(Received for publication, October 19, 1995; and in revised form, January 19, 1996)
From the
The vav proto-oncogene product, p95 or Vav, is primarily expressed in hematopoietic cells and
has been shown to be a substrate for tyrosine kinases. Although its
function is unknown, Vav shares a region of homology with DBL, an
exchange factor for the Rho family of GTP-binding proteins. The
presence of this domain and the observation that cells transformed with
Vav display prominent stress fibers and focal adhesions similar to
those that are observed in RhoA transformed cells suggests that Vav may
play a role in regulating the actin cytoskeleton. We have, therefore,
examined Vav phosphorylation in platelets, which undergo dramatic
cytoskeletal reorganization in response to agonists. Two potent
platelet agonists, thrombin (via its G protein-coupled receptor) and
collagen (via its interaction with the
integrin), caused Vav to become
phosphorylated on tyrosine. Weaker platelet agonists, including ADP,
epinephrine and the thromboxane A
analog, U46619, did not.
The phosphorylation of Vav in response to thrombin was maximal within
15 s and was unaffected by aspirin, inhibitors of aggregation, or the
presence of the ADP scavenger, apyrase. Vav phosphorylation was also
observed when platelets became adherent to immobilized collagen (via
integrin
), fibronectin (via integrin
), and fibrinogen (via integrin
). These results show that Vav
phosphorylation by tyrosine kinases 1) occurs during platelet
activation by potent agonists, 2) also occurs when platelets adhere to
biologically relevant matrix proteins, 3) requires neither platelet
aggregation nor the release of secondary agonists such as ADP and
TxA
, and 4) can be initiated by at least some members of
two additional classes of receptors, G protein-coupled receptors and
integrins, providing further evidence that both of these can couple to
tyrosine kinases.
The vav proto-oncogene product, p95 or Vav, is primarily expressed in cells of hematopoietic
origin(1) . Its oncogenic potential was originally discovered
during gene transfer assays in which a truncated form of Vav was shown
to induce tumor formation in nude mice(1) . Although the
precise function of Vav is unknown, several observations suggest that
it may play a role in signal transduction. First, Vav contains several
structural motifs present in other signaling molecules, including an
SH2 domain, two SH3 domains, a pleckstrin homology domain, a
cysteine-rich domain, and a DBL homology domain(2) . Second,
Vav has been shown to become phosphorylated on tyrosine residues
following the activation of several types of cell surface receptors,
including the T-cell and B-cell antigen receptors in
lymphocytes(3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14) .
Finally, recent studies have shown that lymphoid cells from vav (-/-)-RAG-2 (-/-) chimeric mice are
reduced in both number and proliferative
response(15, 16, 17) .
The biochemical function of Vav has been investigated extensively, but without complete resolution. The presence of a domain homologous to the catalytic region of the DBL protein suggests that Vav may have a function similar to DBL(18) . DBL is a guanine nucleotide exchange factor for RhoA and Cdc42Hs(19, 20) , two members of the Rho family of low molecular weight GTP-binding proteins which are thought to regulate agonist induced cytoskeletal reorganization in fibroblasts(21, 22) . The possibility that Vav may also participate in cytoskeletal reorganization is suggested by its homology to DBL and by the observation that cells transformed with Vav display an increased number of stress fibers and focal adhesions, making them morphologically indistinguishable from cells expressing activated forms of DBL or Rho (23) . However, to date, efforts to demonstrate that Vav has exchange activity for Rho family members have been unsuccessful. Instead, Gulbins et al.(24, 25, 26) have reported that Vav possesses nucleotide exchange activity for Ras, an unexpected observation given that Vav does not contain the CDC25 homology domain present in other mammalian and yeast Ras exchange factors(27) . However, reports describing an association between Vav and Grb-2 support the speculation that this activity may be attributable to an associated protein, rather than Vav itself(28, 29) . Taken together, these observations suggest that Vav could be involved in at least two different pathways involving low molecular weight GTP-binding proteins and may play a role in cytoskeletal organization. However, the precise nature of this role and the molecular basis for Vav's involvement still remain to be determined.
In the
present study, we have examined the role of Vav in platelet activation.
In response to a variety of extracellular agonists, platelets form
multicellular aggregates, secrete the contents of their storage
granules, and adhere and spread on extracellular matrix proteins. This
process is characterized by extensive cytoskeletal reorganization and
involves the loss of their initial discoid shape and the extension of
lamellae and filopodia. These events are typically triggered by
agonists that activate G protein-coupled receptors, such as thrombin,
or by integrin-mediated adhesion of platelets to matrix proteins, such
as collagen. Activation of platelets via these mechanisms has been
shown to result in a dramatic increase in the tyrosine phosphorylation
of multiple platelet proteins, only a few of which have been
identified(30) . Given the evidence that Vav may participate in
pathways leading to cytoskeletal reorganization, the present studies 1)
examine whether Vav is one of the proteins that become
tyrosine-phosphorylated during platelet activation, 2) define the types
of receptors to which Vav phosphorylation is linked, and 3)
characterize its phosphorylation during the process of platelet
activation. The results show that in suspension two strong platelet
agonists, thrombin and collagen, rapidly induce the phosphorylation of
Vav. Phosphorylation occurs on tyrosine residues and does not require
platelet aggregation or the release of secondary agonists such as ADP
and TxA. (
)The results also show that Vav
becomes phosphorylated when platelets undergo integrin-mediated
adhesion and spreading on collagen, fibrinogen, and fibronectin. Thus,
in addition to characterizing Vav phosphorylation in the context of
platelet activation, these results describe two additional classes of
receptors, G protein-coupled receptors and integrins, whose activation
can lead to the phosphorylation of Vav.
Figure 1:
Thrombin-induced tyrosine
phosphorylation of Vav in platelets. Platelets were incubated, while
being stirred, at 37 °C with either 1 unit/ml thrombin (A)
or 50 µM SFLLRN (B) for the times indicated in
the figure, then lysed with an equal volume of 2 RIPA buffer.
In lanes 1-4, the immunoprecipitating antibody was a
rabbit polyclonal anti-Vav antiserum. In lane 5, normal rabbit
serum (NRS) was used as a control. Both immunoblots were
probed with a mixture of two anti-phosphotyrosine antibodies, 4G10 and
PY20.
The cloned thrombin receptor is a G protein-coupled receptor that is activated when thrombin cleaves its N terminus, exposing a tethered ligand for the receptor(33, 34) . Peptides corresponding to the first six residues of the tethered ligand (SFLLRN) have been shown to activate the receptor, mimicking many of the effects of thrombin(35, 36) . In order to determine if the phosphorylation of Vav induced by thrombin was mediated by the cloned thrombin receptor, platelets were incubated with 50 µM SFLLRN. The peptide stimulated Vav phosphorylation with kinetics similar to thrombin (Fig. 1B).
Figure 2: Inhibitors of secondary agonists and aggregation do not inhibit thrombin-induced Vav phosphorylation. Platelets were pretreated with aspirin and apyrase, 200 µM RGDS or 20 µg/ml antibody 7E3 Fab fragments for 30 min as indicated in the figure and then stimulated with 1 unit/ml thrombin for 1 min. Vav was immunoprecipitated with an anti-Vav antibody, and the Western blot was probed with an anti-phosphotyrosine antibody.
Much of the tyrosine
phosphorylation observed in activated platelets has been reported to
occur subsequent to platelet aggregation(30) . Platelet
aggregation is mediated by fibrinogen molecules which bind to
, cross-linking adjacent platelets.
Aggregation can be inhibited by preincubating platelets with a
monoclonal antibody, 7E3, directed toward
, or with the tetrapeptide,
Arg-Gly-Asp-Ser (RGDS), both of which competitively inhibit fibrinogen
binding(37, 38) . Preincubation of platelets with
either RGDS or a Fab fragment of 7E3 inhibited platelet aggregation
(not shown), but had no effect on Vav phosphorylation, even in the
presence of aspirin and apyrase (Fig. 2, lanes 4, 5, and 6). There was no Vav phosphorylation detected
in platelets exposed to RGDS or 7E3 alone (data not shown). These
results demonstrate that platelet aggregation is not required for
thrombin-induced Vav phosphorylation.
Figure 3:
Vav phosphorylation in response to various
platelet agonists. Aspirin-treated platelets were incubated with 1
unit/ml thrombin for 1 min, 30 µg/ml collagen for 3.5 min, 5
µM U46619 for 1 min (A) or 30 µg/ml collagen
for 3.5 min, 20 µM ADP for 2.5 min, or 20 µM epinephrine for 2.5 min as indicated (B). All incubations
were performed at 37 °C under stirred conditions with the
incubation times chosen to allow maximal aggregation induced by each
agonist. Fibrinogen (250 µg/ml) was present in each sample to
support platelet aggregation by all agonists. C, aspirin- and
apyrase-treated platelets were pretreated with buffer or an anti-1
antibody for 30 min as indicated. Platelets were then exposed to 30
µM collagen for an additional 3.5 min. In all cases, Vav
was immunoprecipitated with an anti-Vav antibody, and immunoblots were
probed with an anti-phosphotyrosine
antibody.
Collagen was also
tested for its ability to induce Vav phosphorylation when added to
platelets in suspension. In contrast to U46619, ADP, and epinephrine,
collagen stimulated Vav phosphorylation as well as, if not better than,
thrombin (Fig. 3A, lanes 2 and 3).
The major collagen receptor on the surface of human platelets is
thought to be the integrin, also
known as the glycoprotein Ia-IIa complex(39, 40) ,
although other collagen receptors have been reported as
well(41, 42) . Preincubating the platelets with an
anti-
antibody inhibited collagen-induced Vav
phosphorylation (Fig. 3C) and platelet aggregation (not
shown) by approximately 85%, suggesting that collagen stimulates both
responses via its interaction with
. Fig. 4A shows the time course of Vav phosphorylation in
platelets incubated with collagen. The phosphorylation occurred more
slowly than it did in response to thrombin, as did the onset of
platelet aggregation in response to collagen. Vav phosphorylation was
detected within 1 min and was maximal at 3 min. As with thrombin, Vav
phosphorylation in response to collagen was unaffected by aspirin and
apyrase, which were present throughout this experiment, or by the
addition of the anti-
antibody, 7E3 (Fig. 4B).
Figure 4: Time course of collagen-induced Vav phosphorylation. A, aspirin-treated platelets were incubated with 30 µg/ml collagen for the times indicated (0-3 min). B, aspirin-treated platelets were incubated with 30 µg/ml collagen for 3 min. Where indicated, the platelets were preincubated with Fab fragments from antibody 7E3 (20 µg/ml) for 30 min. In A and B, Vav was immunoprecipitated with an anti-Vav antibody, and the immunoblots were probed with an anti-phosphotyrosine antibody.
Figure 5: Vav phosphorylation induced by adhesion to various extracellular matrix proteins. Platelets were pretreated with buffer or 5 units/ml apyrase for 5 min and then incubated for 1 h on plates coated with BSA, collagen (Coll), fibrinogen (Fb), or fibronectin (FN) as indicated. Platelets were lysed as described under ``Experimental Procedures,'' and protein levels were normalized. Vav was immunoprecipitated, and an immunoblot was probed with an anti-phosphotyrosine antibody.
Figure 6: High affinity adhesion to fibrinogen, but not fibrinogen-mediated platelet aggregation, induces Vav phosphorylation. A, aspirin-treated platelets were incubated with buffer (lanes 1 and 2) or 10 units/ml apyrase (lanes 3 and 4) for 5 min. Next, 150 µg/ml anti-LIBS6 Fab was added (lane 4) for an additional 5 min. Platelets were then incubated for 1 h on plates coated with BSA or fibrinogen as indicated. Vav immunoprecipitates were prepared as described in the legend to Fig. 5, and an immunoblot was probed with an anti-phosphotyrosine antibody. B and C, aspirin- and apyrase-treated platelets in suspension were incubated with 250 µg/ml fibrinogen for 5 min and then 150 µg/ml LIBS6 Fab or 30 µg/ml collagen, as indicated, for an additional 5 min while platelets were being stirred. Platelet lysates were prepared, and sequential immunoprecipitations were performed, first using an anti-Vav antibody (C) and then a polyclonal anti-phosphotyrosine antibody (B). Immunoblots were probed with an anti-phosphotyrosine antibody.
In the presence of LIBS6, platelets in suspension can bind fibrinogen and aggregate. Fibrinogen binding in this manner has been reported to induce the phosphorylation of several 50-70-kDa proteins, a 140-kDa protein, and the tyrosine kinase, Syk(45, 46) . We therefore tested the ability of LIBS6 and soluble fibrinogen to induce Vav phosphorylation in platelets in suspension. LIBS6-mediated fibrinogen binding and aggregation resulted in the phosphorylation of the previously described proteins (Fig. 6B, left); however, Vav was not phosphorylated (Fig. 6B, right). These results make the important distinction that adhesion to immobilized fibrinogen, but not fibrinogen-mediated aggregation, induces Vav phosphorylation. These results are consistent with the lack of Vav phosphorylation observed in aggregated platelets in the presence of U46619, ADP, and epinephrine.
At sites of vascular injury, platelets are exposed to
collagen and other components of the damaged vessel wall in addition to
locally generated thrombin and secreted products such as ADP and
TxA. In response to these agents, platelets adhere to the
subendothelium and form multicellular aggregates. Studies performed in vitro have shown that platelet activation, adhesion, and
aggregation are associated with tyrosine phosphorylation of multiple
platelet proteins. In order to elucidate mechanisms of signal
transduction in platelets, we and others have attempted to identify the
proteins that are phosphorylated on tyrosine during platelet
activation. In this report, we have examined the phosphorylation of
Vav, a protein that may participate in the extensive cytoskeletal
reorganization that occurs during platelet activation.
The
phosphorylation on tyrosine residues of some platelet proteins in
response to thrombin has been characterized as ``aggregation
independent,'' while other proteins are phosphorylated in a manner
dependent on platelet aggregation. The present study shows that Vav can
be placed in the former group of phosphorylated substrates. Taken
together, the data suggest that Vav tyrosine phosphorylation is a
direct consequence of thrombin receptor activation and does not require
a secondary activation process involving released agonists or
aggregation. Since the thrombin receptor, like other G protein-coupled
receptors, does not possess intrinsic kinase activity, these results
provide evidence that G protein-coupled receptors can activate tyrosine
kinases. Accumulating data from platelet studies support this
conclusion. p21-GAP and cortactin are both phosphorylated
in response to thrombin in an aggregation-independent manner in
platelets(31, 47) . Similarly, thrombin induces the
activation of the protein tyrosine kinases Src, Syk, and
Jak2(46, 48, 49, 50) . Several
examples of G protein-coupled receptor-mediated tyrosine
phosphorylation in cells other than platelets have also been described (51, 52, 53, 54) , and studies
utilizing tyrosine kinase inhibitors have demonstrated a functional
requirement for tyrosine kinases in G protein-coupled receptor-mediated
potassium channel regulation(54) , smooth muscle
contraction(55) , stress fiber
formation(56, 57) , and platelet
activation(58, 59, 60) . Taken together,
these observations describe an emerging role for tyrosine
phosphorylation in G protein-coupled receptor-mediated signal
transduction. However, the mechanism by which G proteins induce
tyrosine kinase activation still remains to be defined.
Unexpectedly, adhesion to fibrinogen, in the presence of ADP
released from intracellular stores, fully induced Vav phosphorylation,
while platelet aggregation, even in the presence of exogenous ADP, did
not. Both events involve fibrinogen binding mediated by the activated
form of and in previous reports
have been demonstrated to induce the tyrosine phosphorylation of
similar proteins (32, 62) . It is possible that the
differences observed with Vav phosphorylation are due to the context of
fibrinogen as it binds its receptor. These results, however,
demonstrate a clear difference in signaling initiated by platelet
aggregation versus adhesion and perhaps reflect a mechanism
for mediating differential signals in response to cell-cell versus cell-matrix interactions.
Integrins have been demonstrated to regulate a number of cellular processes including cytoskeletal reorganization, gene expression, differentiation, and cell survival(61) . Furthermore, tyrosine phosphorylation has been observed in numerous cell types following integrin engagement and therefore has been proposed to play a role in mediating these signals(61) . Given that Vav is expressed in hematopoietic cells other than platelets, where integrins are critical for processes such as leukocyte homing and activation(65) , it will be interesting to determine if Vav also participates in these integrin-dependent functions. Lymphocytes from vav (-/-)-RAG-2 (-/-) chimeric mice should be useful in assessing this possibility.
The observation that Vav becomes phosphorylated in response to platelet agonists suggests that it may play a role in platelet activation. We initially began examining Vav in platelets due to its homology with the Rho exchange factor, DBL(18) . Microinjection studies utilizing an activated variant of Rho and C3 exoenzyme, which inhibits Rho activity, have indicated that Rho is involved in the formation of focal adhesions and stress fibers(21, 22) . Rho is also thought to play a role in platelet activation, as C3 exoenzyme inhibits platelet aggregation (67) , although its effects on platelet adhesion and spreading have not been reported. If Vav does regulate Rho or a Rho-related protein, an interesting model emerges whereby thrombin and integrins could transduce a signal to Rho through the tyrosine phosphorylation of Vav, which in turn may affect the cytoskeletal reorganization that occurs during platelet activation. Notably, the agonists which induce Vav phosphorylation in suspension, thrombin and collagen, are classified as ``strong'' agonists and induce maximal platelet aggregation without the need for the synergistic effects of secreted products. The phosphorylation of Vav may be one event that facilitates this process. Additionally, we have shown that Vav becomes phosphorylated as a consequence of integrin-mediated adhesion which initiates the process of platelet spreading and the formation of stress fibers and focal adhesions. Perhaps Vav transduces signals from integrins and the thrombin receptor and in this way participates in the process of spreading and/or full aggregation. Although a direct connection between Vav and Rho has not been established to date, Vav-transformed cells display prominent stress fibers and have been described to be morphologically similar to Rho-transformed cells(23) . Further experiments will be required to determine if Vav can mediate effects on the cytoskeleton through Rho or Rho family members.
Vav has also been reported to
catalyze guanine nucleotide exchange on Ras in a manner which is
enhanced by tyrosine phosphorylation(25) . Although there is
still debate about whether the exchange activity is intrinsic to Vav or
is a function of associated proteins, an interaction with Ras provides
a potential link to the MAP kinase pathway in platelets. Notably, MAP
kinase has been shown to be phosphorylated and activated in
platelets(68) , and we have observed that the time course of
phosphorylation lags slightly in comparison to Vav
phosphorylation, an observation compatible with
mitogen-activated protein kinase activation occurring downstream from
Vav phosphorylation. Future studies are required to dissect this
pathway in platelets.
In summary, we have demonstrated that thrombin treatment and integrin-mediated adhesion induce the tyrosine phosphorylation of Vav in platelets. These receptors represent two additional classes of receptors which couple to Vav. It will be interesting to determine if G protein-coupled receptors and integrins in other cell types will also have an affect on Vav phosphorylation and Vav-mediated signaling. Additionally, our findings support the emerging role for tyrosine kinases in G protein-coupled receptor and integrin-mediated signal transduction. Given Vav's homology to the DBL protein, and the correlation between the phosphorylation state of Vav and the dramatic cytoskeletal reorganization which occurs during platelet activation, perhaps Vav may participate in mediating this process. A better understanding, however, of the biochemical function of Vav is critical for determining the role of Vav in platelet function.