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Oncogene (2001) 20, 6372 ± 6381
2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00
www.nature.com/onc
Vav proteins, adaptors and cell signaling
Xose R Bustelo*,1
1
Centro de InvestigacioÂn del CaÂncer and Instituto de BiologõÂa Molecular y Celular del CaÂncer. CSIC-University of Salamanca,
Campus Unamuno, 37007 Salamanca, Spain
The Vav family is a group of signal transduction
molecules with oncogenic potential that play important
roles in development and cell signaling. Members of this
family are distributed in all animal metazoans but not in
unicellular organisms. Recent genomic studies suggest
that the function of Vav proteins co-evolved with tyrosine
kinase pathways, probably to assure the optimal
conversion of extracellular signals into biological
responses coupled to the cytoskeleton and gene transcription. To date, the best-known function of Vav
proteins is their role as GDP/GTP exchange factors for
Rho/Rac molecules, a function strictly controlled by
tyrosine phosphorylation. Recent publications indicate
that this function is highly dependent on the interaction
of adaptor proteins that aid in the proper phosphorylation of Vav proteins, their interaction with other
signaling molecules, and in modulating the strength of
their signal outputs. In addition to the function of Vav
proteins as exchange factors, there is increasing evidence
suggesting that Vav proteins can mediate other cellular
functions independently of their exchange activities,
probably by working themselves as adaptor molecules.
In this review, we will give a summary of the recent
advances in this ®eld, placing special emphasis on the
non-catalytic roles of Vav and its interaction with other
adaptor molecules. Oncogene (2001) 20, 6372 ± 6381.
Keywords: Vav; exchange factors; Rho/Rac proteins;
tyrosine kinase; adaptor proteins; Ca2+
Introduction
The evolutionary transition of unicellular to multicellular animal organisms coincided with the development of signal transduction pathways regulated by
tyrosine phosphorylation events. With the advent of
membrane and cytoplasmic tyrosine kinases, other
signaling proteins evolved to couple the activation of
those enzymes with downstream e€ectors. These new
cellular functions led in turn to the formation of new
structural domains, one of which, the Src-homology 2
(SH2) domain, was especially successful due to its
¯exibility and adaptability to multiple functions. This
domain is involved in a large number of regulatory
roles, ranging from the tethering of signaling
*Correspondence: XR Bustelo; E-mail: [email protected]
molecules to speci®c subcellular localizations, the
activation/inactivation of proteins, and the facilitation
of homo and heteromerization processes There has
been a traditional classi®cation of SH2-containing
proteins depending on the presence of recognizable
catalytic domains. The proteins lacking them were
termed `adaptor' molecules, since it was assumed they
worked as bridge molecules, allowing the indirect
interaction of tyrosine kinase receptors with e€ector
molecules lacking SH2 domains. The best-known
example of this type of SH2-containing molecules is
Grb2, a protein that binds simultaneously to activated
receptors via its SH2 domain and to the Ras exchange
factor Son-of-Sevenless (SOS) through its SH3
domains. However, as the characterization of the
functional roles of SH2-containing proteins progresses,
the initial distinction between catalytic and adaptor
SH2-containing proteins is blurring. For instance, it is
now known that many enzymes may also work as
adaptor molecules due to their multidomain structure.
On the other hand, some proteins originally regarded
as `adaptor' molecules were found later on to have
enzymatic functions after the discovery of new
catalytic domains.
One group of proteins that best exempli®es the
inadequacy in this classi®cation and the multiple roles
of adaptor molecules in cell signaling is the Vav family.
It is well known now that these proteins act as
guanosine nucleotide exchange factors (GEFs) for
Rho/Rac proteins. However, some biological roles
recently discovered for these proteins cannot be clearly
linked to this function, suggesting that Vav proteins
may also act as adaptor molecules. In addition, there is
accumulating evidence indicating the importance of
adaptor proteins in the regulation of Vav proteins at
di€erent levels. Given the monographic focus of this
issue in the function of adaptor proteins, we will pay
attention to this particular aspect of Vav family
regulation and function. Other areas of Vav family
function were covered extensively in a recent publication (Bustelo, 2000).
Vav proteins: catalytic and adaptor functions?
The Vav family has currently three known members in
mammalian cells (Vav, Vav2, and Vav3), and two in
invertebrates (C. elegans Vav and D. melanogaster
Vav). In addition, cDNA EST clones encoding
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fragments of Vav-like proteins are found in X. laevis,
D. rerio (zebra ®sh), I. punctatus (cat ®sh) and S.
stercoralis (a nematode) (http://www3.ncbi.nlm.nih.
gov/htbin-post/Entrez/query?db= 2&form = 1&term=
vav). Vav proteins are not present in yeast despite the
presence of Rho GTPases and Rho GEFs in these cells.
Vav family members share a similar structure (Figure
1a). Mammalian Vav proteins contain a calponinhomology (CH) domain, an acidic (Ac) region, the
Dbl- (DH) and plekstrin-homology (PH) domains
typical of members of Rho GEFs, a zinc ®nger (ZF)
domain, and two SH3 domains ¯anking a single SH2
region. The nematode Vav protein di€ers mostly at the
C-terminus, where it lacks the two SH3 domains
present in mammalian Vav proteins (Figure 1a). In
Figure 1 (a) Structure of Vav family proteins. The structural
domains have been described in the text. The inhibitory tyrosine
residue involved in Vav inactivation (Y174) is shown. A gray box
indicates the lack of the two cysteine residues in the C-terminus of
the CelVav ZF. Vav-T and Vav3 DN are truncated versions of
Vav and Vav3 generated by alternative splicing in some tissues.
Amino acid numbers are indicated at the bottom of the Figure.
(b) Interacting domains of Vav with GTPases and adaptor
proteins. Dashes indicate proteins interacting with Vav proteins
via other bridge molecules. Shc and p85 are not included since the
region of interaction with Vav proteins is unknown. Other Vavbinding proteins with non-adaptor or unknown functions (Rho
GDI, Cbl-b, Enx, Siah, SHP, Zyxin, Ku70, hnRNP-K, hnRNPC) are not included for simplicity
addition, its ZF region appears to be non-functional
due to the absence of key cysteine residues important
for the folding of the domain. The structure of D.
melanogaster Vav is better conserved, lacking only the
most N-terminal SH3 domain (Figure 1a). Although all
the structural domains present in Vav proteins are
commonly found in many signal transduction molecules, the Vav family is unique in having the DH-PH
cassette coupled to a ZF region and to a SH2 region.
The spatial organization of the C-terminal SH3 and
SH2 of the mammalian Vav proteins is also unusual
among other proteins, being only present in Grb2related proteins such as Grb and Grab/Grap proteins.
The best-characterized function of Vav proteins is
their activity as guanosine nucleotide exchange factors
for Rho/Rac proteins (Crespo et al., 1997; Han et al.,
1997; Movilla and Bustelo, 1999; Schuebel et al.,
1998). This enzyme activity catalyzes the release of
GDP from the inactive GTPases, a step that favors
the subsequent incorporation of GTP and the
acquisition by the GTPases of active conformations
capable of interacting with e€ector molecules. In
addition, this conformational change releases the
inhibitory Rho GDI molecules associated to GDPbound Rho/Rac proteins, an event that allows the
translocation of the GTPases to the plasma membrane. The biological consequences of the exchange
activity of Vav proteins have been reviewed recently
(Bustelo, 2000).
Although there are now more than forty exchange
factors known for Rho/Rac proteins in humans, the
distinctive functional feature of the Vav family is that
their catalytic activity is modulated by direct tyrosine
phosphorylation (Crespo et al., 1997; Han et al., 1997;
Movilla and Bustelo, 1999; Schuebel et al., 1998).
NMR studies have revealed that the structural
mechanism for Vav activation relies on the release of
an autoinhibitory loop nucleated around a tyrosine
residue located in the acidic region of Vav (Y174)
(Aghazadeh et al., 2000). In the non-phosphorylated
state, this residue makes contacts with the side chains
of residues located in the Rac1 binding domain of the
Vav catalytic region (Y209, T212, P320, L325, V328),
leading to the formation of a `closed' con®guration of
the molecule incapable of interacting with its substrate
(Aghazadeh et al., 2000). The phosphorylation of Y174
leads to a steric clash of the incorporated phosphate
group with the side chains of residues present in the
DH region of Vav (R332, Y209), leading to an `open'
con®guration of the exchange factor capable of making
stable interactions with Rac1 (Aghazadeh et al., 2000).
The residue located at position 174 of Vav and its
surrounding amino acid sequences are conserved in the
three mammalian Vav members and in D. melanogaster
Vav, suggesting that this regulatory mechanism is
taking place in most Vav family members. It is not
presently known whether this is the exclusive mechanism for the phosphorylation-dependent activation of
Vav proteins. In this respect, it is possible that other
intramolecular interactions mediated by the most Nterminal CH region could contribute to this auto-
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inhibitory loop. In agreement with this possibility,
deletions in this region can trigger the oncogenic
activation of Vav even when the acidic domain and
the inhibitory residue Y174 are kept intact (Katzav et
al., 1989; Schuebel et al., 1998). Whether phosphorylation plays alternative functional roles in the regulation
of the Vav pathway remains to be fully established. In
this context, it has been suggested that three tyrosine
residues of the Vav acidic domain could also induce the
downmodulation of active Vav, probably by attracting
some inhibitory phosphotyrosine binding proteins
(Billadeau et al., 2000; Kuhne et al., 2000; Lopez-Lago
et al., 2000). Due to this, Vav proteins with Y to F
mutations in residues 174, 140 and 162 are more active
than wild type Vav in a number of biological assays
(Billadeau et al., 2000; Kuhne et al., 2000; Lopez-Lago
et al., 2000). In addition to this putative inhibitory
role, it is possible that other phosphotyrosine residues
could serve as docking sites for other signaling
proteins, thus allowing the engagement of parallel or
synergistic pathways to those activated by Rho/Rac
GTPases. At this moment, however, this alternative
role for tyrosine phosphorylation remains hypothetical.
Vav proteins also become activated by mutations
triggering their latent oncogenic activity. Such mutations include deletions in the CH alone, the CH plus
the acidic domain, or point mutations in the inhibitory
residue Y174 (Katzav et al., 1989; Lopez-Lago et al.,
2000; Schuebel et al., 1998). In this case, there is a
synergistic e€ect when this mutation is combined with
mutations in tyrosine residues located at positions 142
or 160 (numbered according to the sequence of mouse
Vav) (Lopez-Lago et al., 2000). So far, no other genetic
alterations in these proteins were correlated with the
activation of the oncogenic activity of Vav. All these
oncogenic mutants lead to the hyperactivation of the
Rho/Rac pathway, as tested in a number of biological
assays such as cytoskeletal organization, activation of
kinase cascades, and stimulation of transcriptional
factors (Bustelo, 2000).
Interestingly, one of the ®rst roles attributed to Vav
was its function as an adaptor molecule, given the
presence of an SH3-SH2-SH3 cassette and, at that
time, the absence of any recognizable catalytic motif
within its structure. After the discovery of the Vav DH
domain and of its exchange activity towards Rho/Rac
GTPases (Crespo et al., 1997), most of the subsequent
biological studies focused on the role of this protein as
a Rac1 GEF. However, as new biological roles for Vav
proteins are unveiled, it is clear that the stimulation of
the Rho/Rac pathway cannot explain many of the
biological e€ects mediated by Vav proteins. These
additional functions appear to be restricted so far to
the realm of hematopoietic cells. In T- and Blymphocytes, Vav proteins can stimulate the transcription of the interleukin 2 (il-2) gene proximal promoter
via the activation of the transcription factor NF-AT
(an acronym for Nuclear Factor of Activated T-cells).
In addition, Vav proteins induce the generation of
more robust calcium ¯uxes in stimulated lymphocytes.
These functions have been ®rmly established both by
overexpression studies in cell lines and by loss-offunction mutations using gene targeting experiments
(Billadeau et al., 2000; Costello et al., 1999; Fischer et
al., 1995, 1998; Turner et al., 1997; Wu et al., 1995).
These two functions are probably intertwined during
signal transduction, given the known connection
between Ca2+ ¯uxes and NF-AT activation in
lymphocytes (Ser¯ing et al., 2000; Zhu and McKeon,
2000). There is recent evidence indicating that this
biological response includes Rho/Rac-independent
steps. First, oncogenic mutants of Vav with deletions
in the CH region that activate the Rac1 pathway
cannot trigger either of these two responses (LopezLago et al., 2000; Wu et al., 1995). Secondly, Weiss'
group has demonstrated that a catalytically inactive
version of Vav is still capable of inducing NF-AT
activation despite any detectable levels of GTP-bound
Rac1 in these cells (Kuhne et al., 2000). It should be
noted however that this result depends on the type of
DH mutant used, since Vav proteins with a L213Q/F
mutation cannot trigger NF-AT activation (Doody et
al., 2000; our unpublished observations). The reason
for this discrepancy is still unknown. Finally, it is
known that constitutively active versions of Rac1,
RhoA or Cdc42 cannot stimulate NF-AT or Ca2+
¯uxes per se (Holsinger et al., 1998). If the catalytic
domains of Vav proteins are not involved in these two
biological processes, how is this response assembled
during signal transduction in a stimulus-regulated
manner? The answer to this question is dicult due
to the complexity of the NF-AT pathway in T-cells.
This transcriptional factor becomes activated by the
conversion of three independent pathways that mediate
both its subcellular localization and transcriptional
activity (Ser¯ing et al., 2000; Zhu and McKeon, 2000).
First, NF-AT activation requires the generation of
Ca2+ ¯uxes that mediate the activation of calcineurin.
This molecule is a Ca2+-dependent serine phosphatase
that dephosphorylates NF-AT molecules in a cluster of
serine residues, a necessary step for the translocation of
this transcriptional factor to the nucleus. Once in the
nucleus, NF-AT requires the help of converging signals
from the Rac1 and Ras pathways for the transcriptional activation of target genes (Ser¯ing et al., 2000;
Zhu and McKeon, 2000). Fragmentary evidence
indicates that Vav protein can impinge on NF-AT
function at di€erent levels: (i) changes in the
cytoskeleton; (ii) activation of calcium ¯uxes; (iii)
activation of the Ras pathway; (iv) other adaptor
proteins (Figure 2). These levels of regulation are
discussed below.
Using the Rac1 pathway, Vav impacts on the NFAT route by inducing changes in the cytoskeleton that
are subsequently translated into the activation of NFAT by an as yet unknown mechanism (Figure 2). This
action is inferred from experiments performed with Tcells demonstrating that the chemical inhibition of
actin polymerization induces defects in NF-AT responses similar to those found in vav (7/7) cells
(Holsinger et al., 1998). This pathway of NF-AT
activation is independent of Ca2+, since the inhibition
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Figure 2 A model for the function of Vav in lymphoid cells. For simplicity, only the main regulatory steps of the pathways have
been indicated. Dashed arrows indicate uncharacterized pathways. The evidence for some of those interconnections is only
hypothetical and remains to be demonstrated experimentally. See a detailed explanation of each pathway in the text
of actin polymerization does not a€ect the Ca2+
response of stimulated T-cells (Holsinger et al., 1998).
The Vav/Rac1 pathway also induces the activation of
members of the NFkB family (Costello et al., 1999), a
group of transcriptional factors that cooperate at the
promoter level with NF-AT (Figure 2). If this route is
important in NF-AT regulation, it should be expected
that the future analysis of T-cells de®cient for Rho/Rac
GTPases would have similar defects in NF-AT
activation.
Vav appears also to regulate the NF-AT pathway at
the level of nuclear translocation by modulating the
generation of Ca2+ ¯uxes during the stimulation of
lymphocytes (Figure 2). The mechanism by which Vav
promotes Ca2+ in lymphoid cells is uncertain and,
probably, involves cell type-speci®c mechanisms. Using
knock out animals, Tybulewicz's group has shown that
TCR-stimulated vav (7/7) T-cells have impaired
production of inositol triphosphate (IP3), indicating
that the absence of Ca2+ ¯uxes in T-cells is probably
derived from an inecient phospholipase C-g1 (PLCg1) function (Costello et al., 1999). PLC-g1 phosphorylation is normal in these cells, suggesting that Vav
modulates PLC-g1 using a di€erent pathway (Costello
et al., 1999). There are two possible, although not
mutually exclusive, mechanisms for such regulation.
Since Vav proteins can bind to the regulatory subunit
(p85) of phosphatidylinositol-3-OH kinase (PI3-K)
(Weng et al., 1994; Zeng et al., 2000), one possibility
is that Vav could induce the translocation of PLC-g1 to
the plasma membrane via the generation of PI3-K
products. In this respect, it has been shown that
phosphatidylinositol 3,4,5-trisphosphate (PIP3) can
trigger such translocation by interacting with the PH
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domain of PLC-g1 (Falasca et al., 1998). Despite the
obvious attractiveness of this model, there are no data
available demonstrating the association of PI3-K
activity with Vav during cell signaling nor on the
levels of PI3-K activity in stimulated vav (7/7) Tcells. In addition, the interaction of Vav with p85 is not
always detected, indicating that the anity of the
interaction is very weak (our unpublished observations). An alternative mechanism for the regulation of
PLC-g1 is by facilitating the availability of PIP2, the
PLC-g1 substrate. This can be achieved via the
stimulation of PIP-5 kinase, a Rac1 downstream
element (Ren and Schwartz, 1998) (Figure 2). Although
PIP2 is present in the plasma membrane of naõÈ ve cells,
it is assumed that the continuous regeneration of the
PIP2 pool is important to maintain the enzyme reaction
catalyzed by PLC-g1 during the course of cell signaling
(Ren and Schwartz, 1998).
Although all these routes may play a role in the
biological response of Vav protein in T-cells, indirect
evidence indicates that this second messenger-dependent e€ect cannot account for the overall Ca2+ and
NF-AT signal mediated by Vav. Thus, if the Rac1
pathway were the important one for this response, it
could be expected that the oncogenic forms of Vav
would lead to both enhanced Ca2+ production and
NF-AT activation. However, these forms are totally
inactive in these responses despite being capable of
inducing high levels of activation of Rac1 downstream
elements such as JNK (Lopez-Lago et al., 2000; Wu et
al., 1995). Similar results are found when full length
Vav proteins with point mutations in the CH domain
are used (Billadeau et al., 2000). Since these oncogenic
versions lack a functional CH region, it can be
inferred that this domain has some e€ector functions
in the generation of Ca2+ ¯uxes. It is therefore
possible that, in addition to the Rac1 pathway
activated by the DH domain of Vav, the CH region
works as an adaptor motif allowing the binding of a
possible e€ector of Vav which contributes to PLC-g1
activation and the formation of Ca2+ ¯uxes (Figure 2).
This possible mechanism of action raises interesting
questions regarding the regulation of this response. To
assure immune homeostasis, a T-cell should activate
this Vav-mediated response only when it has been
presented with its speci®c antigen. Thus, the possible
e€ector functions of the Vav CH region should be
regulated by upstream signals derived from the
stimulated TCR. One interesting possibility is to
regulate this response by a structural mechanism
similar to that used for the modulation of Vav
enzyme activity (Aghazadeh et al., 2000). If so, it is
possible that the close con®guration of the DH
domain of Vav in the non-phosphorylated state could
also induce the association of the CH region with
other domains of Vav, blocking its interaction with
other e€ector molecules. During T-cell stimulation, the
intramolecular inhibition of the CH region would be
released by the phosphorylation of Vav on tyrosine
residues. This model is attractive because it would
ensure the simultaneous activation of the Rac1 and
Ca2+ signals by Vav. The validity of this regulatory
mechanism remains, however, to be tested experimentally.
Another possible interaction between Vav and the
NF-AT response is in the regulation of the Ras
pathway, a route previously shown to be important
for the activation of this transcriptional factor (Ser¯ing
et al., 2000; Zhu and McKeon, 2000). Ras is also
important for Vav signaling, since the expression of a
dominant negative mutant of Ras inhibits the activation of NF-AT by Vav and Vav2 in lymphocytes
(Doody et al., 2000; Wu et al., 1995). Interestingly, vav
(7/7) T-cells have a defective activation of some
downstream elements of the Ras pathway, such as
ERK2 (Costello et al., 1999). The Vav pathway leading
to ERK2 activation has not been elucidated as yet.
However, an interesting possibility is that Vav could
activate Ras via PLC-g1, perhaps by stimulating the
activity of the diacylglycerol-dependent exchange factor
Ras GRP (Ebinu et al., 1998; Lorenzo et al., 2000)
(Figure 2). Recent knock-out experiments have
indicated that this exchange factor, unlike other Ras
GEFs (such as SOS2, Ras GRF1 and Ras GRF2), is
essential for T-cell signaling (Dower et al., 2000; E.
Santos, personal communication).
Finally, Vav can a€ect the activation of these T-cell
responses by using signals from the SH3-SH2-SH3
domain. In agreement with this possibility, we have
found that a Vav protein lacking the SH3-SH2-SH3
domain and targeted to the plasma membrane by a
farnesylation signal is incapable of stimulating NF-AT
despite having the CH, DH, PH, and ZF regions intact
(unpublished observations). This protein appears to be
functional, since it is capable of inducing high levels of
transformation in ®broblasts (unpublished observations). The mechanism by which the Vav C-terminus
can a€ect T-cell responses is unknown. However, given
the known function of those domains in protein/
protein interactions, it can be assumed that this signal
is given by the binding of proline-rich or phosphorylated proteins to Vav during the responses of
lymphocytes to antigens (Figure 2).
Interestingly, it has been shown that the ability of
Vav family members to activate NF-AT is cell type
speci®c. Thus, Vav promotes activation of NF-AT in
B- and T-cells while Vav2 is only active on B-cells
(Doody et al., 2000). This cannot be explained in the
context of di€erences in the CH region, since these
domains are very similar in these two proteins. This
result adds up an additional level in the complexity of
this biological response. It remains to be determined
whether this di€erential response is mediated by
di€erent phosphorylation kinetics during TCR signaling, by di€erent subcellular localizations in T-cells, or
by the binding of di€erent adaptor molecules that
cooperate in this signal. Interestingly, Vav2 cannot
compensate functionally the absence of Vav in both
immature and mature T-cells (Doody et al., 2001),
indicating that this di€erential response seen with
cultured cell lines is probably a good re¯ection of the
di€erent regulatory or signaling properties of these
Adaptor functions in Vav-mediated signaling
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exchange factors. In summary, the activation of
NF-AT by Vav proteins is a highly complex process
requiring the simultaneous engagement of adaptor
proteins and several signaling pathways regulated by
the catalytic and adaptor functions of Vav proteins.
It is not known whether the SH3-SH2-SH3 region of
Vav may mediate e€ector functions in non-hematopoietic tissues. The overexpression of these C-terminal
domains leads to a dominant negative e€ect in T-cells,
mast cells, and ®broblasts, probably by blocking the
interaction of the autophosphorylated kinases with the
endogenous Vav proteins (Arudchandran et al., 2000;
Wu et al., 1996; unpublished observations). However,
vav and vav3 genes are known to yield by alternative
splicing truncated versions containing only the Cterminal SH3 domain (in the case of Vav) or the SH3SH2-SH3 region (in the case of Vav3) (Okumura et al.,
1997; Trenkle et al., 2000; Zeng et al., 2000; Figure 1a).
Interestingly, the expression of these proteins does not
overlap with the full length proteins (Okumura et al.,
1997; Zeng et al., 2000). These results suggest these
other versions of Vav proteins have functional roles of
their own, probably by working as adaptor proteins in
some speci®c pathways. The availability of cells derived
from the knock outs of vav family genes will help to
solve in the future the role of the possible functions of
the shorter forms of Vav proteins.
Collectively, all these data indicate that Vav proteins
have both GEF-dependent and independent functions.
The activation of GTPase pathways by Vav seems to
be prevalent in non-hematopoietic cells, while lymphoid cells seem to rely more heavily in the combined
and synergistic e€ect of GTPase-dependent and
independent pathways. There is also experimental
evidence suggesting that the GTPase-independent
functions of Vav proteins rely in the association of
these exchange factors with other signaling proteins.
Given the importance of the generation of Ca2+ and
NF-AT signals in lymphoid development and in
immune responses, it is expected that this area of
research will be one of the most predominant in the
Vav ®eld in the near future.
Role of adaptor proteins in the function of Vav proteins
In addition to the possible adaptor roles of Vav
proteins, there is now a large body of data indicating
that the signaling pathways of these proteins are highly
dependent on the cooperation of other adaptor
proteins (Figure 1b). The role of these proteins can
be classi®ed in several, not mutually exclusive, types: (i)
Proteins that modulate the phosphorylation levels of
Vav proteins (CD19, CD28, LAT); (ii) Proteins that
help in the coordination of the transduction of Vavdependent signals (CD19, LAT, Slp76, BLNK); (iii)
Proteins that provide parallel pathways that synergize
with those activated by Vav (Slp76, BLNK); (iv)
Proteins of unknown function in the signal transduction of Vav proteins (Shc); (v) Pathogen-encoded
proteins that regulate the Vav pathway in an
adaptor-like fashion (Nef). Below, we will summarize
the role of each of those proteins in the pathways of
Vav proteins.
Although it was initially thought that the SH2mediated interaction of Vav proteins was sucient for
assuring its phosphorylation by protein tyrosine
kinases, it is now known that this process only occurs
eciently with the aid of a number of ancillary
proteins, at least in the case of hematopoietic cells.
These adaptor proteins are membrane receptors or,
alternatively, cytoplasmic proteins containing SH2,
SH3 or phosphotyrosine residues. In the case of Blymphocytes, both Vav and Vav2 require the interaction with the cytoplasmic tail of the CD19 receptor for
optimal tyrosine phosphorylation, a step that facilitates
the proximity of these exchange factors with the IgMassociated Syk (Li et al., 1997; O'Rourke et al., 1998;
Weng et al., 1994; Figure 3a). At least in the case of
Vav, a second bene®t of this type of complex is the
localization of Vav in close proximity to Lyn, a Src
family kinase that binds to the cytoplasmic tail of
CD19 (Fujimoto et al., 1999). The binding of Vav
proteins to CD19 is accomplished by the direct
interaction of a Y391EEP motif present in the
cytoplasmic tail of this receptor with the SH2 domain
of Vav proteins (O'Rourke et al., 1998; Figure 3a).
Mutation of this site abrogates Vav binding and CD19mediated responses, indicating that this interaction
mediates both Vav phosphorylation and the activation
of downstream signaling events (Li et al., 1997;
O'Rourke et al., 1998). The phosphorylation of the
CD19 Y391 site is probably mediated by Syk, since this
tyrosine is only phosphorylated after BCR crosslinking (O'Rourke et al., 1998).
The case of T-lymphocytes is more complex because
the optimal phosphorylation of Vav relies in the
simultaneous utilization of di€erent adaptor molecules.
One way of favoring the interaction of Vav with the Tcell speci®c Zap70 kinase is the use of CD28, a
receptor stimulated at the same time that the TCR in
non anergic T-cell responses (Figure 3a). In good
agreement with such role, the co-engagement of the
TCR with CD28 yields levels of Vav tyrosine
phosphorylation much higher than when each receptor
is cross-linked independently. However, unlike the
interaction with CD19, this interaction appears to be
established indirectly by using Grb2 as bridge molecule
(Kim et al., 1998; Figure 3a). In this complex, Grb2
binds simultaneously to the phosphorylated tail of the
CD28 receptor and the Vav N-terminal SH3 domain
using its SH2 domain and C-terminal SH3 domain,
respectively (Figure 3a).
The optimal phosphorylation of Vav is also impaired
when the adaptor molecule LAT is missing in T-cells
(Finco et al., 1998; Figure 3b). LAT is an adaptor
protein localized in rafts of the plasma membrane rich
in cholesterol and glycolipids (Zhang et al., 1998a,b;
see review by Watson, this issue). LAT does not have
any recognizable catalytic motifs but contains various
tyrosine residues consensus for both the phosphorylation by cytoplasmic kinases and for the binding of SH2
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Figure 3 Interaction of Vav with transmembrane (a) and cytoplasmic (b) adaptor proteins during lymphocyte signaling. L, Ligand;
KD, kinase domain; Ac, acidic domain; P, phosphorylated tyrosine residue. Transmembrane domains are indicated as gray circles.
Proteins are not shown in scale
domains (Zhang et al., 1998a,b). Upon cell stimulation,
LAT is phosphorylated by activated Syk/Zap70
proteins on tyrosine residues, leading to the creation
of binding sites for SH2-containing proteins (Zhang et
al., 1998a). As a consequence, several SH2/SH3containing proteins move into these membrane rafts
during cell signaling (Zhang et al., 1998a,b). Since the
Vav SH2 domain cannot bind to phosphorylated LAT
(Zhang et al., 1998a), the interaction between these two
proteins is probably mediated by Grb2, a molecule that
binds to phosphorylated LAT via its SH2 domain
(Zhang et al., 1998a; Figure 3b). The use of Grb2 for
the Vav/LAT and Vav/CD28 interaction is probably a
functional stratagem to facilitate the subsequent
binding of the unoccupied Vav SH2 domain with the
Oncogene
activated, tyrosine phosphorylated Zap70 (Figure 3b).
Despite the type of interaction and adaptor molecule
used, the ®nal result is the same: the translocation of
Vav proteins to speci®c regions of the plasma
membrane where they will be localized close to the
upstream kinase. Whether Vav2 and Vav3 utilize CD28
and LAT for the interaction with Zap70 in T-cells is
unknown yet.
Current evidence indicates that the role of adaptor
proteins in the Vav pathway does not end at the
modulation of the phosphorylation levels of Vav
protein. Since the result of the interaction of Vav with
CD28, CD19, and LAT is to promote the translocation
of Vav to lipid rafts, these interactions should promote
a more ecient signaling output due to the close
Adaptor functions in Vav-mediated signaling
XR Bustelo
proximity of the activated proteins to their more direct
e€ector proteins (Rho/Rac proteins), Rho/Rac downstream elements (PIP-5 K, PAK) and other signaling
proteins (Slp76, see below). Indeed, the inclusion of a
farnesylation signal at the C-terminus of full-length
Vav allowing its constitutive localization in the plasma
membrane induces the hyperactivation of the NF-AT
pathway in T-cells (Billadeau et al., 2000).
It is not clear at present whether there is a functional
complex in T-cells similar to the Vav proteins/PIP-5
kinase/CD19 system of B-cells. However, B- and T-cells
share another type of regulation of Vav function: its
interaction with members of the BLNK/Slp76 family.
Slp76 and BLNK are proteins whose structure contains
an N-terminal SAM domain, a hydrophilic region with
several phosphorylation sites, a proline rich region, and
a SH2 domain (for a review, see Koretzky, this issue).
Slp76 is a signaling protein expressed in T- and myeloid
cells while BLNK is con®ned to B-cells (see review by
Koretzky, this issue). Slp76 plays essential roles in Tcell signaling, coupling the activation of the TCR with
increases in Ca2+, the stimulation of the Ras/MAPK
pathway, and the activation of il-2 gene transcription.
Similar roles have been attributed to BLNK in B-cell
signaling (see Koretzky, this issue). Since Slp76 and
BLNK do not have catalytic domains, their biological
functions are due to their interaction with other
signaling molecules (see Koretzky, this issue). In the
context of Vav-mediated signaling, it has been shown
that the co-expression of Vav with Slp76 induces a
synergistic response, leading to enhanced activation of
the NF-AT transcriptional activator (Wu et al., 1996).
Interestingly, Vav and Slp76 form a very stable
complex during T-cell stimulation, an association
mediated by the binding of the Vav SH2 domain to
phosphorylated residues located in the acidic domain of
Slp76 (Onodera et al., 1996; Raab et al., 1997; Tuosto
et al., 1996; Wu et al., 1996). The role of Slp76 in the
Vav pathway remains controversial. It was proposed
initially that the function of Slp76 in this interaction
was to favor the optimal activation of some of the
downstream elements of Rac1 during lymphocyte
signaling. According to these results, Slp76 was shown
to nucleate a heteromolecular complex composed of the
Rac1/Vav and Nck/PAK pairs (Bubeck Wardenburg et
al., 1998; Figure 4). In this complex, Vav and Nck bind
via their SH2 domains to di€erent phosphotyrosine
residues of Slp76. At the same time, Rac1 binds to the
DH domain of Vav and PAK to the SH3 domains of
Nck. The ®nal outcome of this multimeric complex is
the close proximity between activated Rac1 and its
e€ector PAK and, consequently, a more e€ective
stimulation of this kinase during signal transduction
(Bubeck Wardenburg et al., 1998; Figure 4). It should
be noted however that recent experiments indicate that
the activation of PAK in T-cells is totally independent
of the presence of Slp76 (Ku et al., 2001). Moreover,
Koretzky's group has reported that the synergistic
response between Vav and Slp76 occurs also with
mutants of Slp76 that cannot bind to Vav (Fang and
Koretzky, 1999). Taken together, these results suggest
6379
Figure 4 A model for the interaction of Vav with Slp76 and Nef in
T-lymphocytes. PRR, proline-rich region; RR, arginine motif; P,
phosphorylated tyrosine residue. Proteins are not shown in scale
that the function of the Vav/Slp76 complex is not
related with the optimal activation of the Rac1/PAK
route. There are at least three other possibilities for the
cross talk between Vav and Slp76 in the absence of
physical association. Thus, Slp76 and Vav converge in
the generation of Ca2+ ¯uxes by favoring the
phosphorylation and activity of PLC-g1, respectively
(Costello et al., 1999; Yablonski et al., 1998). Slp76 and
Vav are also important for the stimulation of ERK
during T-cell signaling (Costello et al., 1999; Yablonski
et al., 1998). Finally, Vav and Slp76 converge in the
translocation and transcriptional activation of NF-AT
(Musci et al., 1997; Wu et al., 1996). The detailed
understanding of the Vav/Slp76 synergy will have to
wait for the characterization of the Slp76 binding
proteins that mediate each of the biological responses
attributed to this adaptor protein. In addition to the
functional cooperativity with Vav, Slp76 has Vavindependent functions, because the phenotype of slp76de®cient mice is more severe than the observed in vav
(7/7) and vav (7/7)/vav2 (7/7) mice (M Turner,
personal communication). Given the structural similarity between Slp76 and BLNK, it is likely these
synergistic pathways are conserved in B-cells.
In addition to the hematopoietic proteins discussed
above, there are other ubiquitous proteins forming
complexes with Vav proteins. All mammalian Vav
proteins can bind to Shc (Movilla and Bustelo, 1999;
Zeng et al., 2000), a known SH2/PTB-containing
Oncogene
Adaptor functions in Vav-mediated signaling
XR Bustelo
6380
protein that has been implicated in the Ras pathway
(for a review, see Ravichandran, this issue). Shc binds
to Vav proteins during the signaling of many
membrane protein tyrosine kinases (Movilla and
Bustelo, 1999; Zeng et al., 2000). However, it is not
yet known whether this interaction is mediated by the
Vav SH2 domain, the SH2/PTB domain of Shc, or
indirectly by the simultaneous binding of these two
proteins to the autophosphorylated receptor. The
functional signi®cance of the Vav/Shc interaction is
unknown as yet. Grb2 is also found constitutivelyassociated with Vav proteins in non-hematopoietic cells
(Ye and Baltimore, 1994; Zeng et al., 2000). It is not
yet clear the functional implications of the Grb2/Vav
association in non-hematopoietic cells. Co-transfection
of Grb2 with Vav does not alter the transforming
activity of this protein when tested in NIH3T3 cells,
indicating that Grb2 has no major e€ects in the
signaling output of these proteins in ®broblasts
(unpublished observations). p85, the regulatory subunit
of PI3-K has also been shown to associate with Vav3
during the stimulation of a number of non-hematopoietic membrane receptors (Zeng et al., 2000).
However, the precise role of p85 in the pathways of
Vav and Vav3 remains to be elucidated.
From what we have seen so far, adaptor proteins
exert mostly auxiliary roles in the pathways of Vav
proteins. However, at least one human pathogen, the
human immunode®ciency virus (HIV), has learnt that
the use of adaptor proteins to interfere with the Vav
pathway is a good strategy to alter the signaling
properties of the host cell for its own bene®t. To this
end, the HIV has invented an adaptor protein of its
own, the viral Nef protein. Nef is a 27 to 35 kDa
myristylated protein essential for optimal viral replication and in the progression to AIDS (Herna Remkema
and Saksela, 2000). This protein appears to have
pleiotropic functions. Thus, Nef downmodulates
CD4, the TCR/CD3 complex, the major histocompatibility class I antigens, and the CD28 co-receptor on
the surface of infected cells (Skowronski et al., 1999;
Swigut et al., 2001). These actions are mediated by the
interaction of Nef with the endocytic machinery of the
host cell (Skowronski et al., 1999). Nef also a€ects
apoptotic pathways, both by inducing the Fasmediated killing of non infected cells and by inhibiting
the apoptotic response in the host cell (Roshal et al.,
2001). Finally, Nef perturbs the signaling responses of
the host cells including, among others, the stimulation
of tyrosine kinases, GTPases (Rac1 and Cdc42), and
serine/threonine kinases (PAK) (Herna Remkema and
Saksela, 2000). The result of this interference is the
generation of cytoskeletal changes (Fackler et al.,
1999), enhanced calcium signaling (Manninen et al.,
2000), and activation of transcriptional factors such as
NF-AT (Manninen et al., 2000). The perturbation of
these signaling pathways appear to be due to the work
of Nef as a truly adaptor protein. Thus, a proline-rich
region present in the N-terminus of Nef can form
complexes with tyrosine kinases of the Src family,
leading to enhanced kinase activities (Briggs et al.,
1997). What it is more important for us in this review,
Nef also nucleates a Slp76-like complex in T-cells that
leads to activation of PAK1 by Vav/Rac1 (Fackler et
al., 1999, 2000; Figure 4). To accomplish this, Nef
binds via its proline-rich region to the C-terminal SH3
domain of Vav and, simultaneously, to PAK using the
Nef diarginine motif (Fackler et al., 1999, 2000). The
outcome of this complex is the close proximity of Vav/
Rac1 to PAK1, inducing the ecient activation of this
kinase (Fackler et al., 1999, 2000; Figure 4). In
addition, Nef seems to activate the exchange activity
of Vav, probably by driving this molecule to the
plasma membrane (Fackler et al., 1999). Thus, in this
case, an adaptor protein is used to mimic the normal
cycle of Vav during cell stimulation, allowing the
activation of this protein even when the upstream
receptors have been downmodulated by the virus.
It can be inferred from what we have summarized in
these pages that we have now a much better understanding of the function of Vav proteins during cell
signaling. However, many interesting questions remain,
especially in regard to the GEF-independent functions
of Vav proteins and the precise role of the adaptor
proteins that bind to them during cell signaling. We
will need to clarify also the cross-talk probably existing
between the GEF-dependent and independent routes
triggered by Vav proteins. Ultimately, the elucidation
of all these pending questions will allow to have a
precise knowledge of the multiple regulatory and
e€ector steps mediating the signaling of membrane
receptors coupled to tyrosine kinase pathways.
Acknowledgments
The author likes to thank Drs Eugenio Santos, Victor
Tybulewicz and Martin Turner for communicating results
prior to publication, and Drs FaÂtima NuÂnÄez and Mercedes
Dosil for their comments on the manuscript. XR Bustelo is
a CSIC member whose work is supported by the US
National Cancer Institute (CA7373501), the British Association for International Cancer Research (00-061), the
Programa General del Conocimiento of the Spanish
Ministry of Science and Technology (PM99-0093), and
the Programa FEDER of the Spanish Ministry of Education/European Union (1FD97-2116).
References
Aghazadeh B, Lowry WE, Huang XY and Rosen MK.
(2000). Cell, 102, 625 ± 633.
Arudchandran R, Brown MJ, Peirce MJ, Song JS, Zhang J,
Siraganian RP, Blank U and Rivera J. (2000). J. Exp.
Med., 191, 47 ± 60.
Oncogene
Billadeau DD, Mackie SM, Schoon RA and Leibson PJ.
(2000). J. Immunol., 164, 3971 ± 3981.
Briggs SD, Sharkey M, Stevenson M and Smithgall TE.
(1997). J. Biol. Chem., 272, 17899 ± 17902.
Adaptor functions in Vav-mediated signaling
XR Bustelo
Bubeck Wardenburg J, Pappu R, Bu JY, Mayer B, Cherno€
J, Straus D and Chan AC. (1998). Immunity, 9, 607 ± 616.
Bustelo XR. (2000). Mol. Cell. Biol., 20, 1461 ± 1477.
Costello PS, Walters AE, Mee PJ, Turner M, Reynolds LF,
Prisco A, Sarner N, Zamoyska R and Tybulewicz VL.
(1999). Proc. Natl. Acad. Sci. USA, 96, 3035 ± 3040.
Crespo P, Schuebel KE, Ostrom AA, Gutkind JS and
Bustelo XR. (1997). Nature, 385, 169 ± 172.
Doody GM, Billadeau DD, Clayton E, Hutchings A,
Berland R, McAdam S, Leibson PJ and Turner M.
(2000). EMBO J., 19, 6173 ± 6184.
Doody GM, Bell SE, Vigorito E, Clayton E, McAdam S,
Toozie R, Fernandez C, Lee IJ and Turner M. (2001). Nat.
Immunol., 6, 482 ± 484.
Dower NA, Stang SL, Bottor€ DA, Ebinu JO, Dickie P,
Ostergaard HL and Stone JC. (2000). Nat. Immunol., 1,
317 ± 321.
Ebinu JO, Bottor€ DA, Chan EY, Stang SL, Dunn RJ and
Stone JC. (1998). Science, 280, 1082 ± 1086.
Fackler OT, Lu X, Frost JA, Geyer M, Jiang B, Luo W, Abo
A, Alberts AS and Peterlin BM. (2000). Mol. Cell. Biol.,
20, 2619 ± 2627.
Fackler OT, Luo W, Geyer M, Alberts AS and Peterlin BM.
(1999). Mol. Cell., 3, 729 ± 739.
Falasca M, Logan SK, Lehto VP, Baccante G, Lemmon MA
and Schlessinger J. (1998). EMBO J., 17, 414 ± 422.
Fang N and Koretzky GA. (1999). J. Biol. Chem., 274,
16206 ± 16212.
Finco TS, Kadlecek T, Zhang W, Samelson LE and Weiss A.
(1998). Immunity, 9, 617 ± 626.
Fischer KD, Kong YY, Nishina H, Tedford K, Marengere
LE, Kozieradzki I, Sasaki T, Starr M, Chan G, Gardener
S, Nghiem MP, Bouchard D, Barbacid M, Bernstein A
and Penninger JM. (1998). Curr. Biol., 8, 554 ± 562.
Fischer KD, Zmuldzinas A, Gardner S, Barbacid M,
Bernstein A and Guidos C. (1995). Nature, 374, 474 ± 477.
Fujimoto M, Poe JC, Jansen PJ, Sato S and Tedder TF.
(1999). J. Immunol., 162, 7088 ± 7094.
Han J, Das B, Wei W, Van Aelst L, Mosteller RD, KhosraviFar R, Westwick JK, Der CJ and Broek D. (1997). Mol.
Cell. Biol., 17, 1346 ± 1353.
Herna Remkema G and Saksela K. (2000). Front Biosci., 5,
D268 ± D283.
Holsinger LJ, Graef IA, Swat W, Chi T, Bautista DM,
Davidson L, Lewis RS, Alt FW and Crabtree GR. (1998).
Curr. Biol., 8, 563 ± 572.
Katzav S, Martin-Zanca D and Barbacid M. (1989). EMBO
J., 8, 2283 ± 2290.
Kim HH, Tharayil M and Rudd CE. (1998). J. Biol. Chem.,
273, 296 ± 301.
Ku GM, Yablonski D, Manser E, Lim L and Weiss A.
(2001). EMBO J., 20, 457 ± 465.
Kuhne MR, Ku G and Weiss A. (2000). J. Biol. Chem., 275,
2185 ± 2190.
Li X, Sandoval D, Freeberg L and Carter RH. (1997). J.
Immunol., 158, 5649 ± 5657.
Lopez-Lago M, Lee H, Cruz C, Movilla N and Bustelo XR.
(2000). Mol. Cell. Biol., 20, 1678 ± 1691.
Lorenzo PS, Beheshti M, Pettit GR, Stone JC and Blumberg
PM. (2000). Mol. Pharmacol., 57, 840 ± 846.
Manninen A, Herma Renkema G and Saksela K. (2000). J.
Biol. Chem., 275, 16513 ± 16517.
Movilla N and Bustelo XR. (1999). Mol. Cell. Biol., 19,
7870 ± 7885.
Musci MA, Motto DG, Ross SE, Fang N and Koretzky GA.
(1997). J. Immunol., 159, 1639 ± 1647.
O'Rourke LM, Tooze R, Turner M, Sandoval DM, Carter
RH, Tybulewicz VL and Fearon DT. (1998). Immunity, 8,
635 ± 645.
Okumura K, Kaneko Y, Nonoguchi K, Nishiyama H, Yokoi
H, Higuchi T, Itoh K, Yoshida O, Miki T and Fujita J.
(1997). Oncogene, 14, 713 ± 720.
Onodera H, Motto DG, Koretzky GA and Rothstein DM.
(1996). J. Biol. Chem., 271, 22225 ± 22230.
Raab M, da Silva AJ, Findell PR and Rudd CE. (1997).
Immunity, 6, 155 ± 164.
Ren XD and Schwartz MA. (1998). Curr. Opin. Genet. Dev.,
8, 63 ± 67.
Roshal M, Zhu Y and Planelles V. (2001). Apoptosis, 6, 103 ±
116.
Schuebel KE, Movilla N, Rosa JL and Bustelo XR. (1998).
EMBO J., 17, 6608 ± 6621.
Ser¯ing E, Berberich-Siebelt F, Chuvpilo S, Jankevics E,
Klein-Hessling S, Twardzik T and Avots A. (2000).
Biochim. Biophys. Acta, 1498, 1 ± 18.
Skowronski J, Greenberg ME, Lock M, Mariani R, Salghetti
S, Swigut T and Iafrate AJ. (1999). Cold Spring Harb.
Symp. Quant. Biol., 64, 453 ± 463.
Swigut T, Shohdy N and Skowronski J. (2001). EMBO J., 20,
1593 ± 1604.
Trenkle T, McClelland M, Adlkofer K and Welsh J. (2000).
Gene, 245, 139 ± 149.
Tuosto L, Michel F and Acuto O. (1996). J. Exp. Med., 184,
1161 ± 1166.
Turner M, Mee PJ, Walters AE, Quinn ME, Mellor AL,
Zamoyska R and Tybulewicz VL. (1997). Immunity, 7,
451 ± 460.
Weng WK, Jarvis L and LeBien TW. (1994). J. Biol. Chem.,
269, 32514 ± 32521.
Wu J, Katzav S and Weiss A. (1995). Mol. Cell. Biol., 15,
4337 ± 4346.
Wu J, Motto DG, Koretzky GA and Weiss A. (1996).
Immunity, 4, 593 ± 602.
Yablonski D, Kuhne MR, Kadlecek T and Weiss A. (1998).
Science, 281, 413 ± 416.
Ye ZS and Baltimore D. (1994). Proc. Natl. Acad. Sci. USA,
91, 12629 ± 12633.
Zeng L, Sachdev P, Yan L, Chan JL, Trenkle T, McClelland
M, Welsh J and Wang LH. (2000). Mol. Cell. Biol., 20,
9212 ± 9224.
Zhang W, Sloan-Lancaster J, Kitchen J, Trible RP and
Samelson LE. (1998a). Cell, 92, 83 ± 92.
Zhang W, Trible RP and Samelson LE. (1998b). Immunity, 9,
239 ± 246.
Zhu J and McKeon F. (2000). Cell Mol. Life Sci., 57, 411 ±
420.
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