Correspondence to Sanford J. Shattil: sshattil{at}ucsd.edu
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
IIbß3 mediates fibrinogen-dependent platelet aggregation and spreading on damaged vascular surfaces, whereas
Vß3 promotes osteoclast adhesion to vitronectin or osteopontin (Byzova et al., 1998; Shattil and Newman, 2004). Genetic deficiency of
IIbß3 and
Vß3 leads to defects in hemostasis and bone remodeling, respectively (Hodivala-Dilke et al., 1999; Feng et al., 2001). Adhesive ligand binding to ß3 integrins leads to c-Src activation and tyrosine phosphorylation of c-Src substrates in platelets and osteoclasts (Feng et al., 2001; Obergfell et al., 2002; Arias-Salgado et al., 2003). The close relationship between ß3 integrins and c-Src is underscored by defective spreading of platelets that are deficient in multiple Src family kinases (Obergfell et al., 2002) and by overlapping bone remodeling phenotypes in mice that are deficient in c-Src or ß3 (Soriano et al., 1991; Hodivala-Dilke et al., 1999; McHugh et al., 2000). Consequently, attention is now focused on how ß3 integrins regulate c-Src to initiate outside-in signaling.
c-Src is maintained in an autoinhibited state by concerted intramolecular interactions of the SH2 domain with a COOH-terminal motif centered at phosphotyrosine 529 and of the SH3 domain with a polyproline sequence in the linker region between the SH2 and kinase domains (Sicheri and Kuriyan, 1997; Young et al., 2001; Harrison, 2003). As c-Src appears to associate constitutively with ß3 integrins via the c-Src SH3 domain (Arias-Salgado et al., 2003), considerable reliance may be placed on the SH2phosphotyrosine 529 interaction to help maintain low c-Src activity in nonadherent platelets. Thus, disruption of the SH2phosphotyrosine 529 interaction by dephosphorylation of c-Src tyrosine 529 should facilitate c-Src activation during cell adhesion. Phosphorylation of c-Src tyrosine 529 is catalyzed by Csk, which is associated with the IIbß3c-Src complex in resting platelets (Okada et al., 1991; Obergfell et al., 2002; Arias-Salgado et al., 2003). However, the identity of the proteintyrosine phosphatase (PTP) that dephosphorylates c-Src tyrosine 529 to promote initiation of ß3 integrin signaling has remained unknown. In this study, we used biochemical and genetic approaches to unambiguously identify PTP-1B, which is a ubiquitous nonreceptor tyrosine phosphatase, as a phosphatase that is required for dephosphorylation of c-Src tyrosine 529 and for c-Src activation downstream of
IIbß3. Moreover, we demonstrate that PTP-1B is required for outside-in signaling in platelets and for normal platelet thrombus formation in living mice.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
To establish whether PTP-1B is required for integrin activation of c-Src, platelets from knockout mice that were deficient in PTP-1B (PTP-1B/) and wild-type (PTP-1B+/+) littermates were studied (Klaman et al., 2000). Incubation of wild-type platelets with MnCl2 and fibrinogen caused an increase in the tyrosine phosphorylation of numerous proteins. In contrast, PTP-1B/ platelets showed markedly reduced fibrinogen-dependent tyrosine phosphorylation (Fig. 1 c). Because several of the phosphorylated proteins, including Syk (72 kD) and adhesion and degranulation-promoting adaptor protein (130 kD), are substrates of c-Src during outside-in IIbß3 signaling, the catalytic activity of c-Src in
IIbß3 immunoprecipitates was assessed indirectly by monitoring the phosphorylation of activation loop tyrosine 418. Whereas fibrinogen binding to PTP-1B+/+ platelets stimulated phosphorylation of c-Src tyrosine 418, this response was minimal or absent in PTP-1B/ platelets (Fig. 1, c and e). Platelets from heterozygous (PTP-1B+/) littermates responded normally (not depicted). The defective responses of PTP-1B/ platelets could not be explained by reduced surface expression of
IIbß3 receptors (Fig. 1 d). These results suggest that PTP-1B/ platelets have a fundamental defect in
IIbß3 activation of c-Src.
To determine whether PTP-1B is required for fibrinogen-dependent dephosphorylation of c-Src tyrosine 529, the phosphorylation state of tyrosine 529 was monitored with an antibody specific for nonphosphorylated tyrosine 529. Whereas fibrinogen binding to wild-type platelets stimulated dephosphorylation of c-Src tyrosine 529 (as indicated by increased immunoreactivity of the dephosphotyrosine 529 antibody), no such dephosphorylation was observed in PTP-1B/ platelets. In fact, the level of tyrosine 529 phosphorylation paradoxically increased upon fibrinogen binding (Fig. 1 e). Thus, PTP-1B is required for IIbß3-dependent dephosphorylation of c-Src tyrosine 529, likely explaining the defective activation of c-Src in PTP-1B/ platelets.
The finding of relatively increased phosphorylation of c-Src tyrosine 529 in fibrinogen-bound PTP-1B/ platelets suggested that PTP-1B may play some unexpected role in the phosphorylation of tyrosine 529 by Csk. Csk is normally associated with the IIbß3c-Src complex in resting platelets and dissociates from it upon fibrinogen binding (Obergfell et al., 2002). However, Csk failed to fully dissociate from
IIbß3 and c-Src after fibrinogen binding to PTP-1B/ platelets (Fig. 1 f). Thus, PTP-1B may not only dephosphorylate c-Src tyrosine 529 upon fibrinogen binding to
IIbß3 (Arregui et al., 1998; Dadke and Chernoff, 2002) but may also regulate the interaction between Csk and the
IIbß3c-Src complex.
Mechanism of PTP-1BIIbß3c-Src interactions
To better understand the basis for interactions between PTP-1B, IIbß3, and c-Src during outside-in
IIbß3 signaling, mouse fibroblasts that were deficient in the ubiquitous Src family kinases c-Src, c-Yes, and Fyn (SYF cells; Klinghoffer et al., 1999) were stably transfected with
IIbß3. These
IIbß3-SYF cells express PTP-1B endogenously, enabling examination of PTP-1B interactions after transient transfection of c-Src. As observed with platelets,
IIbß3-SYF cells expressing c-Src showed a fibrinogen-inducible association of PTP-1B with c-Src and
IIbß3 (Fig. 2 a). However, PTP-1B failed to associate with c-Src in cells lacking
IIbß3 or with
IIbß3 in cells lacking c-Src. Thus, the fibrinogen-dependent interaction of PTP-1B with
IIbß3 or c-Src requires both integrin and tyrosine kinase.
|
To establish what regions of PTP-1B are required for these interactions, HA-tagged PTP-1B was cotransfected with c-Src into IIbß3-SYF cells. Wild-type PTP-1B and two different phosphatase-inactive "substrate-trapping" mutants (C215S and D181A) each interacted with
IIbß3 and c-Src (Fig. 3 a). Interestingly, the interaction with c-Src was somewhat greater with the D181A PTP-1B mutant, which is known to exhibit a higher affinity for binding to PTP-1B substrates than the C215S mutant (Flint et al., 1997). These data are consistent with a direct dephosphorylation of c-Src tyrosine 529 by PTP-1B. In contrast to substrate-trapping mutants, the double mutation of proline 309 and 310 to alanine prevented PTP-1B interaction with
IIbß3 and c-Src, as did the double mutation of tyrosine 152 and 153 to phenylalanine. These amino acid residues may help to mediate interactions of PTP-1B with one or more members of the integrin signaling complex during the early phase of outside-in signaling (Dadke and Chernoff, 2002). In addition, they may enable the phosphorylation of PTP-1B by c-Src because PTP-1B can phosphorylate c-Src in vitro (Jung et al., 1998), and fibrinogen binding to
IIbß3-SYF cells (Fig. 3 b) or platelets (Fig. 3 c) stimulated tyrosine phosphorylation of PTP-1B in a Src-dependent manner.
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although PTPs frequently exert negative regulation of signaling pathways, positive regulation has also been described previously (Neel et al., 2003; Tonks, 2003). In fact, receptor tyrosine phosphatases such as RPTP- or nonreceptor phosphatases such as Shp2 promote outside-in integrin signaling in fibroblasts, in some cases by dephosphorylating c-Src tyrosine 529 or the equivalent residue in another Src family kinase (Oh et al., 1999; Su et al., 1999). Although PTP-1B has been implicated in ß1 integrindependent c-Src activation, this has been observed only in immortalized fibroblasts and not in a primary cell type (Cheng et al., 2001), raising the question as to its physiological significance. Our data establish the in vivo relevance of PTP-1B activation of c-Src downstream of a ß3 integrin. PTP-1B may also exert negative regulation of integrin signaling by dephosphorylating c-Src substrates such as p130 Cas (Arregui et al., 1998; Liu et al., 1998; Cheng et al., 2001). Multiple substrates for PTP-1B may exist in platelets, although our results indicate that the dominant action of PTP-1B is the positive regulation of
IIbß3 signaling through activation of integrin-associated c-Src. In contrast to these results for PTP-1B, platelets from motheaten viable mice that were deficient in Shp1 catalytic function displayed normal
IIbß3-dependent activation of c-Src (unpublished data) and a morphology upon attachment to fibrinogen that is similar to wild-type platelets (Lin et al., 2004; unpublished data).
The fibrinogen-dependent association of PTP-1B with IIbß3 was observed in human and mouse platelets and in a fibroblast model system, enabling examination of its structural basis. PTP-1B recruitment to
IIbß3 required catalytic competence and the SH3 domain of c-Src (Figs. 1 and 2). Moreover, specific proline (proline 309 and 310) and tyrosine (tyrosine 152 and 153) residues in PTP-1B were necessary (Fig. 3), suggesting that a protein (or proteins) with SH3, SH2, and/or phosphotyrosine-binding domains is involved in mediating linkage of PTP-1B to the integrin complex. Although the linker protein in platelets could be c-Src itself, there is no evidence that the c-Src SH2 domain binds to PTP-1B, and the c-Src SH3 domain may not be available to PTP-1B when it engages the integrin ß3 cytoplasmic domain (Arias-Salgado et al., 2003). Thus, a model is proposed in which PTP-1B is localized in resting platelets to internal membranes (Frangioni et al., 1993). Then, fibrinogen binding induces
IIbß3 oligomerization (Simmons et al., 1997; Buensuceso et al., 2003), triggering transautophosphorylation of integrin-associated c-Src. This event might not be sufficient for full c-Src activation (Harrison, 2003), but low level activation might enable c-Src to phosphorylate a protein that is capable of recruiting PTP-1B to the
IIbß3 complex. After recruitment, PTP-1B may become a substrate for c-Src (Fig. 3, b and c; Jung et al., 1998) and induce further c-Src activation by promoting Csk dissociation from the integrin complex and dephosphorylation of tyrosine 529 (Fig. 1 f).
Although additional studies will be required to determine the mode of PTP-1B linkage to the IIbß3 complex in platelets, work in other cells has implicated scaffold or adaptor molecules, such as SHPS-1, PAG/Cbp, and Dok-1, in mediating interactions between Src kinases, PTPs, and/or Csk in response to growth factors or cell adhesion (Timms et al., 1999; Dube et al., 2004; Zhang et al., 2004). However, SHPS-1 is poorly expressed in platelets, and PAG/Cbp does not interact with
IIbß3 (Wonerow et al., 2002). Intriguingly, Dok-1 contains a phosphotyrosine-binding domain and potential SH2-binding sites and is a substrate for PTP-1B (Dube et al., 2004). Furthermore, Dok-1 binds directly to Csk (Shah and Shokat, 2002) and may associate with integrin ß cytoplasmic domains (Calderwood et al., 2003). Dok-2, a homologue of Dok-1, is expressed in platelets (Garcia et al., 2004). In preliminary studies, we have found that Dok-2 coimmunoprecipitates with PTP-1B from resting platelets. In addition, fibrinogen binding to platelets stimulates tyrosine phosphorylation of Dok-2, dissociation of Dok-2 from PTP-1B, and its association with Csk (unpublished data). However, a role for Dok-2 or any other Csk-binding protein (Thomas et al., 1999) in regulating PTP-1B recruitment to
IIbß3 and outside-in signaling remains to be determined.
Altogether, these studies have established a new function for PTP-1B by uncovering requirements for PTP-1B in integrin-dependent c-Src activation, platelet spreading on fibrinogen, and clot retraction and platelet thrombus formation. Defects in both platelet spreading and clot retraction may be adequately explained by the c-Src activation defect in PTP-1B/ platelets because both responses require outside-in IIbß3 signaling (Phillips et al., 2001; Shattil and Newman, 2004). However, although thrombus formation under flow conditions depends on signaling inputs from multiple platelet receptors, including
IIbß3 (Ruggeri, 2002; Jackson et al., 2003), it is legitimate to ask whether the impairment of c-Src activation in PTP-1B/ platelets is the cause of defects in platelet calcium mobilization and thrombus formation, which were observed in the microcirculation of PTP-1B/ mice (Fig. 6). Although we cannot exclude the possibility of additional unstudied signaling pathways that are affected by a deficiency of PTP-1B, we found no defect in agonist-induced
IIbß3 activation, platelet aggregation, or agonist costimulation of platelet spreading. Furthermore, the ligation of
IIbß3 is known to induce calcium transients that are required for the formation of stable platelet aggregates under conditions of flow and wall shear stress, which are typical within arterioles (Mazzucato et al., 2002; Nesbitt et al., 2002). In particular, IP3 production and calcium mobilization are triggered by Src-dependent activation of phospholipase C
during outside-in
IIbß3 signaling (Wonerow et al., 2003). Thus, links between defective integrin activation of c-Src and reduced calcium mobilization provide a plausible explanation for the reduced thrombus formation observed in PTP-1B/ mice.
In contrast to the reduced platelet thrombus formation in cremasteric vessels of PTP-1B/ mice, there was no spontaneous bleeding, although rebleeding from tail bleeding time wounds was more frequent than in control mice. Thus, the degree of any abnormality in hemostasis imposed by PTP-1B deficiency may be dictated by the type, location, and extent of vascular injury. The same might be true in the case of pharmacological inhibition of PTP-1B. Interestingly, one PTP-1B antagonist of questionable specificity has been shown to reverse platelet aggregation that is stimulated by cross-linking the FcRIIa receptor (Ragab et al., 2003). Although the selectivity of PTP-1B antagonists is still an issue, they are being evaluated for the treatment of type 2 diabetes and obesity because PTP-1B negatively regulates insulin and leptin receptor signaling in nonhematopoietic tissues (Elchebly et al., 1999; Klaman et al., 2000; Cheng et al., 2002; Zabolotny et al., 2002; Tonks, 2003; Hooft van Huijsduijnen et al., 2004). Assuming that PTP-1B antagonists with appropriate selectivity and toxicity profiles can be developed, current studies indicate that these compounds should be analyzed for their effects on platelet outside-in
IIbß3 signaling.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mouse strains
PTP-1B+/+ and PTP-1B/ mice (SV129/C57BL6/J) were described previously (Klaman et al., 2000). Age- and sex-matched littermates were used for each experiment. Mice were housed and handled in accordance with institutional guidelines.
Cell lines, plasmids, and transfections
SYF cells (mouse embryonic fibroblasts deficient in c-Src, Fyn, and c-Yes) were obtained from American Type Tissue Collection. Cells were maintained at 37°C with 6% CO2 in DME supplemented with 10% FBS, L-glutamine, and antibiotics. The vector pCDM8/IIb has been described previously (Hughes et al., 1995). Integrin ß3 cDNA was subcloned into HindIII-XhoI sites of pcDNA3.1/Zeo (Invitrogen). Expression vectors containing human
IIb and ß3 subunits were cotransfected into SYF cells with LipofectAMINE (Invitrogen). Stable transfectants (
IIbß3-SYF cells) were isolated by selective growth in medium containing 125 µg/ml Zeocin (Invitrogen), and clones expressing
IIbß3 were isolated by single cell sorting. A single clone (A29) was used for the studies reported in this article, but similar results were obtained with three other independent clones.
Expression vectors for wild-type and mutant c-Src (K295R, Y529F, 90144, and
150246) and HA-tagged PTP-1B have been described previously (Sells and Chernoff, 1995; Arias-Salgado et al., 2003). Mutant PTP-1B constructs (C215S, D181A, P309/310A, and Y152/153F) were generated using the Site-Directed Mutagenesis Kit (Stratagene), and mutations were confirmed by direct DNA sequencing. Transient transfections of SYF cells were performed with LipofectAMINE. After 24 h, cells were serum starved in 0.5% FBS and were cultured for an additional 24 h before further use.
Platelet isolation and functional assays
Human and mouse platelets were obtained from fresh anticoagulated whole blood, washed, and resuspended to 3 x 108 cells/ml in a platelet incubation buffer (Law et al., 1999b). A pool of platelets from at least four mice was used for each experiment. FITC-fibrinogen binding to platelets and platelet aggregation were measured as described previously (Law et al., 1999b). Surface expression of IIbß3 in mouse platelets was monitored with a FITC-conjugated antimouse ß3 antibody. Platelet spreading was assessed by confocal microscopy after plating cells on immobilized fibrinogen (100 µg/ml of coating concentration) for 4090 min. Fluorescence images were acquired with a laser scanning confocal microscope (model MRC 1024; Bio-Rad Laboratories) using a 60 x oil immersion objective (Nikon). Platelet surface areas were measured using Image Pro Plus software (Media Cybernetics, Inc.). Platelet adhesion was quantified by an acid phosphatase assay after incubating 1.5 x 106 cells (50 µl) for 1 h at RT in fibrinogen-coated microtiter wells (Law et al., 1999b). The percentage of adherent platelets was determined by calculating the ratio of bound/maximal signal at 405 nm, with maximal signal obtained from wells with platelets not subjected to washing. Fibrin clot retraction was studied by incubating 150 µl of mouse platelet-rich plasma (2.2 x 107 platelets) in the presence of 1 U/ml thrombin and 3 mM CaCl2 for 2 h at RT in an aggregometer cuvette. A paper clip was added to facilitate clot removal at the termination of the experiment. The volume of residual clot-free plasma was determined, and clot volume was taken as 150 µl minus this value. Clot volume was expressed as a percentage of the original 150-µl plasma volume.
Immunoprecipitation and immunoblotting
Cells were lysed in buffer containing 1% NP-40, 150 mM NaCl, 50 mM Tris, pH 7.4, 1 mM sodium vanadate, 0.5 mM sodium fluoride, 1 mM leupeptin, and complete protease inhibitor cocktail (Roche Applied Science). Lysates were clarified by centrifugation at 13,000 g for 10 min at 4°C, and 100500 µg of protein from the soluble fraction was immunoprecipitated using a relevant primary antibody and protein A or protein GSepharose beads. Immunoprecipitates were subjected to SDS-PAGE and immunoblotting, and immunoreactive bands were detected by enhanced chemiluminescence (SuperSignal West Pico Substrate; Pierce Chemical Co.).
Platelet thrombus formation in vivo
Fluorescence and brightfield microscopy was used to capture real-time digital images of Fura 2-AMlabeled platelets in developing thrombi of living mice after a laser-induced injury to the arteriole wall in the cremaster muscle (Falati et al., 2002). In brief, platelets from PTP-1B/ and PTP-1B+/+ mice were loaded with Fura 2-AM. Then, 250300 x 106 labeled platelets, corresponding to 20% of the total endogenous platelet number, were infused into the circulation of an anesthetized mouse. PTP-1B/ and PTP-1B+/+ donor platelets were infused into PTP-1B/ or PTP-1B+/+ recipient mice as indicated in each particular experiment. Vascular injury was induced 10 min after platelet infusion. Real-time multichannel intravital microscopy was used to monitor two fluorescence channels and one brightfield channel almost simultaneously. The accumulation of labeled platelets within a developing thrombus was monitored at 510 nm after excitation at 380 nm, and calcium mobilization within those platelets was monitored at 510 nm after excitation at 340 nm. Mouse tail bleeding times and the occurrence of rebleeding from tail wounds were assessed as described previously (Law et al., 1999a).
Online supplemental material
Videos show thrombus formation in a cremasteric arteriole of a living PTP-1B+/+ (Video 1) or PTP-1B/ (Video 2) mouse at three frames/s for 3 min. PTP-1B+/+ and PTP-1B/ platelets were labeled with Fura 2, an arteriole in a recipient cremaster muscle was subjected to laser injury, and the accumulation of fluorescent platelets into the developing thrombus was assessed as described above and in Fig. 6 a. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200503125/DC1.
![]() |
Acknowledgments |
---|
This work was supported by grants DK60838, HL56595, HL57900, HL77645, and HL77817 from the National Institutes of Health.
Submitted: 23 March 2005
Accepted: 26 July 2005
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Arias-Salgado, E.G., S. Lizano, S. Sarker, J.S. Brugge, M.H. Ginsberg, and S.J. Shattil. 2003. Src kinase activation by a novel and direct interaction with the integrin ß cytoplasmic domain. Proc. Natl. Acad. Sci. USA. 100:1329813302.
Arregui, C.O., J. Balsamo, and J. Lilien. 1998. Impaired integrin-mediated adhesion and signaling in fibroblasts expressing a dominant-negative mutant PTP1B. J. Cell Biol. 143:861873.
Brunton, V.G., I.R.J. MacPherson, and M.C. Frame. 2004. Cell adhesion receptors, tyrosine kinases and actin modulators: a complex three-way circuitry. Biochim. Biophys. Acta. 1692:121144.[Medline]
Buensuceso, C., M. De Virgilio, and S.J. Shattil. 2003. Detection of integrin IIbß3 clustering in living cells. J. Biol. Chem. 278:1521715224.
Byzova, T.V., R. Rabbani, S. D'Souza, and E.F. Plow. 1998. Role of integrin Vß3 in vascular biology. Thromb. Haemost. 80:726734.[Medline]
Calderwood, D.A., Y. Fujioka, J.M. de Pereda, B. Garcia-Alvarez, T. Nakamoto, B. Margolis, C.J. McGlade, R.C. Liddington, and M.H. Ginsberg. 2003. Integrin beta cytoplasmic domain interactions with phosphotyrosine-binding domains: a structural prototype for diversity in integrin signaling. Proc. Natl. Acad. Sci. USA. 100:22722277.
Cheng, A., G.S. Bal, B.P. Kennedy, and M.L. Tremblay. 2001. Attenuation of adhesion-dependent signaling and cell spreading in transformed fibroblasts lacking protein tyrosine phosphatase-1B. J. Biol. Chem. 276:2584825855.
Cheng, A., N. Uetani, P.D. Simoncic, V.P. Chaubey, A. Lee-Loy, C.J. McGlade, B.P. Kennedy, and M.L. Tremblay. 2002. Attenuation of leptin action and regulation of obesity by protein tyrosine phosphatase 1B. Dev. Cell. 2:497503.[CrossRef][Medline]
Dadke, S., and J. Chernoff. 2002. Interaction of protein tyrosine phosphatase (PTP) 1B with its substrates is influenced by two distinct binding domains. Biochem. J. 364:377383.[CrossRef][Medline]
de Virgilio, M., W.B. Kiosses, and S.J. Shattil. 2004. Proximal, selective and dynamic interactions between integrin IIbß3 and protein tyrosine kinases in living cells. J. Cell Biol. 165:305311.
Dube, N., A. Cheng, and M.L. Tremblay. 2004. The role of protein tyrosine phosphatase 1B in Ras signaling. Proc. Natl. Acad. Sci. USA. 101:18341839.
Elchebly, M., P. Payette, E. Michaliszyn, W. Cromlish, S. Collins, A.L. Loy, D. Normandin, A. Cheng, J. Himms-Hagen, C.C. Chan, et al. 1999. Increased insulin sensitivity and obesity resistance in mice lacking the protein tyrosine phosphatase-1B gene. Science. 283:15441548.
Falati, S., P. Gross, G. Merrill-Skoloff, B.C. Furie, and B. Furie. 2002. Real-time in vivo imaging of platelets, tissue factor and fibrin during arterial thrombus formation in the mouse. Nat. Med. 8:11751181.[CrossRef][Medline]
Faruqi, T.R., E.J. Weiss, M.J. Shapiro, W. Huang, and S.R. Coughlin. 2000. Structure-function analysis of protease-activated receptor 4 tethered ligand peptides. Determinants of specificity and utility in assays of receptor function. J. Biol. Chem. 275:1972819734.
Feng, X., D.V. Novack, R. Faccio, D.S. Ory, K. Aya, M.I. Boyer, K.P. McHugh, F.P. Ross, and S.L. Teitelbaum. 2001. A Glanzmann's mutation in beta 3 integrin specifically impairs osteoclast function. J. Clin. Invest. 107:11371144.
Flint, A.J., T. Tiganis, D. Barford, and N.K. Tonks. 1997. Development of "substrate-trapping" mutants to identify physiological substrates of protein tyrosine phosphatases. Proc. Natl. Acad. Sci. USA. 94:16801685.
Frangioni, J.V., A. Oda, M. Smith, E.W. Salzman, and B.G. Neel. 1993. Calpain-catalyzed cleavage and subcellular relocation of protein phosphotyrosine phosphatase 1B (PTP-1B) in human platelets. EMBO J. 12:48434856[Abstract]
Garcia, A., S. Prabhakar, S. Hughan, T.W. Anderson, C.J. Brock, A.C. Pearce, R.A. Dwek, S.P. Watson, H.F. Hebestreit, and N. Zitzmann. 2004. Differential proteome analysis of TRAP-activated platelets: involvement of DOK-2 and phosphorylation of RGS proteins. Blood. 103:20882095.
Haimovich, B., L. Lipfert, J.S. Brugge, and S.J. Shattil. 1993. Tyrosine phosphorylation and cytoskeletal reorganization in platelets are triggered by interaction of integrin receptors with their immobilized ligands. J. Biol. Chem. 268:1586815877.
Harrison, S.C. 2003. Variation on a Src-like theme. Cell. 112:737740.[CrossRef][Medline]
Hodivala-Dilke, K.M., K.P. McHugh, D.A. Tsakiris, H. Rayburn, D. Crowley, M. Ullman-Cullere, F.P. Ross, B.S. Coller, S. Teitelbaum, and R.O. Hynes. 1999. Beta3-integrin-deficient mice are a model for Glanzmann thrombasthenia showing placental defects and reduced survival. J. Clin. Invest. 103:229238.
Hooft van Huijsduijnen, R., W.H. Sauer, A. Bombrun, and D. Swinnen. 2004. Prospects for inhibitors of protein tyrosine phosphatase 1B as antidiabetic drugs. J. Med. Chem. 47:41424146.[CrossRef][Medline]
Hughes, P.E., T.E. O'Toole, J. Ylanne, S.J. Shattil, and M.H. Ginsberg. 1995. The conserved membrane-proximal region of an integrin cytoplasmic domain specifies ligand-binding affinity. J. Biol. Chem. 270:1241112417.
Hynes, R. 2002. Integrins: bidirectional, allosteric signaling machines. Cell. 110:673687.[CrossRef][Medline]
Jackson, S.P., W.S. Nesbitt, and S. Kulkarni. 2003. Signaling events underlying thrombus formation. J. Thromb. Haemost. 1:16021612.[CrossRef][Medline]
Juliano, R.L., P. Reddig, S. Alahari, M. Edin, A. Howe, and A. Aplin. 2004. Integrin regulation of cell signalling and motility. Biochem. Soc. Trans. 32:443446.[CrossRef][Medline]
Jung, E.J., Y.S. Kang, and C.W. Kim. 1998. Multiple phosphorylation of chicken protein tyrosine phosphatase 1 and human protein tyrosine phosphatase 1B by casein kinase II and p60c-src in vitro. Biochem. Biophys. Res. Commun. 246:238242.[CrossRef][Medline]
Klaman, L.D., O. Boss, O.D. Peroni, J.K. Kim, J.L. Martino, J.M. Zabolotny, N. Moghal, M. Lubkin, Y.B. Kim, A.H. Sharpe, et al. 2000. Increased energy expenditure, decreased adiposity, and tissue-specific insulin sensitivity in protein-tyrosine phosphatase 1B-deficient mice. Mol. Cell. Biol. 20:54795489.
Klinghoffer, R.A., C. Sachsenmaier, J.A. Cooper, and P. Soriano. 1999. Src family kinases are required for integrin but not PDGFR signal transduction. EMBO J. 18:24592471.
Law, D.A., F.R. DeGuzman, P. Heiser, K. Ministri-Madrid, N. Killeen, and D.R. Phillips. 1999a. Integrin cytoplasmic tyrosine motif is required for outside-in aIIbb3 signalling and platelet function. Nature. 401:808811.[CrossRef][Medline]
Law, D.A., L. Nannizzi-Alaimo, K. Ministri, P. Hughes, J. Forsyth, M. Turner, S.J. Shattil, M.H. Ginsberg, V. Tybulewicz, and D.R. Phillips. 1999b. Genetic and pharmacological analyses of Syk function in aIIbb3 signaling in platelets. Blood. 93:26452652.
Lin, S.Y., S. Raval, Z. Zhang, M. Deverill, K.A. Siminovitch, D.R. Branch, and B. Haimovich. 2004. The protein-tyrosine phosphatase SHP-1 regulates the phosphorylation of alpha-actinin. J. Biol. Chem. 279:2575525764.
Litvinov, R.I., C. Nagaswami, G. Vilaire, H. Shuman, J.S. Bennett, and J.W. Weisel. 2004. Functional and structural correlations of individual IIbß3 molecules. Blood. 104:39793985.
Liu, F., M.A. Sells, and J. Chernoff. 1998. Protein tyrosine phosphatase 1B negatively regulates integrin signaling. Curr. Biol. 8:173176.[CrossRef][Medline]
Lowell, C.A. 2004. Src-family kinases: rheostats of immune cell signaling. Mol. Immunol. 41:631643.[CrossRef][Medline]
Mazzucato, M., P. Pradella, M.R. Cozzi, L. De Marco, and Z.M. Ruggeri. 2002. Sequential cytoplasmic calcium signals in a 2-stage platelet activation process induced by the glycoprotein Ibalpha mechanoreceptor. Blood. 100:27932800.
McHugh, K.P., K. Hodivala-Dilke, M.H. Zheng, N. Namba, J. Lam, D. Novack, X. Feng, F.P. Ross, R.O. Hynes, and S.L. Teitelbaum. 2000. Mice lacking ß3 integrins are osteosclerotic because of dysfunctional osteoclasts. J. Clin. Invest. 105:433440.
Neel, B.G., H. Gu, and L. Pao. 2003. The Shping news: SH2 domain-containing tyrosine phosphatases in cell signaling. Trends Biochem. Sci. 28:284293.[CrossRef][Medline]
Nesbitt, W.S., S. Kulkarni, S. Giuliano, I. Goncalves, S.M. Dopheide, C.L. Yap, I.S. Harper, H.H. Salem, and S.P. Jackson. 2002. Distinct glycoprotein Ib/V/IX and integrin IIbß3-dependent calcium signals cooperatively regulate platelet adhesion under flow. J. Biol. Chem. 277:29652972.
Obergfell, A., K. Eto, A. Mocsai, C. Buensuceso, S.L. Moores, J.S. Brugge, C.A. Lowell, and S.J. Shattil. 2002. Coordinate interactions of Csk, Src, and Syk kinases with IIbß3 initiate integrin signaling to the cytoskeleton. J. Cell Biol. 157:265275.
Oh, E.S., H.H. Gu, T.M. Saxton, J.F. Timms, S. Hausdorff, E.U. Frevert, B.B. Kahn, T. Pawson, B.G. Neel, and S.M. Thomas. 1999. Regulation of early events in integrin signaling by protein tyrosine phosphatase SHP-2. Mol. Cell. Biol. 19:32053215.
Okada, M., S. Nada, Y. Yamanashi, T. Yamamoto, and H. Nakagawa. 1991. CSK: a protein-tyrosine kinase involved in regulation of src family kinases. J. Biol. Chem. 266:2424924252.
Phillips, D.R., K.S. Prasad, J. Manganello, M. Bao, and L. Nannizzi-Alaimo. 2001. Integrin tyrosine phosphorylation in platelet signaling. Curr. Opin. Cell Biol. 13:546554.[CrossRef][Medline]
Ragab, A., S. Bodin, C. Viala, H. Chap, B. Payrastre, and J. Ragab-Thomas. 2003. The tyrosine phosphatase 1B regulates linker for activation of T-cell phosphorylation and platelet aggregation upon FcgammaRIIa cross-linking. J. Biol. Chem. 278:4092340932.
Ruggeri, Z.M. 2002. Platelets in atherothrombosis. Nat. Med. 8:12271234.[CrossRef][Medline]
Sells, M.A., and J. Chernoff. 1995. Epitope-tag vectors for eukaryotic protein production. Gene. 152:187189.[CrossRef][Medline]
Shah, K., and K.M. Shokat. 2002. A chemical genetic screen for direct v-Src substrates reveals ordered assembly of a retrograde signaling pathway. Chem. Biol. 9:3547.[CrossRef][Medline]
Shattil, S.J., and P.J. Newman. 2004. Integrins: dynamic scaffolds for adhesion and signaling in platelets. Blood. 104:16061615.
Sicheri, F., and J. Kuriyan. 1997. Structures of Src-family tyrosine kinases. Curr. Opin. Struct. Biol. 7:777785.[CrossRef][Medline]
Simmons, S.R., P.A. Sims, and R.M. Albrecht. 1997. IIbß3 redistribution triggered by receptor cross-linking. Arterioscler. Thromb. Vasc. Biol. 17:33113320.
Soriano, P., C. Montgomery, R. Geske, and A. Bradley. 1991. Targeted disruption of the c-src proto-oncogene leads to osteopetrosis in mice. Cell. 64:693702.[CrossRef][Medline]
Su, J., M. Muranjan, and J. Sap. 1999. Receptor protein tyrosine phosphatase activates Src-family kinases and controls integrin-mediated responses in fibroblasts. Curr. Biol. 9:505511.[CrossRef][Medline]
Suen, P.W., D. Ilic, E. Caveggion, G. Berton, C.H. Damsky, and C.A. Lowell. 1999. Impaired integrin-mediated signal transduction, altered cytoskeletal structure and reduced motility in Hck/Fgr deficient macrophages. J. Cell Sci. 112:40674078.
Thomas, S.M., M. Hagel, and C.E. Turner. 1999. Characterization of a focal adhesion protein, Hic-5, that shares extensive homotogy with paxillin. J. Cell Sci. 112:181190.
Timms, J.F., K.D. Swanson, A. Marie-Cardine, M. Raab, C.E. Rudd, B. Schraven, and B.G. Neel. 1999. SHPS-1 is a scaffold for assembling distinct adhesion-regulated multi-protein complexes in macrophages. Curr. Biol. 9:927930.[CrossRef][Medline]
Tonks, N.K. 2003. PTP1B: from the sidelines to the front lines! FEBS Lett. 546:140148.[CrossRef][Medline]
Wonerow, P., A. Obergfell, J.I. Wilde, R. Bobe, N. Asazuma, T. Brdicka, A. Leo, B. Schraven, V. Horejsi, S.J. Shattil, and S.P. Watson. 2002. Differential role of glycolipid-enriched membrane domains in glycoprotein VI- and integrin-mediated phospholipase Cgamma2 regulation in platelets. Biochem. J. 364:755765.[CrossRef][Medline]
Wonerow, P., A.C. Pearce, D.J. Vaux, and S.P. Watson. 2003. A critical role for phospholipase Cgamma2 in IIbß3-mediated platelet spreading. J. Biol. Chem. 278:3752037529.
Young, M.A., S. Gonfloni, G. Superti-Furga, B. Roux, and J. Kuriyan. 2001. Dynamic coupling between the SH2 and SH3 domains of c-Src and Hck underlies their inactivation by C-terminal tyrosine phosphorylation. Cell. 105:115126.[CrossRef][Medline]
Zabolotny, J.M., K.K. Bence-Hanulec, A. Stricker-Krongrad, F. Haj, Y. Wang, Y. Minokoshi, Y.B. Kim, J.K. Elmquist, L.A. Tartaglia, B.B. Kahn, and B.G. Neel. 2002. PTP1B regulates leptin signal transduction in vivo. Dev. Cell. 2:489495.[CrossRef][Medline]
Zhang, S.Q., W. Yang, M.I. Kontaridis, T.G. Bivona, G. Wen, T. Araki, J. Luo, J.A. Thompson, B.L. Schraven, M.R. Philips, and B.G. Neel. 2004. Shp2 regulates SRC family kinase activity and Ras/Erk activation by controlling Csk recruitment. Mol. Cell. 13:341355.[CrossRef][Medline]