul of kinase buffer for three
times, then were resuspended in 40 ul of IX SDS PAGE sample buffer. The above SDS
PAGE sample was denatured at 95oC for 5 minutes, 20 ul of the sample was loaded on a
10% polyacrylamide gel for electrophoresis. After completion of SDS PAGE, the gel was
dried and exposed to a film (KODAK X-OMAT film) for detection.
Western blotting and immunodetection of proteins
After completion of SDS PAGE, the proteins in the polyacrylamide gel were
transferred to PVDF membrane (Bio-Rad Inc.) by mean of the wet transfer method.
Transfer was done in a Bio-Rad Mini Trans-blot Transfer Cell filled with transfer buffer
(50 mM Tris.HCl, 40 mM glycine, 0.037% SDS, and 20% methanol) at 100V for one
hour at room temperature. Following transfer, the PVDF membrane was blocked for one
hour in IX TBS buffer (20 mM Tris.HCl, 500 mM NaCl, pH 7.5) containing 5% non-fat
milk. The membrane was incubated in IX TBST buffer (IX TBS + 0.05% Tween-20)
containing the primary antibody (1:1000 diluted in IX TBST) for one hour at room
temperature with slowly shaking. The membrane was washed three times with IX TBST,
each time for 10-15 minutes, then incubated with the alkaline phosphatase conjugated
secondary antibody (1:3000 diluted in IX TBST, Santa Cruz Biotechnology Inc.) for 45
minutes at room temperature. The membrane was washed three times with IX TBST
buffer, each time for 10-15 minutes. Finally, the membrane was incubated with Immuno-
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star chemiluminescence substrate (Bio-Rad Inc.) for S minutes, and then it was wrapped
in Saran-Wrap and exposed to a film (KODAK X-OMAT film) for detection.
Expression and purification of fusion proteins in E Coli expression system
1) Preparation of Ni2+ agarose bound his-tagged vavL, his-tagged vavL mutants, and
his-tagged vav/Y-DH proteins.
The plasmids of pRSET/vavL, pRSET/vavL mutants (h,c,b,m), and
pRSET/vav/Y-DH were transformed into the E. Coli BL-21 (DE3) strain. A single colony
of transformed cells was inoculated into 50 ml of LB medium (10 g Bacto tryptone, 5 g
bacto-yeast extract, and 10 g NaCl, pH 7.5/per Liter medium) containing 100 ug/ml
ampicillin. The bacterial culture was grown overnight at 30°C with shaking at 250 rpm.
Next morning the overnight culture was spun down at 3,000 g for 5 minutes at 4°C, then
the pellet of bacteria was resuspended into 600 ml of fresh LB medium with 100 ug/ml
ampicillin. The culture was incubated at 37°C with shaking at 250 rpm. When the O D eoo
value of the bacterial culture reached 0.4, IPTG was added to the culture to a final
concentration of 2-3 mM to induce the desired proteins. The culture was grown at 37°C
for further 2-3 hrs with shaking at 300 rpm. After completion of protein induction, the
culture was chilled on ice and then the bacteria were collected by centrifugation at 1,500
g for 20 minutes at 4°C. The bacterial pellet was resuspended in 10 ml of ice-cold IX
PBS, spun down again, and stored at -20 °C for further protein purification.
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The pellet of 600 ml culture was thawed and then resuspended in 10 ml of lysis buffer
(20 mM Tris.HCl, pH 7.8, 500 mM NaCl, 5 mM Imidazole, 1% Triton-XlOO, 10 uM
lysosome, and 1 mM PMSF). The above lysate was sonicated 3X 30sec at 20 output on
ice (BRANSON. SONIFIER 250). The suspension was centrifuged at 18,000 g for 45
minutes at 4°C. 200 ul of 50% Ni2* agarose slurry was added to the supemant, then the
mixture was rotated at 4°C for 2 hrs. The agarose beads were washed three times with
washing buffer (20 mM Tris.HCl, pH 7.8, 500 mM NaCl, 60 mM Imidazole, and 1%
Triton-XlOO.) and once with binding buffer (20 mM Tris.HCl, pH 7.8, 500 mM NaCl,
and 5 mM Imidazole.). After washing, the protein bound beads were resuspended in 300
ul of binding buffer and stored at 4°C for further use. The agarose bead bound proteins
were quantitated on SDS PAGE gel by coomassie blue staining.
2) Preparation of soluble GST-Rac, GST-Ras, and GST-Raf proteins
The plasmids of pGEX4T/Racl, pGEX4T/Ras, and pGEX4T/Raf were
transformed into the E. Coli DH-5a strain. A single colony of transformed cells was
inoculated into 50 ml of LB medium containing 100 ug/ml ampicillin. The bacterial
culture was grown overnight at 37°C with shaking at 250 rpm. Next morning the
overnight culture was spun down at 3,000 g for 5 minutes at 4°C, then the pellet of
bacteria was resuspended into 600 ml of fresh LB medium with 100 ug/ml ampicillin.
The culture was grown at 37°C with shaking at 250 rpm. When the OD6oo value of the
bacterial culture reached 0.6, IPTG was added to the culture to a final concentration of 2-
3 mM to induce the desired proteins. The culture was grown at 37°C for further 3-4 hrs
51
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with shaking at 300 rpm. After completion of protein induction, the culture was chilled
on ice and then the bacteria were collected by centrifugation at 1,500 g for 20 minutes at
4°C. The bacterial pellet was resuspended in 10 ml of ice-cold IX PBS, spun down again,
and stored at -20 °C for further protein purification.
The pellet of 600 ml culture was thawed and then resuspended in 10 ml of lysis
buffer (IX PBS, 1% Triton-XlOO, 5 mM MgCfe, 10 uM lysosome, and 1 mM PMSF).
The above lysate was sonicated 3X 30sec at 20 output on ice. The suspension was
centrifuged at 18,000 g for 45 minutes at 4°C. 200 ul of 50% GSH-agarose slurry was
added to the supemant, then the mixture was rotated at 4°C for 2 hrs. The agarose beads
were washed three times with washing buffer (IX PBS, pH 7.4, and 1% Triton-XlOO.)
and twice with IX PBS. After washing, the protein bound beads were eluted by adding 1
ml of elution buffer (50 mM Tris.HCl, pH 7.8, 150 mM NaCl, 5 mM MgCb, 10 mM
reduced Glutathione, and 1 mM DTT.) and rotating at 4°C for 15 minutes. The beads
were pelleted by centrifugation and the supemant containing GST-fusion proteins was
collected. The above elution step was repeated once more and the supemants were put
together for dialysis. The eluted GST-fusion protein supemant was transferred into a
molecularporous membrane tubling (MWCO: 3,500. Spectrum Laboratories, Inc.). Then
the tubling was dialyzed against one liter of dialysis buffer (50 mM Tris.HCl, pH 7.8, 150
mM NaCl, 10 mM MgCb, and 20 mM KC1.) for overnight at 4°C. Next morning the
eluted proteins were continued to be dialyzed for 3-4 more hrs with one liter of fresh
dialysis buffer. Then the dialyzed proteins were concentrated by passing the dialysate
through a Centricon-30 membrane (Amicon Inc.). The proteins were stored in 30-50%
52
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glycerol at -70°C for further use. The soluble proteins were quantitated on SDS PAGE
gel by coomassie blue staining.
Preparation of nucleotide free GST-GTPases
The purified soluble GST-GTPases were mixed with an equal volume of 2X
buffer A (50 mM TrisHCl, pH 7.6, 20 mM KC1, I mM DTT, and 2.5 mM EDTA) and
incubated at room temperature for 30 minutes. The protein solution was transferred to
Microcon-30 (Amiicon Inc.) and concentrated by centrifugation. Thereafter, the protein
was diluted in 1 ml of buffer G and concentrated again. This step was repeated two more
times to obtain the nucleotide free GTPases.
In vitro binding reaction between GST-Rac or GST-Ras and his-tagged vav/Y-DH
100 pmoles of Ni3^ agarose beads bound his-tagged vav/Y-DH was added to
buffer G (without DTT) and reacted with 300-400 pmoles of soluble GST-Rac or GST-
Ras. 500 ul/ml BS A and 5 mM Imidazole were added to the above reactions. The binding
reaction volume was 200 ul and the reaction mixture was rotated at 4°C for one hour. The
Ni3 ~ agarose beads were pelleted by centrifugation at full speed for 10 Sec, and washed
three times with buffer G (without DTT) containing 1% Triton-XlOO and 60 mM
Imidazole, then washed two more times with buffer G (without DTT). The beads were
resuspended in 40 ul of IX SDS PAGE sample buffer and denatured at 95°C for 5
minutes. 20 ul of the above SDS sample was loaded on a 10% polyacrylamide gel and
53
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followed by electrophoresis, western blotting and immunodetection of bound GST-
GTPases. The anti-Rac antibody (Santa Cruz Biotechnology) was used to detect the
interaction of GST-Rac and his-tagged vav/Y-DH, whereas the anti-GST (Santa Cruz
Biotechnology) for the interaction of GST-Ras and his-tagged vav/Y-DH.
In vitro GDP (GTP) releasing assay
The nucleotide free GST-Rac or GST-Ras was added to buffer A (SO mM
Tris.HCl, pH 7.5, 20 mM KC1, I mM DTT, and 2.5 mM EDTA) containing 3 H-
GDP(GTP) (10 Ci/mmole, 1 mCi/ml, NEN-Dupont). The GTPase and JH-GDP(GTP)
were mixed in the molar ratio of 1:10. The coupling reaction was incubated at 37°C for
10 minutes. After 10 minutes incubation, MgC12 was added to a final concentration of 10
mM, and the coupling reaction was continued for 5 more minutes. The sample of
coupling of GTPase to 3 H-GDP(GTP) was placed on ice for further experimental use.
The small amount of the coupling sample was taken to estimate the coupling efficiency
by filtration on nitrocellulose filters and liquid scintillation counting.
After the coupling efficiency of 3 H-GDP(GTP)- GTPase was measured. The 3H-
GDP(GTP) coupled GST/Rac or GST/Ras was added to the Ni2 + agarose beads bound
his-tagged vav/Y-DH or his-tagged vav/Ptyr-DH respectively in buffer G (50 mM
Tris.HCl, pH 7.8, 150 mM NaCl, 5 mM MgCl2 , 20 mM KC1, and 1 mM DTT). The cold
GTP was then added to the above reaction solution at final concentration of 0.5 mM. The
molar ratio of GST-Rac or GST-Ras to his-tagged vav/Y-DH in the reaction was 1:2 or
54
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2:3 respectively. The reaction was incubated at 37°C with rotating. The aliquots
representing 10 pmoles of GTPases were removed at 0, 20, and 40 minutes time points of
the reaction and the radioactivity associated with the GTPases was measured by filtration
onto nitrocellulose filters and liquid scintillating.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure S.l Construction o f Dbl-homology domain of vav. Hind III and Xho I indicate
the cloning site, pRSET A is the His-tagged fusion protein expression vector, and vav/Y-
DH represents Dbl-homology domain of vav including Y-174 residue.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Vav/Y-DH ( M l 72-402)
(H is).
H in d I I I
Xho
pRSET A
2 .9 kb
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

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Creator Xia, Yi (author)

Core Title Dual functions of Vav in Ras-related small GTPases signaling regulation

School Graduate School

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag biology, molecular,health sciences, oncology,OAI-PMH Harvest

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Advisor Broek, Daniel (committee chair), [illegible] (committee member), Stallcup, Michael R. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c16-290583

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