Identification of a novel integrin signaling pathway involving the kinase Syk and the guanine nucleotide exchange factor Vav1

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Identification of a novel integrin signaling pathway involving the kinase Syk and the guanine nucleotide exchange factor Vav1 Cindy K. Miranti*, Lijun Leng†, Petra Maschberger†‡, Joan S. Brugge* and Sanford J. Shattil†§ Background: Integrins induce the formation of large complexes of cytoskeletal and signaling proteins, which regulate many intracellular processes. The activation and assembly of signaling complexes involving focal adhesion kinase (FAK) occurs late in integrin signaling, downstream from actin polymerization. Our previous studies indicated that integrin-mediated activation of the nonreceptor tyrosine kinase Syk in hematopoietic cells is independent of FAK and actin polymerization, and suggested the existence of a distinct signaling pathway regulated by Syk.

Addresses: *Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA. Departments of †Vascular Biology and §Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037, USA.

Results: Multiple proteins were found to be activated by Syk, downstream of engagement of the platelet/megakaryocyte-specific integrin αIIbβ3. The guanine nucleotide exchange factor Vav1 was inducibly phosphorylated in a Sykdependent manner in cells following their attachment to fibrinogen. Together, Syk and Vav1 triggered lamellipodia formation in fibrinogen-adherent cells and both Syk and Vav1 colocalized with αIIbβ3 in lamellipodia but not in focal adhesions. Additionally, Syk and Vav1 cooperatively induced activation of Jun N-terminal kinase (JNK), extracellular-signal-regulated kinase 2 (ERK2) and the kinase Akt, and phosphorylation of the oncoprotein Cbl in fibrinogen-adherent cells. Activation of all of these proteins by Syk and Vav1 was not dependent on actin polymerization.

Correspondence: Joan S. Brugge E-mail: [email protected]

Present address: ‡Institut für Prophylaxe und Epidemiologie der Kreislaufkrankheiten Klinikum Innenstadt, Pettenkoferstrasse 9, D-80336 München, Germany.

Received: 27 August 1998 Revised: 15 October 1998 Accepted: 3 November 1998 Published: 16 November 1998 Current Biology 1998, 8:1289–1299 http://biomednet.com/elecref/0960982200801289 © Current Biology Ltd ISSN 0960-9822

Conclusions: Syk and Vav1 regulate a unique integrin signaling pathway that differs from the FAK pathway in its proximity to the integrin itself, its localization to lamellipodia, and its activation, which is independent of actin polymerization. This pathway may regulate multiple downstream events in hematopoietic cells, including Rac-induced lamellipodia formation, tyrosine phosphorylation of Cbl, and activation of JNK, ERK2 and the phosphatidylinositol 3′-kinase-regulated kinase Akt.

Background Integrins are heterodimeric receptors that mediate cell–matrix and cell–cell adhesion. Following ligand engagement, the cytoplasmic domains of integrins associate with multiple intracellular protein complexes to form large protein networks. These networks of cytoskeletal and signaling proteins stabilize cell adhesion, induce alterations in cell shape and movement, and transduce signals that regulate cell proliferation, survival and differentiation [1,2]. The dissection of these protein networks to delineate signaling pathways responsible for specific integrin-induced events represents a significant challenge in this area. The best characterized signaling complex activated by integrins involves the focal adhesion kinase (FAK). This protein localizes to focal adhesion sites, functions as a protein tyrosine kinase, and serves as a scaffold protein that binds to multiple cytoskeletal and signaling proteins, including integrins, talin, paxillin, Src kinases, phosphatidylinositol (PI)

3′-kinase, Cas, Graf and Grb2 [2]. FAK regulates pathways that control cell migration, growth and survival through activation of extracellular-signal-regulated kinases (ERKs) of the mitogen-activated protein (MAP) kinase family, PI 3′-kinase and multiple small GTPases. The activation of FAK and organization of FAK complexes is inhibited by cytochalasin D, which prevents actin polymerization following integrin engagement. These results suggest that pathways regulated by FAK are activated at a relatively late stage of integrin signaling, downstream from events that regulate actin polymerization. Studies of the integrin αIIbβ3 in platelets and in a reconstituted model system in chinese hamster ovary (CHO) cells have provided evidence for the existence of a distinct integrin-regulated pathway involving a hematopoieticcell-specific protein tyrosine kinase, Syk. Syk activation in response to fibrinogen binding to αIIbβ3 is not inhibited by cytochalasin D and can be induced by experimental

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dimerization of αIIbβ3, even in the absence of fibrinogen binding [3–6]. Moreover, unlike FAK activation, Syk activation requires the activity of one or more Src family kinases and the presence of both the αIIb and β3 cytoplasmic tails [5]. Consequently, the activation of Syk may represent an event more proximal to αIIbβ3 engagement, and independent of FAK. Furthermore, the studies in CHO cells have recapitulated many of the features of ‘outside–in’ signaling typical of platelets, most notably the relative time courses of Syk and FAK tyrosine phosphorylation after cell adhesion to fibrinogen, and the different sensitivities of these two tyrosine kinases to cytochalasin D [5]. On the basis of these considerations, we reasoned that the CHO cell model system might be useful in characterizing events downstream of Syk activation in an integrin signaling pathway. Therefore, we transiently expressed Syk and other relevant signaling molecules in the A5 CHO cell line, which stably expresses αIIbβ3, and evaluated the morphological and biochemical consequences during cell adhesion to fibrinogen. The results define a previously uncharacterized pathway downstream of Syk and the guanine nucleotide exchange factor Vav1 that regulates multiple membrane-proximal signaling events and lamellipodia formation.

Results Integrin aIIbb3 and Syk cooperate to stimulate tyrosine

become tyrosine-phosphorylated. When Vav1 was coexpressed with Syk, however, there was slight tyrosine phosphorylation of Vav1 in suspended cells but prominent tyrosine phosphorylation of Vav1 in adherent cells. The amounts of Syk and Vav1 recovered in the immunoprecipitates of suspended and adherent cells were similar, confirming that cell adhesion had increased the levels of tyrosine phosphorylation of these proteins (Figure 1). The phosphorylation response of Vav1 paralleled that of Syk over time — responses to adhesion were already maximal by 15 minutes, the earliest time point studied, and persisted for at least 45 minutes (data not shown). These results establish that Vav1 can become tyrosine phosphorylated in response to cell adhesion mediated by αIIbβ3, but only under conditions in which Syk is also expressed. Integrin-induced tyrosine phosphorylation of Syk is associated with increased Syk kinase activity [3,5,9]. To evaluate whether Syk kinase activity was required for integrin-dependent phosphorylation of Vav1, the experiments above were repeated using a kinase-inactive form of Syk (lysine 402 residue mutated to arginine; K402R). In contrast to the results with wild-type Syk, no tyrosine phosphorylation of Syk K402R or Vav1 was observed during A5 CHO cell adhesion to fibrinogen, despite levels of expression of Syk K402R similar to those of wild-type Syk (Figure 1). Thus, integrin-dependent tyrosine phosphorylation of Vav1 requires Syk catalytic function.

phosphorylation of Vav1

Syk becomes tyrosine phosphorylated and activated during integrin-dependent adhesion of hematopoietic cells [3,4,7–9]. To better define the role of Syk in outside–in signaling through αIIbβ3, we studied the adhesive and signaling responses of A5 CHO cells stably expressing αIIbβ3 after transient transfection of Syk and other potentially relevant signaling molecules. As the expression of αIIbβ3 and Syk is restricted to hematopoietic cells, we reasoned that some of their downstream effectors might be similarly restricted. One such candidate effector is Vav1, a hematopoietic-specific Rho family guanine nucleotide exchange factor, which can couple with Syk and multiple downstream effectors [10,11] and is tyrosine phosphorylated in integrin-stimulated myeloid cells, neutrophils and platelets [8,12,13]. To examine whether αIIbβ3 induces tyrosine phosphorylation of Vav1 in a Syk-dependent manner, A5 CHO cells transiently expressing Syk and/or Vav1 were placed in suspension or plated onto fibrinogen-coated dishes. After 30 minutes, tyrosine phosphorylation of Syk and Vav1 was assessed on immunoblots. As reported previously [5], fibrinogen-adherent cells transfected with Syk exhibited prominent tyrosine phosphorylation of Syk, whereas the same cells maintained in suspension did not (Figure 1). In contrast, when Vav1 was expressed alone, it failed to

Figure 1 Vav1

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Syk-dependent regulation of Vav1 through αIIbβ3. A5 CHO cells were transfected with Vav1 alone or along with wild-type (WT) or the K402R mutant of Syk. The ability of Syk and Vav1 to be inducibly tyrosine phosphorylated after plating on fibrinogen (FBGN) for 30 min was measured by immunoblotting with anti-phosphotyrosine antibody (pTyr blot) following immunoprecipitation (IP) with the corresponding antibody. Levels of total Syk and Vav1 in the immunoprecipitates were measured by immunoblotting with appropriate antibody.

Research Paper Integrin signaling through Syk and Vav1 Miranti et al.

Src family kinases are activated following platelet stimulation [14], and the ability of Syk to be activated in fibrinogen-adherent A5 CHO cells requires Src activation [5]. Inhibition of endogenous Src kinases by transient expression of a kinase-inactive, dominant-negative Src mutant (lysine 295 residue mutated to arginine; K295R) greatly reduced adhesion-dependent tyrosine phosphorylation of both Syk and Vav1 (data not shown). Thus, one or more Src family kinases are required for Vav1 tyrosine phosphorylation, possibly through Src activation of Syk.

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Figure 2

GFP + Syk + Vav1

Integrin aIIbb3, Syk and Vav1 signal to Rac

Tyrosine phosphorylation of Vav1 has been shown to lead to an increase in its guanine nucleotide exchange activity toward Rho family GTPases, particularly Rac [10]. Two prominent responses to activated Rac in adherent cells are lamellipodia formation [15] and activation of Jun N-terminal kinase (JNK) [16]. To address whether Syk and/or Vav1 expression enhances lamellipodia formation following αIIbβ3 engagement, A5 CHO cells were transfected with Syk, Vav1 and green fluorescent protein (GFP, as a marker for transfection) and plated on fibrinogen-coated coverslips. Adherent cells were fixed, stained with rhodamine–phalloidin to mark filamentous actin (F-actin) and studied by confocal microscopy. Cell attachment to the coverslips was entirely dependent on αIIbβ3 because it was prevented by preincubation of cells with a functionblocking anti-αIIbβ3 antibody (A2A9, 10 µg/ml) or by an αIIbβ3-selective cyclic peptide (integrilin, 10 µM). Following attachment to fibrinogen, there was progressive cell spreading, lamellipodia extension and focal adhesion formation. Lamellipodia were identified by their characteristic appearance on the ventral surface of the cells and by positive membrane-proximal staining in these locations for αIIbβ3 and F-actin (Figure 2). Expression of Syk and Vav1 caused a dramatic increase in lamellipodia formation. Of the cells transfected with GFP, Syk and Vav1, 52% exhibited one or more lamellipodia and many cells contained several of these structures (Figure 2). In contrast, only a minority of untransfected (GFP-negative) cells on the same coverslip exhibited lamellipodia. Only 15% of control cells transfected with GFP alone or with GFP and Syk (but not Vav1) displayed lamellipodia, and the difference between these cells and the GFP+Syk+Vav1 transfectants was statistically significant (p < 0.0001). In the case of cells expressing GFP and Vav1 (but not Syk), 22% contained one or more lamellipodia; this was significantly greater than that seen with GFP or GFP+Syk transfectants but significantly less than that observed with GFP+Syk+Vav1 transfectants (p < 0.0001). Cotransfection with Syk, Vav1 and a dominant-inhibitory form of Rac, RacN17, reduced lamellipodia formation to levels below that of untransfected cells (data not shown), indicating that Rac or a closely related small GTPase is required for Syk and Vav1 enhancement of lamellipodia formation in fibrinogen-adherent cells.

GFP + Syk + Vav1 (untransfected cells)

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Effect of Syk and Vav1 on lamellipodia formation in fibrinogen-adherent A5 CHO cells. Cells were transfected with GFP alone (bottom panel) or with a combination of GFP, Syk and Vav1 (top and middle panels); 24 h later, the cells were plated onto fibrinogen-coated coverslips and incubated for 18 h. Adherent cells were fixed, permeabilized and stained with rhodamine–phalloidin to mark F-actin, and studied by confocal microscopy. Positive green fluorescence of cells due to expression of GFP was taken as an indication of successful transfection. Cells subjected to the transfection procedure with GFP, Syk and Vav1 but which failed to express GFP (middle panel) were taken as ‘untransfected’ cells. The cells shown in each panel are representative of predominant morphologies from all five experiments so performed. Bar = 10 µm.

After 18 hours of adhesion on fibrinogen, Syk transfectants exhibited Syk staining throughout the cell. Nonetheless, in most cells, Syk frequently colocalized with αIIbβ3 and Factin along the membranes of lamellipodia. Similar results were obtained with cells plated on fibrinogen for only 15 minutes (Figure 3a–c). In Vav1 transfectants, Vav1 was also present throughout the cell, but colocalization with αIIbβ3 or F-actin was much less apparent and observed only in an occasional lamellipodium in a minority of the cells (Figure 3e–g). In addition to lamellipodia, most Syk/Vav1 transfectants also contained numerous focal adhesions that stained with an antibody to αIIbβ3 and were connected to actin stress fibers that stained with rhodamine–phalloidin. No Syk or Vav1 was observed in these structures, however (Figure 3d,h). These results indicate that A5 CHO cell adhesion through αIIbβ3 triggers lamellipodia formation in a manner that is dependent on Syk

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Figure 3 (a)

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Partial colocalization of Syk and Vav1 with αIIbβ3 and F-actin in the lamellipodia of fibrinogen-adherent A5 CHO cells. (a–d) Cells were transfected with Syk and plated 24 h later onto fibrinogen-coated coverslips for either (a,c) 15 min or (b,d) 18 h. Cells were doublestained with an antibody to Syk (green, a–d), and rhodamine–phalloidin (red; a,b) or an antibody to αIIb (red; c,d). Lamellipodia are present in (a–c) and yellow areas representing colocalization of Syk and F-actin, or Syk and αIIb, can be observed at the periphery of these structures (arrowheads). In contrast, the cell in (d) exhibits many red, integrin-rich focal adhesions and there is no Syk colocalization to focal adhesions. (e–h) Cells were transfected with Syk and Vav1 and plated for 18 h onto fibrinogen-coated coverslips. The cells were double-stained with antibodies to the Myc epitope tag of Vav1 (green) and to αIIb (red). Occasional areas of colocalization of αIIb and Vav1 can be seen (yellow, arrowheads). (h) Vav1 does not colocalize to focal adhesions (red). Bars = 10 µm. These results are representative of three separate experiments.

RacN17 (Figure 4b). Taken together, these results indicate that Rac can serve as a downstream effector of αIIbβ3 in a pathway that includes Src, Syk and Vav1.

(f)

Integrin aIIbb3, Syk and Vav1 signal to ERK2

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and Vav1, consistent with the hypothesis that tyrosinephosphorylated Vav1 participates in the activation of Rac but does not function within focal adhesions. To determine whether Syk and Vav1 regulate JNK activation through Rac, A5 CHO cells were transiently transfected with hemagglutinin (HA) epitope-tagged JNK, plated on fibrinogen for 45 minutes, and the enzymatic activity in JNK immunoprecipitates was assessed using a glutathione S-transferase (GST)–c-Jun fusion protein as a substrate. Fibrinogen-adherent cells cotransfected with Syk and Vav1 exhibited a significant increase in JNK activity (7.5 ± 2.6-fold, p < 0.005). No increase was observed in cells transfected with Syk alone, and a modest increase was seen with Vav1 alone (2.9 ± 0.9-fold; Figure 4a). JNK activation was reduced 66% by coexpression of dominant-negative

The ERKs are activated following engagement of integrins in many cell types [2,17]. Whereas the best defined integrin pathways leading to activation of ERK involve Src kinases in association with either FAK or caveolin [18,19], Syk has been shown to regulate ERK activation in other receptor pathways [20,21]. The constitutive association of Vav1 with Grb2 and other components of the ERK pathway [22] suggests that Vav1 can contribute to activation of ERK in hematopoietic cells. Therefore, we studied the effect of A5 CHO cell adhesion to fibrinogen on the activity of HA-tagged ERK2, using myelin basic protein as a substrate. In cells transfected with ERK2, Syk and Vav1, cell adhesion to fibrinogen increased ERK2 activity 4 ± 0.9-fold. Syk alone did not enhance ERK2 activity, whereas Vav1 slightly increased ERK2 activation (2 ± 0.5-fold; Figure 5a). Unlike JNK, however, ERK2 activation was not blocked by expression of dominant-negative RacN17 (data not shown), but was blocked by expression of dominant-negative RasN17 (Figure 5b). Thus, αIIbβ3, Syk and Vav1 can collaborate to activate both ERK2 and JNK, albeit by routes which differ in their dependency on Rac. Integrin aIIbb3, Syk and Vav1 signal to c-Cbl

Cbl is a multi-domain protein that associates with many other cellular proteins through its Src homology 2 (SH2)domain-like phosphotyrosine-binding domain, proline-rich SH3-domain-binding motifs and tyrosine-phosphorylated SH2-binding sites [23]. We were interested in whether Syk and Vav1 might couple with Cbl downstream of αIIbβ3 as Cbl is phosphorylated on tyrosine following engagement of integrins, and associates with Syk and Vav1 in activated T lymphocytes [24–26]. Cbl also associates with the p85 subunit of PI 3′-kinase, raising the possibility that Cbl could couple Syk and αIIbβ3 to PI 3′-kinase activity [27].

Research Paper Integrin signaling through Syk and Vav1 Miranti et al.

Figure 4

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Figure 5

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Effects of Syk and Vav1 on JNK activity in A5 CHO cells. (a) Cells were transfected with Syk, Vav1 and HA-tagged JNK in the indicated combinations; a dash indicates cells that were not transfected. After 48 h, cells were placed in suspension on bovine serum albumin (BSA)coated plates, or plated on fibrinogen (FBGN) for 45 min, and the levels of JNK kinase activity were measured in HA–JNK immunoprecipitates using GST–c-Jun as the substrate. Total levels of JNK in the kinase assay were monitored by immunoblotting with antiJNK antibody (JNK blot). (b) A dominant-negative mutant of Rac (RacN17) was cotransfected with Syk, Vav1 and HA–JNK and its effect on JNK kinase activity measured as described in (a).

To examine Cbl phosphorylation and its association with p85, HA-tagged c-Cbl was transiently expressed in A5 CHO cells. The adhesion of these cells to fibrinogen resulted in a Syk-dependent increase in tyrosine phosphorylation of Cbl and this response was dramatically enhanced by the combination of Syk and Vav1 (Figure 6). Furthermore, p85 could be selectively recovered in Cbl immunoprecipitates from fibrinogen-adherent cells that expressed both Syk and Vav1 (Figure 6). Thus, an αIIbβ3–Syk–Vav1 pathway may promote assembly of a multimolecular signaling complex that includes Cbl and the activated p85 subunit of PI 3′-kinase. Integrin aIIbb3, Syk and Vav1 signal to PI 3¢-kinase

The activity of endogenous PI 3′-kinase was studied indirectly by determining the activity of an HA-tagged PI 3′-kinase effector, Akt, using histone 2B as a substrate. Akt is a serine/threonine kinase that is dependent on PI 3′-kinase for activation following integrin stimulation [28,29]. Adhesion of A5 CHO cells to fibrinogen induced a 2 ± 0.5-fold increase in Akt activity in a manner that was dependent on both Syk and Vav1 expression (Figure 7a). This response was not inhibited by coexpression of

ERK blot:

–HA–ERK2 Current Biology

Effect of Syk and Vav1 on ERK2 activity in A5 CHO cells. (a) Cells were transfected with Syk, Vav1 and HA-tagged ERK2 in the indicated combinations. After 48 h, cells were left in suspension or plated on fibrinogen (FBGN) for 45 min and the levels of ERK2 kinase activity were measured in HA–ERK2 immunoprecipitates using myelin basic protein (MBP) as a substrate. Total levels of ERK2 in the kinase assay were measured by immunoblotting with anti-ERK antibody (ERK blot). (b) A dominant-negative mutant of Ras (RasN17) was cotransfected with Syk, Vav1 and HA-tagged ERK2 and its effect on ERK2 activity was measured as in (a).

dominant-negative RacN17, but was blocked by dominant-negative Ras (data not shown). Akt activation was also inhibited by pretreatment of the cells with 100 nM wortmannin, a PI 3′-kinase inhibitor, consistent with the idea that Syk and Vav1 enhancement of Akt activation was mediated through activation of PI 3′-kinase (Figure 7b). As PI 3′-kinase has been implicated in numerous postligand binding events during outside–in integrin signaling in platelets [29,30], the effect of 100 nM wortmannin on other Syk-dependent and Vav1-dependent responses in fibrinogen-adherent A5 CHO cells was studied. In two experiments, wortmannin decreased the percentage of control-GFP-transfected cells that have lamellipodia from an average of 14.3% to 8.0%, whereas it decreased the ability of Syk and Vav1 to induce lamellipodia formation from 52.3% to 20.5%. Wortmannin also blocked the ability of Syk and Vav1 to enhance adhesion-dependent Akt activation (Figure 7b), but it had no effect on their ability to enhance JNK or ERK2 activation (data not shown).

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Syk and Vav1 regulate Cbl phosphorylation and association with PI 3′-kinase. A5 CHO cells were transfected with Syk, Vav1 and HA-tagged Cbl. After 48 h, the levels of tyrosine phosphorylation of Cbl after plating on fibrinogen (FBGN) for 45 min were monitored by immunoblotting with anti-phosphotyrosine antibody (pTyr blot) following immunoprecipitation (IP) with anti-Cbl antibody. The ability of the p85 subunit of PI 3′-kinase to associate with Cbl was detected by immunoblotting the Cbl immunoprecipitates with anti-p85 antibody (p85 blot). Total levels of Cbl in the IP were measured by immunoblotting with anti-Cbl antibody (Cbl blot).

Integrin-αIIbβ3-mediated tyrosine phosphorylation of Syk, Vav1 and Cbl were also unaffected by wortmannin (data not shown). Taken together, these data indicate that activation of PI 3′-kinase is required for Rac-mediated lamellipodia formation downstream of Syk and Vav1, but it is not required for other events regulated by these proteins. Sensitivity of Syk-regulated and Vav1-regulated events to inhibition of actin polymerization

Assembly and reorganization of the actin cytoskeleton are required for many biological responses of cells to integrinmediated adhesion [31,32]. Tyrosine phosphorylation of Syk in fibronectin-adherent monocytes or fibrinogen-adherent A5 CHO cells is not sensitive to cytochalasin D, an inhibitor of actin polymerization [5,7]. Similarly, cytochalasin D failed to inhibit either tyrosine phosphorylation of Vav1 in fibrinogen-adherent A5 CHO cells (Figure 8a) or the ability of Src to enhance Syk and Vav1 tyrosine phosphorylation (data not shown). These results are consistent with the idea that Src, Syk and Vav1 activation are proximal events in αIIbβ3 signaling that require neither actin polymerization nor cytoskeletal reorganization. Cytochalasin D also failed to inhibit several other Syk and Vav1 responses in fibrinogen-adherent cells, including Cbl tyrosine phosphorylation and association with p85 (Figure 8a), and activation of JNK, ERK2 and Akt

Effect of Syk and Vav1 on Akt activity in A5 CHO cells. (a) Cells were transfected with Syk, Vav1 and HA-tagged Akt. After 48 h, cells were maintained in suspension or plated on fibrinogen (FBGN) for 45 min and the levels of Akt kinase activity were measured in HA–Akt immunoprecipitates using histone 2B (H2B) as a substrate. Preimmune serum (PS) was added as a control. Total levels of Akt in the kinase assay were measured by immunoblotting with anti-Akt antibody (Akt blot). (b) The level of PI 3′-kinase-dependent activation of HA–Akt was monitored by inhibition with 100 nM wortmannin.

(Figure 8b). Yet in these same experiments, cytochalasin D fully inhibited adhesion-dependent FAK tyrosine phosphorylation (Figure 8c) and cell spreading (data not shown). Together these data suggest that Syk and Vav1 assemble a signaling complex downstream of αIIbβ3 that is independent of actin polymerization and distinct from the FAK pathway.

Discussion The present studies were carried out to identify and characterize signaling pathways and cellular responses triggered by platelet integrin αIIbβ3 and propagated through the hematopoietic-cell-specific tyrosine kinase Syk. The major conclusions are as follows. First, adhesion of αIIbβ3expressing A5 CHO cells to fibrinogen triggers a series of responses that are dependent on kinase-active Syk and Vav1. Second, these responses include lamellipodia formation, activation of the MAP kinases ERK2 and JNK, and PI 3′-kinase, and tyrosine phosphorylation of Cbl. Third, all of the biochemical responses occurred in the presence of cytochalasin D, indicating that they are independent of actin polymerization and distinct from signaling pathways involving FAK. Fourth, one major effector in the αIIbβ3–Syk–Vav1 pathway is Rac, which mediates lamellipodia formation and JNK activation. Other effectors must be involved, however, as Vav1-dependent activation of ERK2 and PI 3′-kinase was independent of Rac. Fifth,

Research Paper Integrin signaling through Syk and Vav1 Miranti et al.

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Figure 8 Syk and Vav1 activate downstream signals independent of actin polymerization. A5 CHO cells were transfected with Syk and Vav1 along with (a) HA–Cbl, (b) HA–JNK, HA–ERK2, or HA–Akt. Cells were plated on fibrinogen (FBGN) in the absence or presence of cytochalasin D (CytoD), and the levels of (a) Syk, Vav1 and Cbl phosphorylation and association with the p85 subunit of PI 3′-kinase, or (b) JNK, ERK2, and Akt kinase activity were measured as described in Figures 1, 4–7. Total levels of each protein in the immunoprecipitates were measured by immunoblotting with the appropriate antibody. (c) FAK immunoprecipitates (IP) were immunoblotted with anti-phosphotyrosine antibody (pTyr blot) to monitor levels of FAK phosphorylation.

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colocalization and coprecipitation studies indicated that Syk and Vav1 may regulate the assembly of an integrinproximal, multimolecular signaling complex that would be unique to platelets and possibly other hematopoietic cells.

potential role of Syk and Vav1 in integrin signaling that is not possible with human platelets. Integrin signaling to Vav1

These conclusions were derived from studies in a CHO cell model system but are likely to be directly relevant to αIIbβ3 signaling in platelets. As in A5 CHO cells, Syk and Vav1 become phosphorylated on tyrosine residues when platelets adhere to fibrinogen [3,12]. Induction of phosphorylation in both cell types is rapid, consistent with a role for Syk and Vav1 during initial events triggered by platelet adhesion, such as actin polymerization and lamellipodial extension [30,33]. Furthermore, just as in CHO cells, integrin-dependent activation of Syk in hematopoietic cells is not prevented by cytochalasin D, whereas FAK activation is [7].

The rapid and marked increase in tyrosine phosphorylation of Vav1 in fibrinogen-adherent A5 CHO cells required coexpression and tyrosine phosphorylation of Syk and Vav1, and the kinase activity of Syk. This suggests that Vav1 phosphorylation may be mediated directly by Syk, an idea consistent with the following observations: first, adhesion of A5 CHO cells and platelets to fibrinogen increases Syk kinase activity [3,5]; second, a dominantnegative form of Src, which inhibits Syk activation in A5 cells [5], also inhibited Vav1 phosphorylation; and, third, Syk and Vav1 are capable of a binary interaction involving the SH2 domain of Vav1 and tyrosine residues within the linker region of Syk [11].

The enhanced activation of multiple signaling pathways by Syk and Vav1 does not appear to be the consequence of a non-specific increase in all αIIbβ3-induced signaling events as αIIbβ3-dependent FAK and SHPS-1 [34] phosphorylation is not enhanced by Syk and Vav1 expression (data not shown). Furthermore, each of the signaling pathways activated by Syk and Vav1 coexpression are differentially regulated by distinct subsets of downstream signals. For example, Rac is specifically required for JNK activation, but not for ERK2 or Akt activation, whereas PI 3′-kinase is required for Akt activation and lamellipodia formation, but not for JNK or ERK2 activation. Thus, the strong parallels between platelets and the reconstituted CHO cell system and the specificity of the effects indicate that the A5 CHO cells provide a means to dissect the

In addition to platelets, neutrophils and hematopoietic cell lines exhibit tyrosine phosphorylation of Syk and Vav1 following engagement of β1 or β2 integrins [7–9,13]. The existence of an integrin pathway with at least two components specific to hematopoietic cells suggests that it might promote specialized adhesive functions, including hematopoietic cell development, platelet spreading on vascular matrices, or leukocyte migration across the endothelium. It remains to be determined, however, whether an integrin–Syk–Vav1 pathway is essential for a subset of integrin-dependent responses in hematopoietic cells or whether it plays an adjunctive role. For example, Rac figures prominently in the αIIbβ3–Syk–Vav1 pathway. On the other hand, integrins can facilitate the activation of Rac in non-hematopoietic cells in the

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absence of Syk and Vav1 [35], and receptors other than integrins may activate Rac in hematopoietic cells. Integrin aIIbb3 signaling to Rac

Studies in fibroblasts and other cells have established that integrins collaborate with growth factor, cytokine and G-protein-linked receptors to effect a sequence of actindriven events at the cell periphery, including the formation of filopodia, lamellipodia and focal adhesions. These responses are mediated, in part, by Cdc42, Rac and Rho, respectively [36,37]. What is the relationship of integrin signaling to these small GTPases? The present studies provide support for the idea that αIIbβ3 may signal to Rac. First, Syk, and to a lesser extent Vav1, localized with αIIbβ3 and F-actin at the periphery of lamellipodia, but neither Syk nor Vav1 were ever observed within focal adhesions. Second, filopodia were not prominent in A5 CHO cells in these experiments, but when Syk and Vav1 were coexpressed, these cells exhibited a marked increase in lamellipodia formation. Third, the adhesion of A5 CHO cells to fibrinogen stimulated the activation of JNK, a known downstream effector of Rac [16], and JNK activation and lamellipodia formation were inhibited by dominant-negative RacN17. We propose that αIIbβ3 and Syk act in concert to activate Rac, in part by stimulating tyrosine phosphorylation of Vav1, which is known to increase the Rac exchange activity of the Vav1 Dbl homology (DH) domain [10]. An analogous series of events may take place in platelets given the fact that tyrosine phosphorylation of Syk and Vav1 [3,12] and lamellipodial extension [38] occur soon after cells adhere to fibrinogen. Furthermore, platelet adhesion stimulates actin polymerization [39], a response that can be triggered directly by addition of constitutively active Rac to detergent-permeabilized platelets [40]. Integrin aIIbb3 signaling to PI 3¢-kinase

Expression of Syk and Vav1 in A5 CHO cells was associated with an adhesion-dependent increase in PI 3′-kinase activity, as assessed by enzymatic activity of Akt, a serine/threonine kinase regulated by the lipid products of PI 3′-kinase [41]. This response was inhibited by wortmannin, an irreversible inhibitor of PI 3′-kinase. In platelets, agonists appear to stimulate at least three PI 3′-kinase isoforms, generating PI 3,4,5-trisphosphate and PI 3,4-bisphosphate, leading to two waves of Akt activation. Since some of these responses require αIIbβ3 engagement [29], we propose that they may also be dependent on Syk and Vav1. Of the downstream effectors of Syk and Vav1 studied, only PI 3′-kinase and lamellipodia formation were inhibited by wortmannin. As the Rac exchange activity of Vav1 is activated when PI 3,4,5-trisphosphate binds to the Vav1 pleckstrin homology (PH) domain [42], wortmannin may

inhibit Rac by preventing generation of this lipid. Wortmannin exerted no inhibitory influence on the activation of JNK, however. As lamellipodia formation and JNK activation were both inhibited by dominant-negative Rac, it is likely that both events are regulated by Rac or a closely related protein. Although it is difficult to reconcile how these two Rac-regulated events are differentially sensitive to wortmannin, Rac may be activated by distinct exchange proteins with different sensitivity to wortmannin or the wortmannin-sensitive step in lamellipodia formation may be downstream of Rac. Integrin aIIbb3 signaling to ERK2

Activation of ERK2 through αIIbβ3 displayed the same dependence on Syk and Vav1 as did activation of JNK. Whereas JNK activation was dependent on activation of Rac, ERK2 activation was dependent on Ras. One established means by which integrins can activate ERKs is through the activation of Src and FAK, resulting in the tyrosine phosphorylation of binding sites for the SH2 domain of Grb2/Sos and activation of the Ras/Raf pathway [2,18]. The present results suggest an additional route for integrin activation of ERK2 in hematopoietic cells that involves Src, Syk, Vav1 and Ras. For example, Vav1 may associate with and influence the function of Grb2 and other components of the ERK pathway [13,20–22]. Integrin aIIbb3 signaling to Cbl

The strong induction of Cbl phosphorylation in adherent A5 CHO cells expressing Syk and Vav1 adds to the mounting evidence that Cbl is involved in integrin signaling. Cbl is phosphorylated on tyrosine after stimulation of integrins [24,26,43], and oncogenic forms of Cbl induce anchorage-independence in NIH-3T3 cells [26]. In addition, reduction of Cbl expression in macrophages using antisense Cbl oligonucleotides inhibits cell spreading on fibronectin [44]. Like Vav1, Cbl can function as a scaffold for the assembly of signaling complexes though its multiple protein-binding sites [23]. For example, the association of Cbl with the p85 subunit of PI 3′-kinase observed in the present study and others may be one way to recruit PI 3′-kinase activity to receptor complexes [24,44]. Here, the 3′ polyphosphoinositide (3′PPI) products of this enzyme could regulate the activity of other cellular proteins such as Vav1 and Akt. Cbl could be phosphorylated by one of several protein tyrosine kinases activated by integrin engagement. Cbl has been shown to associate with Src kinases as well as Syk, and reduction or elimination of the activity of Src family enzymes results in reduction in tyrosine phosphorylation of Cbl [27,45]. As Syk activation is dependent on Src kinase catalytic activity in adherent A5 CHO cells [5], it is difficult at the present time to distinguish which enzyme is responsible for Cbl phosphorylation in these cells.

Research Paper Integrin signaling through Syk and Vav1 Miranti et al.

Figure 9

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activation by fibronectin integrin receptors was found to be resistant to cytochalasin D [18].

Integrin

Src Syk

Vinculin Zyxin Talin

p85

PI3K p110

Cbl ?

Vav1

Grb2

Unlike integrin-mediated activation of FAK and its downstream effectors, αIIbβ3-induced Syk and Vav1 tyrosine phosphorylation, and Syk- and Vav1-dependent activation of ERK2, JNK, Akt and Cbl phosphorylation were found to be insensitive to inhibition of actin polymerization. We believe that this sensitivity to cytochalasin D is dependent on Syk and Vav1 expression as, in the absence of Syk and Vav1, integrin-induced ERK2 activation in A5 CHO cells is inhibited by cytochalasin D (data not shown). These results indicate that FAK and Syk are activated by αIIbβ3 through distinct mechanisms that differ in their dependence on actin polymerization and that Syk may regulate integrin-mediated signaling events through distinct signaling pathways.

Akt

Rac

Conclusions ERK

Lamellipodia

CH Dbl

JNK

PH SH3

SH2

3′PPI

PTK

PO42– Current Biology

Model for possible protein interactions involved in Syk and Vav1 regulation of integrin-αIIbβ3-mediated signaling based on known binding interactions of the components. Src and Syk activation by αIIbβ3 engagement could lead to phosphorylation of Cbl and Vav1. Cbl can interact with a phosphotyrosine site on Syk through its SH2like domain and, in turn, the Vav1 SH2 domain can bind to a phosphotyrosine site on Cbl. Cbl might also recruit the p85 and p110 subunits of PI 3′-kinase (PI3K) to the integrin complex. In addition to activation of Akt, the 3′PPI products of PI 3′-kinase might interact with the PH domain of tyrosine phosphorylated Vav1, promoting Rac exchange activity. ERK2 activation might result from the interaction of Grb2 with the SH3 domain of Vav1. Vav1 localization in lamellipodia may be stabilized by interaction with talin and vinculin as those proteins have been shown to be constitutively associated with Vav1 in T cells [49]. CH, calponin homology domain; PTK, protein tyrosine kinase.

Integrin aIIbb3 signaling independent of actin polymerization

Previous studies of Src kinases in integrin signaling have focused on the role of Src interactions with FAK following actin polymerization and contractility. Here, we report that Src enhancement of Syk and Vav1 phosphorylation in fibrinogen-adherent A5 CHO cells is not inhibited by cytochalasin D, indicating that Src activation by αIIbβ3 is not dependent on actin polymerization. Thus, in hematopoietic cells, Src may be activated by events proximal to the integrin tail and then participate either with Syk to regulate events in lamellipodia or with FAK to regulate events in focal adhesions. Additionally, Src

We propose that platelets and other hematopoietic cells have a unique integrin signaling pathway that includes a Src family kinase, Syk and Vav1 (Figure 9). This pathway would differ in certain key respects from the ‘classical’ integrin–Src–FAK pathway, most notably by its relative proximity, both spatially and temporally, to the integrin and by its ability to promote assembly of an integrin signaling complex that is initially independent of actin polymerization. Indeed, one function of this pathway may be to promote actin polymerization and cytoskeletal rearrangements through tyrosine phosphorylation of adaptors such as Vav1 and Cbl and activation of Rac, MAP kinases and PI 3′-kinase. The multiple outputs from this pathway would be expected to collaborate with other signaling pathways to effect the full range of anchorage-dependent responses of platelets and other hematopoietic cells.

Materials and methods Cell lines, plasmids and transfections

The A5 CHO cell line stably expressing the αIIbβ3 integrin was described previously [5]. The pEMCV–Myc–Syk and pEMCV–Myc–Syk K402R constructs were generated by placing a Myc epitope tag at the 5′ end of Syk using PCR. A point mutation in the ATP-binding site of Syk (K402R) was generated by site-directed mutagenesis. Additional plasmids were obtained as follows: pEF–neo–Myc–Vav1 was obtained from A. Altman; pCAGGS–HA–Cbl was from S. Burakoff; pCMV–Myc–RacN17 was from M. Simon; pHA–JNK was obtained from M. Renshaw; pHA–ERK2 was from M. Weber; and pcCMV–HA–Akt was from P. Tsichlis [46]. For transfections, 2 × 106 cells on 100 mm tissue culture plates were transfected with 4 µg total DNA using the Lipofectamine reagent (Gibco). The amount of plasmid DNA for each construct was as follows: 0.5 µg Syk, 1 µg Vav1, 0.5 µg c-Cbl, 0.2 µg HA–ERK2, 0.5 µg HA–JNK, 0.02 µg HA–Akt and 0.25 µg RacN17. After transfection (24 h), cells were serum starved in 0.1% FBS and, 24 h later, used for adhesion assays.

Adhesion assays Cells were washed with PBS and trypsinized for 10 min at 37°C. Cells were harvested in PBS/5 mM EDTA and 1 mg/ml soybean trypsin

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inhibitor, washed once and resuspended in DMEM. Cells were incubated in suspension for 40 min at 37°C prior to replating. When indicated, cytochalasin D (10 µM) was added to cells 40 min prior to plating and wortmannin (100 nM) 15 min prior to plating. Cells were placed in 100 mm fibrinogen-coated plates (100 µg/ml) and allowed to adhere for 15–45 min. Suspension cells were held in a small volume of DMEM or placed in BSA-coated plates. Non-adherent cells were removed from fibrinogen-coated plates and adherent cells were lysed in 500 µl lysis buffer. Suspension cells were either lysed in an equal volume of 2× lysis buffer, or were collected from the BSA plates, pelleted and lysed in 500 µl 1× lysis buffer. All extracts were clarified by centrifugation and total protein levels were determined using Coomassie dye reagent (BioRad) or BCA (Pierce).

Immunoprecipitation and immunoblotting Syk and Vav1 were immunoprecipitated after lysis in Src kinase lysis buffer [14] using rabbit anti-Syk antibody generated to the Spacer B region of Syk, and rabbit anti-Vav1 antibody (Santa Cruz). Cbl was immunoprecipitated after lysis in Cbl lysis buffer [27] using rabbit-antiCbl antibody (Santa Cruz). Precipitates were collected on protein A–agarose beads (Pierce) and washed 3× with lysis buffer before solubilizing in 2× SDS sample buffer and separation by SDS–PAGE. Gels were transferred to PVDF membranes and blocked in 5% BSA. Blots were probed with anti-phosphotyrosine antibody (4G10, Tom Roberts), immunoprecipitating antibody or rabbit-anti p85 PI 3′-kinase antibody (Upstate Biochemical). Blots were probed with HRP-conjugated secondary antibody (BioRad) and visualized by chemiluminescence (NEN).

Kinase assays HA-tagged ERK2, Akt and JNK were immunoprecipitated with mouse anti-HA 12CA5 monoclonal antibody prepared as previously described [47]. Immunoprecipitates were processed for in vitro kinase assays as follows: ERK2 using myelin basic protein (Gibco) as a substrate [48]; Akt using histone 2B (Boehringer Mannheim) as a substrate [28]; and JNK immunoprecipitates using GST–c-Jun (Upstate Biochemical) as a substrate [16]. After separation by SDS–PAGE, gels were transferred to PVDF membranes. After exposing blots to film for autoradiography and to a Fuji phosphoimager for quantitation, blots were blocked and probed with mouse anti-ERK antibody (Transduction Labs), rabbit antiAkt antibody [46] or with rabbit anti-JNK1 antibody (Santa Cruz).

Confocal microscopy

Cells were transfected with GFP alone (pEGFP, 0.02 µg, Clontech) or with a combination of GFP, Syk and Vav1 as described above; 24 h later, the cells were plated onto fibrinogen-coated coverslips and incubated an additional 18 h. Then adherent cells were fixed, permeabilized with Triton X-100 and stained with rhodamine–phalloidin to mark Factin. Lamellipodia formation in fibrinogen-adherent cells was quantitated and expressed as the percentage of 100 transfected cells containing more than one lamellipodium. For Syk colocalization experiments, cells were transfected with Syk and, 24 h later, plated onto fibrinogen-coated coverslips for either 15 min or 18 h. After fixation and permeabilization, cells were double-stained with a polyclonal antibody to Syk and either rhodamine–phalloidin or a polyclonal antibody to αIIb. For Vav1 colocalization studies, cells were transfected with Syk (nonepitope tagged) and Vav1 and plated for 18 h onto fibrinogen-coated coverslips. After fixation and permeabilization, the cells were doublestained with an antibody to the Myc tag (Vav1) and an antibody to αIIb. Cells were analyzed with an MRC 1024 Bio-Rad laser scanning confocal imaging system attached to a Leitz Diaplan microscope.

Acknowledgements The authors are grateful to A. Altman, M. Renshaw, M. Schwartz, M. Simon, S. Burakoff, M. Weber, and P. Tsichlis for generous gifts of plasmid reagents. We are also thankful to M. Ginsberg for the A5 CHO cell line, and K. Zoller and I. MacNeil for the Syk antibody and the Myc–Syk and Myc–Syk K402R expression plasmids. These studies were supported in part by grants from the National Institutes of Health (HL56595, HL57900 to S.J.S.), (CA27951, CA78773 to J.S.B.) and (CA72203 to C.K.M.).

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