Article |
Address correspondence to Sanford J. Shattil, Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Rd., VB-5, La Jolla, CA 92037. Tel.: (858) 784-7148. Fax: (858) 784-7422. E-mail: shattil{at}scripps.edu
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Abstract |
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Key Words: integrin; signaling; Src; Syk; tyrosine kinase
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Introduction |
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Blood platelets are frequently used to study outside-in signaling because they represent a physiologically important cell, and the platelet-specific integrin, IIbß3, is required for hemostasis. Outside-in signaling in platelets is triggered when fibrinogen or von Willebrand factor binds to
IIbß3, and it is dependent on close functional, if not physical, relationships between
IIbß3 and the signaling machinery of the cell (Shattil et al., 1998). Signals transduced by
IIbß3 regulate platelet filopodial extension, spreading, aggregation, and granule secretion (Shattil et al., 1998; Phillips et al., 2001). Since these responses involve polymerization and rearrangements of actin filaments (Fox, 1993; Hartwig et al., 1999),
IIbß3 signals presumably regulate actin dynamics.
Specific nonreceptor tyrosine kinases have been implicated in IIbß3 outside-in signaling (Jackson et al., 1996; Shattil et al., 1998; Phillips et al., 2001). For example, Syk becomes activated within seconds of fibrinogen binding to platelets, whereas FAK becomes activated later during platelet aggregation and spreading (Lipfert et al., 1992; Clark et al., 1994). Syk activation does not require actin polymerization since it is unaffected by inhibitors such as cytochalasin D, whereas FAK activation requires actin polymerization (Lipfert et al., 1992; Clark et al., 1994). Thus, activation of Syk, but not FAK, may be a key early event in outside-in signaling. This idea is supported by several recent observations. First, platelet adhesion to fibrinogen stimulates a direct interaction of Syk with the cytoplasmic tail of ß3, correlating with Syk activation (Woodside et al., 2001). Second, Syk heterologously expressed in CHO cells is activated in response to clustering of
IIbß3 complexes (Hato et al., 1998). Third, adhesion of platelets or
IIbß3-CHO cells to fibrinogen induces Syk-dependent tyrosine phosphorylation of proteins implicated in cytoskeletal regulation, including Vav1 and SLP-76 (Cichowski et al., 1996; Judd et al., 2000; Obergfell et al., 2001). The biological relevance of these interactions for outside-in signaling in platelets is suggested by studies of mice deficient in SLP-76, which exhibit a bleeding diathesis and, among other platelet abnormalities, defective spreading on fibrinogen (Clements et al., 1999; Judd et al., 2000).
In addition to Syk and FAK, platelets contain several Src family members (Src, Fyn, Fgr, Hck, Lyn, Yes), with Src itself being the most abundant (Golden et al., 1986; Huang et al., 1991; Stenberg et al., 1997). Studies using chemical cross-linking and immunoprecipitation techniques have suggested that one or more Src kinases may be associated with IIbß3, either before or after thrombin-induced platelet activation (Dorahy et al., 1995; Kralisz and Cierniewski, 1998). Furthermore, Src becomes activated and partitions to the detergent-insoluble actin cytoskeleton in thrombin-aggregated platelets, suggesting that it is regulated by fibrinogen binding to
IIbß3 (Horvath et al., 1992; Clark and Brugge, 1993; Fox et al., 1993).
Based on these considerations, we used biochemical and genetic approaches here to determine how outside-in signaling is initiated in platelets. The results establish that Src is constitutively associated with IIbß3 and that platelet adhesion to fibrinogen selectively activates this pool of Src, possibly by inducing the dissociation of an Src-regulatory kinase, Csk, from the
IIbß3 complex. Furthermore, Src kinases are required for
IIbß3-dependent activation of Syk, and both Src and Syk are required to initiate cytoskeletal events responsible for platelet spreading on fibrinogen.
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Results |
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To determine whether platelet adhesion to fibrinogen affects the activation state of Src, Western blots of IIbß3 immunoprecipitates were probed with phospho-specific antibodies to Src tyrosine residues 418 and 529. Autophosphorylation of Tyr-418 within the kinase activation loop is a marker of Src activation. On the other hand, phosphorylation of Tyr-529 (the target residue of Csk) promotes intramolecular interactions of the Src COOH terminus with the SH2 domain, effectively inhibiting kinase activity (Xu et al., 1999; Young et al., 2001). Thus, phosphorylation at Tyr-529 is a marker of Src suppression. Platelet adhesion to fibrinogen for 45 min caused a simultaneous increase in the phosphorylation of Src Tyr-418 and a decrease in the phosphorylation Tyr-529 (Fig. 2 A). This effect was observed within 15 min, the earliest time point at which sufficient numbers of adherent platelets could be analyzed (unpublished data). Src activation in fibrinogen-adherent platelets was unaffected by 10 µM cytochalasin D, implying that it did not require actin polymerization (Fig. 2 A). Adhesion-dependent tyrosine phosphorylation of FAK was blocked by cytochalasin D, as reported previously (Fig. 2 B) (Lipfert et al., 1992). In contrast to these results for integrin-associated Src, the bulk of the Src in platelets that did not coprecipitate with
IIbß3 failed to become activated during platelet adhesion to fibrinogen (Fig. 2 C).
To analyze these results quantitatively, Src "activity" was expressed arbitrarily as the ratio of the phospho-specific Tyr-418 and Tyr-529 immunoblot signals after normalization for the amount of Src in the ß3 immunoprecipitates (Fig. 2, D and E). Platelet adhesion to fibrinogen caused a mean 35-fold increase in Src activity as assessed by this method (P < 0.02). This effect was confirmed by direct measurements of Src kinase activity in ß3 immunoprecipitates (P < 0.001) (Fig. 2 F).
In contrast to platelet adhesion to immobilized fibrinogen, binding of soluble fibrinogen to platelets requires prior activation of IIbß3. Therefore, to investigate whether soluble fibrinogen binding is sufficient to activate Src, platelets were incubated in the presence of 250 µg/ml fibrinogen, and 0.5 mM MnCl2 was added to directly activate
IIbß3 (Bazzoni and Hemler, 1998). Fibrinogen binding caused both the dissociation of Csk from the
IIbß3 complex and the increased phosphorylation of Src Tyr-418 (Fig. 3, Fib + Mn2+ lane). These responses were observed as early as 1 min, were stable for at least 20 min, and were blocked by 2 mM RGDS, which inhibits fibrinogen binding to
IIbß3. Interestingly, MnCl2 or RGDS each induced a small amount Csk dissociation and Src Tyr-418 phosphorylation, suggesting that both integrin activation and ligation contribute to Src activation (Fig. 3). Together with the data for adherent platelets, these results establish that fibrinogen binding to
IIbß3 causes dissociation of Csk from the
IIbß3 complex at the same time that the integrin-associated pool of Src becomes activated. These responses require neither actin polymerization nor tyrosine phosphorylation of FAK.
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To identify potential downstream effectors of IIbß3 and Src, the effect of PP2 or SU6656 on adhesion-dependent tyrosine phosphorylation of several platelet proteins was examined. The results with PP2 are shown in Fig. 7 and are identical to those obtained with SU6656. PP2, but not PP3, inhibited adhesion-dependent tyrosine phosphorylation of Syk (Fig. 7 A), tyrosine phosphorylation of putative Syk substrates Vav1, Vav3, and SLP-76, and tyrosine phosphorylation of SLAP-130, an adaptor that binds to SLP-76 (Fig. 7 B) (Judd et al., 2000; Obergfell et al., 2001). On the other hand, PP2 had no effect on the adhesion-dependent association of Syk with
IIbß3 (Fig. 7 C). Thus, one or more Src family kinases appear to be required for
IIbß3-dependent activation of Syk and for tyrosine phosphorylation of Syk substrates implicated in cytoskeletal regulation (Judd et al., 2000; Obergfell et al., 2001).
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Discussion |
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Human- and gene-targeted mouse platelets were used here to define the relationships between IIbß3, Src, and Syk in outside-in signaling. The major new findings are: (a) Src and its regulatory kinase, Csk, are constitutively associated with
IIbß3 in resting platelets. (b) Upon soluble fibrinogen binding to
IIbß3 or platelet adhesion to immobilized fibrinogen, Csk dissociates from
IIbß3 and Src becomes activated, independent of actin polymerization. Activated Src localizes to the periphery of spreading platelets, including filopodia. (c) The activity of Src is required for
IIbß3-dependent tyrosine phosphorylation of Syk and for platelet spreading on fibrinogen. (d) Syk is also required for platelet spreading but not for Src activation. Thus, outside-in signaling in platelets is initiated by the sequential activation of Src and Syk in proximity to
IIbß3, providing a molecular basis for signal generation from
IIbß3 to the actin cytoskeleton.
Immunoprecipitation analysis of NP-40 detergent extracts revealed that Src and the related kinases, Fyn and Yes, were associated with IIbß3, both before and after platelet adhesion to fibrinogen. These interactions are specific because no association was observed between
IIbß3 and two other nonreceptor tyrosine kinases, Pyk2 and Btk. Furthermore, Src did not coprecipitate with GP Ib
, another abundant platelet membrane adhesion receptor (Fig. 1). These results are consistent with a brief report that Src coprecipitates with
IIbß3 from Triton X-100 lysates of resting platelets (Kralisz and Cierniewski, 1998). In another study, Dorahy et al. (1995) detected association of Src (and Lyn) with
IIbß3, but only in the presence of a cell-permeable chemical cross-linker which simultaneously activated the platelets. Our ability to detect Src, Fyn, and Yes in
IIbß3 immunoprecipitates without cross-linkers reflects differences in the conditions used for platelet preparation and analysis. Although we did not analyze
IIbß3 immunoprecipitates for all Src family members expressed in platelets, these results indicate that Src associates with and is regulated by
IIbß3, and this functional relationship may extend to some other Src family members.
These studies do not resolve whether the association of Src with IIbß3 is direct or indirect. Several proteins are capable of binding directly to the cytoplasmic tails of
IIb or ß3 in vitro, and one of these might serve to link Src to
IIbß3 in platelets (Shattil et al., 1998; Liu et al., 2000; Phillips et al., 2001). FAK and Syk warrant discussion in this regard. FAK can bind to peptides derived from integrin ß tails (Schaller et al., 1995), and we found it to be associated with
IIbß3 in platelet lysates (Fig. 1). Furthermore, when FAK becomes auto-phosphorylated at Tyr-397 in adherent fibroblasts, it forms a bimolecular complex with Src (Guan, 1997; Polte and Hanks, 1997; Schaller et al., 1999; Schaller, 2001). However, a FAKSrc complex cannot mediate the interaction we observed between
IIbß3 and Src in platelets because, in contrast to FAK activation, the
IIbß3/Src association was neither adhesion-dependent nor inhibited by cytochalasin D (Fig. 2 B). Syk is also unlikely to serve as a necessary link between Src and
IIbß3 because it coprecipitated with the integrin only after platelet adhesion (Fig. 1), even in Syk-null platelets (Fig. 10) (Woodside et al., 2001). The precise mode of interaction between
IIbß3 and Src remains to be determined.
How does fibrinogen binding to IIbß3 lead to activation of Src? Src is stabilized in an inactive conformation by intramolecular interactions of the SH2 domain with pTyr-529 and the SH3 domain with a polyproline helix in the SH2 kinase linker region (Xu et al., 1999; Young et al., 2001). Tyr-529 is likely maintained in the phosphorylated state by Csk (Okada et al., 1991; Latour and Veillette, 2001), a kinase that was associated with
IIbß3 in nonadherent platelets. Fibrinogen interaction with platelets resulted in the dissociation of Csk (but not Src) from
IIbß3 (Figs. 1 and 3), suggesting that Src may become activated at integrin adhesion sites following its physical separation from Csk. This idea is consistent with the localization of activated Src to filopodia and edges of fibrinogen-adherent platelets (Fig. 4), and with the observation that only the integrin-associated pool of Src became activated in such platelets (Fig. 2). In T lymphocytes, the proximity of Csk to Src kinases is influenced by specific transmembrane proteins, such as PAG/Cbp, which are enriched in lipid rafts and bind Csk when tyrosine-phosphorylated (Brdicka et al., 2000; Kawabuchi et al., 2000). PAG/Cbp is also present in platelets (Watson et al., 2001), but additional studies will be required to determine if it is involved in regulating the association of Csk with
IIbß3. Fibrinogen binding to platelets might also activate Src by influencing the localization or activity of a protein tyrosine phosphatase that can dephosphorylate Src Tyr-529. Three such phosphatases have been implicated in integrin signaling in other cell types, including receptor-like protein-tyrosine phosphatase-
, PTP-1B, and SHP-2 (Oh et al., 1999; Su et al., 1999; Cheng et al., 2001). In theory, Src might also be subject to regulation in platelets by proteins within nascent adhesion sites that engage the Src SH2 or SH3 domains (Xu et al., 1999; Young et al., 2001).
The results with PP2 and SU6656 strongly suggest that IIbß3-dependent tyrosine phosphorylation of Syk and platelet spreading on fibrinogen are controlled by an Src kinase. The defect in spreading of murine platelets lacking Src, Fgr, Hck, and Lyn confirms this assessment, and the normal spreading of platelets lacking Fgr, Hck, and Lyn suggests that Src itself plays a dominant role. However, the current studies do not exclude the involvement of other Src family members in specific phases of outside-in signaling.
PP2 and SU6656 blocked adhesion-dependent tyrosine phosphorylation of the Rac exchange factors, Vav1 and Vav3, and the molecular adaptor, SLP-76, all of which have been implicated in cytoskeletal regulation downstream of integrins in hematopoietic cells (Fig. 7) (Cichowski et al., 1996; Judd et al., 2000; Moores et al., 2000). Since these proteins are direct substrates of Syk, the results imply that Src kinases lie upstream of Syk in an IIbß3 pathway, possibly directly upstream. Although not studied, other substrates of Src kinases and Syk, such as cortactin and tubulin, might also couple
IIbß3 to cytoskeletal events (Gallet et al., 1999; Faruki et al., 2000). Unlike Syk activation by immune response receptors that contain ITAM domains,
IIbß3 activation of Syk is ITAM-independent (Gao et al., 1997; Turner et al., 2000; Woodside et al., 2001). Based on the current results, we speculate that fibrinogen binding to platelets induces clustering of
IIbß3 complexes, leading to activation of Src kinases, interaction of Syk with the cytoplasmic tail of ß3, and activation of Syk by the Src kinases. Activation of additional Syk molecules might then proceed by autophosphorylation in trans.
Syk-/- mouse platelets have a very subtle defect in agonist-induced fibrinogen binding (Law et al., 1999), and a profound defect in aggregation induced by collagen (Watson and Gibbins, 1998). These abnormalities cannot explain the spreading defect we observed in fibrinogen-adherent syk-/- platelets (Fig. 8) because no agonists were added in this experiment, and agonists are not required for platelet attachment to immobilized fibrinogen via IIbß3 (Savage et al., 1992; Law et al., 1999). By the same token, neither Src nor Syk were required for agonist enhancement of platelet spreading on fibrinogen (Fig. 8). Like platelets, Syk-deficient neutrophils exhibit defective integrin-dependent responses, such as the respiratory burst (unpublished data).
Although integrins and Src kinases are ubiquitous, Syk was thought until recently to be confined to hematopoietic cells. However, Syk is more widely distributed and may regulate the anchorage-dependent growth of epithelial and endothelial cells (Coopman et al., 2000; Inatome et al., 2001; Tsujimura et al., 2001; Yamada et al., 2001). Moreover, Syk can interact with integrin ß1 and ß2 tails as well as ß3 (Woodside, D., and M. Ginsberg, personal communication), and our preliminary studies indicate that Src is associated with ß1 integrins in platelets. Consequently, some of the functions attributable to Syk in hematopoietic and nonhematopoietic cells may require coordinated interactions between integrins, Src, and Syk. Thus, the paradigm delineated here for initiation of outside-in IIbß3 signaling in platelets may be relevant to integrins in a variety of biological contexts.
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Materials and methods |
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Bone marrow chimeras
src+/- mice (Soriano et al., 1991) were bred to the hck-/-/fgr-/-/lyn-/- background (Meng and Lowell, 1997) and mated to obtain hck-/-/fgr-/-/lyn-/-/src-/- mutant embryos. hck-/-/fgr-/-/lyn-/-/src+/+ embryos were generated from the same pregnancy. syk+/- mice (Turner et al., 1995) were mated to obtain Syk-/- embryos. Fetuses were genotyped by PCR. Bone marrow chimeras were generated by injecting mutant or wild-type littermate fetal liver cells into lethally irradiated C57BL/6 recipients. Repopulation of the hematopoietic compartment by transplanted cells was confirmed by absence of the relevant tyrosine kinase(s) in platelets, as determined by Western blotting, and by absence of peripheral B-cells in the syk-/- chimeras, as determined by flow cytometry. Platelets from chimeras were studied 68 wk after transplantation. In some experiments, syk+/- rather than syk+/+ chimeras were used as controls, with no differences in results from these two sources.
Interactions of platelets with fibrinogen
Washed human platelets were obtained from fresh, anticoagulated whole blood and resuspended to 3 x 108 cells/ml in a platelet incubation buffer (Leng et al., 1998). To test the effects of soluble fibrinogen binding to IIbß3, platelets were incubated at room temperature for 15 min with 250 µg/ml fibrinogen in the presence or absence of 0.5 mM MnCl2 (to activate integrins) (Bazzoni and Hemler, 1998) and 2 mM RGDS (to block fibrinogen binding). Platelets were then sedimented at 180 g for 5 min, washed once with phosphate-buffered saline, and solubilized for 10 min on ice in a buffer containing 0.5% NP-40, 50 mM NaCl, 50 mM Tris, pH 7.4 and inhibitors (1 mM sodium vanadate, 0.5 mM sodium fluoride, 0.5 mM leupeptin, 0.25 mg/ml Pefabloc, 100 µg/ml aprotinin). Lysates were routinely clarified by sedimentation at 10,000 rpm in a 4° microcentrifuge, and subjected to immunoprecipitation and Western blotting. Identical results were obtained if lysates were clarified at 100,000 g for 30 min. For studies of platelet adhesion to fibrinogen, 100 mm bacterial tissue culture plates were precoated with 5 mg/ml BSA or 100 µg/ml fibrinogen (Haimovich et al., 1993). After blocking with heat-denatured BSA, 4.5 x 108 platelets in 1.5 ml were added and incubated for the indicated periods of time at 37°C in a CO2 incubator. Nonadherent cells from the BSA plates were sedimented at 10,000 rpm for 3 s in a microcentrifuge and lysed immediately. Platelets adherent to fibrinogen were gently rinsed twice in phosphate-buffered saline, lysed on the plates directly and processed for immunoprecipitation and Western blotting.
For studies of mouse platelets, anticoagulated mouse blood was obtained by cardiac puncture (Judd et al., 2000). Platelet preparation and experimental procedures were similar to those with human platelets.
Immunoprecipitation and Western blotting
500 µl aliquots of each platelet lysate containing equal amounts of protein (ranging from 500750 µg between experiments) were immunoprecipitated with the indicated antibodies and protein A. Samples were electrophoresed in 7.5% SDS-polyacrylamide gels, transferred onto nitrocellulose and subjected to Western blotting (Gao et al., 1997; Miranti et al., 1998). Immunoreactive bands were detected by enhanced chemiluminescence with reaction times ranging from 5 s to 5 min. Blots were scanned in a Hewlett-Packard ScanJet 5300C scanner, and labeled bands were quantified by calibrated densitometry using NIH Image software.
Src Kinase Assay
IIbß3 was immunoprecipitated from platelet lysates with antibody #8053, and the protein A beads were washed twice. Beads were resuspended in kinase buffer and Src kinase activity was measured with a peptide substrate (KVEKIGEGTYGVVYK) according to the manufacturer's instructions (Src Assay Kit, Upstate Biotechnology Inc.)
Confocal microscopy
Washed human or mouse platelets were plated on fibrinogen-coated coverslips for 45 min at room temperature. Adherent cells were fixed in 3.7% formaldehyde, permeabilized with 0.1% Triton X-100, and stained with primary and FITC- or Texas redconjugated secondary antibodies as indicated. Rhodamine-phalloidin was used to stain F-actin. Single images were acquired with a Leica fluorescence microscope equipped with a laser scanning confocal system (MRC 1024; Bio-Rad Laboratories). Images were processed in Adobe Photoshop. Surface areas of at least 100 platelets per sample were measured using Image-Pro Plus Software (Media Cybernetics, Inc.). Statistical analyses were performed using Student's t test.
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Footnotes |
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Acknowledgments |
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These studies were supported by grants CA78773, DK58066, HL5447, and HL57900 from the National Institutes of Health and by an American Heart Association post-doctoral fellowship (A. Obergfell).
Submitted: 21 December 2001
Revised: 21 February 2002
Accepted: 1 March 2002
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