The Y’s that bind: negative regulators of Src family kinase activity in platelets
2009; Elsevier BV; Volume: 7; Linguagem: Inglês
10.1111/j.1538-7836.2009.03369.x
ISSN1538-7933
Autores Tópico(s)Eosinophilic Disorders and Syndromes
ResumoSummaryMembers of the Src family of protein tyrosine kinases play important roles in platelet adhesion, activation, and aggregation. The purpose of this review is to summarize current knowledge regarding how Src family kinase activity is regulated in general, to describe what is known about mechanisms underlying SFK activation in platelets, and to discuss platelet proteins that contribute to SFK inactivation, particularly those that use phosphotyrosine-containing sequences to recruit phosphatases and kinases to sites of SFK activity. Members of the Src family of protein tyrosine kinases play important roles in platelet adhesion, activation, and aggregation. The purpose of this review is to summarize current knowledge regarding how Src family kinase activity is regulated in general, to describe what is known about mechanisms underlying SFK activation in platelets, and to discuss platelet proteins that contribute to SFK inactivation, particularly those that use phosphotyrosine-containing sequences to recruit phosphatases and kinases to sites of SFK activity. All SFKs share a similar modular domain structure (Fig. 1A) that comprises, in order, from the amino (N)- to the carboxy (C)-terminus: (i) a unique N-terminal sequence, also referred to as the Src homology (SH) 4 domain, that contains myristoylation and sometimes also palmitoylation sites that influence sub-cellular localization, (ii) the SH3 domain that binds proline-rich sequences, (iii) a short connector that joins the SH3 and the SH2 domains, (iv) the SH2 domain that binds phosphotyrosine (pY)-containing sequences, (v) the SH2-kinase linker sequence, (vi) a kinase, or SH1, domain that is responsible for enzymatic activity and is comprised of a smaller N-terminal catalytic lobe that is connected to a larger C-terminal regulatory lobe by a flexible activation loop that contains a regulatory tyrosine (Y) residue (Y416in human Src), and (vii) a C-terminal tail that also contains a regulatory Y residue (Y527in human Src) [1Sicheri F. Kuriyan J. Structures of Src-family tyrosine kinases.Curr Opin Struct Biol. 1997; 7: 777-85Crossref PubMed Scopus (329) Google Scholar]. Maintenance of SFKs in an inactive conformation requires synergy amongst a number of intra-molecular interactions that involve the SH3 and SH2 domains [2Brown M.T. Cooper J.A. Regulation, substrates and functions of Src.Biochim Biophys Acta. 1996; 1287: 121-49Crossref PubMed Scopus (1080) Google Scholar, 3Ingley E. Src family kinases: regulation of their activities, levels and identification of new pathways.Biochim Biophys Acta. 2008; 1784: 56-65Crossref PubMed Scopus (252) Google Scholar]. In fully assembled, inactive SFKs (Fig. 1A), the ligand-binding surface of the SH3 domain is occupied by a polyproline type II-like sequence within the SH2-kinase linker, whereas the SH2 domain is occupied by a phosphorylated tyrosine residue located in the C-terminal tail. In addition, the SH3 domain and SH2-kinase linker of inactive SFKs contact residues in the N lobe of the kinase domain, whereas the SH2 domain interacts with residues of C lobe. Together, these interactions inhibit kinase activity by forcing the N and C lobes of the kinase domain together, which displaces the activation loop of the N lobe away from the position it occupies in active kinases and leads to rearrangement of residues that would otherwise participate in catalysis. To paraphrase Anna Karenina, whereas all inactiveSFKs are alike, activekinases can be active in different ways. Fully active, fully disassembled SFKs (Fig. 1B) are characterized by: (i) disruption of intra-molecular interactions in which both the SH3 and SH2 domains participate, (ii) the presence of a phosphotyrosine (pY) residue within the activation loop of the kinase domain (which arises as a consequence of autophosphorylation), and (iii) the presence of an unphosphorylated Y residue within the C-terminal tail of the enzyme [2Brown M.T. Cooper J.A. Regulation, substrates and functions of Src.Biochim Biophys Acta. 1996; 1287: 121-49Crossref PubMed Scopus (1080) Google Scholar, 3Ingley E. Src family kinases: regulation of their activities, levels and identification of new pathways.Biochim Biophys Acta. 2008; 1784: 56-65Crossref PubMed Scopus (252) Google Scholar]. However, SFKs need not be fully disassembled to be active. Thus, dissociation of the SH3 domain from its inhibitory intra-molecular interactions is alone sufficient to activate an SFK. Such dissociation can be driven by artificial or naturally occurring mutations, within either the SH3 domain, the SH2-kinase linker, or the kinase domain [2Brown M.T. Cooper J.A. Regulation, substrates and functions of Src.Biochim Biophys Acta. 1996; 1287: 121-49Crossref PubMed Scopus (1080) Google Scholar, 3Ingley E. Src family kinases: regulation of their activities, levels and identification of new pathways.Biochim Biophys Acta. 2008; 1784: 56-65Crossref PubMed Scopus (252) Google Scholar]. In addition, because the proline-rich sequence in the SH2-kinase linker (PX4PX12P) binds to the SH3 domain with low affinity, it can be displaced by inter-molecular interaction of the SH3 domain with higher affinity proline-rich sequences (e.g., PXXPXR) in other proteins (Fig. 1C) [2Brown M.T. Cooper J.A. Regulation, substrates and functions of Src.Biochim Biophys Acta. 1996; 1287: 121-49Crossref PubMed Scopus (1080) Google Scholar, 3Ingley E. Src family kinases: regulation of their activities, levels and identification of new pathways.Biochim Biophys Acta. 2008; 1784: 56-65Crossref PubMed Scopus (252) Google Scholar]. Interestingly, SFKs that are activated by disruption of their SH3 domain-dependent intra-molecular interactions may retain the intra-molecular interaction between the C-terminal pY residue and the SH2 domain, because the latter interaction is required for inhibition, but need not be disrupted for activation, of the SFK [4Lerner E.C. Smithgall T.E. SH3-dependent stimulation of Src-family kinase autophosphorylation without tail release from the SH2 domain in vivo.Nat Struct Biol. 2002; 9: 365-9PubMed Google Scholar]. Dissociation of the SH2 domain from its intra-molecular interaction with the inhibitory C-terminal pY residue, which is accompanied by dissociation of the SH3 domain from its intra-molecular interactions [5Ayrapetov M.K. Wang Y.H. Lin X. Gu X. Parang K. Sun G. Conformational basis for SH2-Tyr(P)527 binding in Src inactivation.J Biol Chem. 2006; 281: 23776-84Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar], is also sufficient to activate SFKs (Fig. 1D). Although such dissociation has been artificially driven by substitution of the C-terminal Y residue with phenylalanine [6Hunter T. A tail of two src's: mutatis mutandis.Cell. 1987; 49: 1-4Abstract Full Text PDF PubMed Google Scholar] or by elimination of the C-terminal tail of the enzyme altogether [7Takeya T. Hanafusa H. Structure and sequence of the cellular gene homologous to the RSV src gene and the mechanism for generating the transforming virus.Cell. 1983; 32: 881-90Abstract Full Text PDF PubMed Scopus (298) Google Scholar], this interaction can be disrupted upon dephosphorylation of the C-terminal pY residue [Fig. 1D(i)], which can be accomplished by a number of different phosphatases, including proline-enriched tyrosine phosphatase (PEP), T-cell protein tyrosine phosphatase (TCPTP), tandem SH2 domain-containing protein tyrosine phosphatase-1 (SHP-1), and CD45 [3Ingley E. Src family kinases: regulation of their activities, levels and identification of new pathways.Biochim Biophys Acta. 2008; 1784: 56-65Crossref PubMed Scopus (252) Google Scholar]. In addition, as is the case with the SH3 domain, the pY-containing sequence in the C-terminal tail (pYQPG, pYQQQ, or pYQPQ) binds to the SH2 domain with relatively low affinity, and can be easily overcome by pY-containing sequences (e.g. pYEEI) in other proteins for which the SH2 domain has higher affinity [Fig. 1D(ii)] [2Brown M.T. Cooper J.A. Regulation, substrates and functions of Src.Biochim Biophys Acta. 1996; 1287: 121-49Crossref PubMed Scopus (1080) Google Scholar, 3Ingley E. Src family kinases: regulation of their activities, levels and identification of new pathways.Biochim Biophys Acta. 2008; 1784: 56-65Crossref PubMed Scopus (252) Google Scholar]. In this latter case, the active SFK may, but need not, be dephosphorylated on its C-terminal Y residue [8Nika K. Tautz L. Arimura Y. Vang T. Williams S. Mustelin T. A weak Lck tail bite is necessary for Lck function in T cell antigen receptor signaling.J Biol Chem. 2007; 282: 36000-9Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar]. In this context, it is clear that a complete understanding of how SFKs regulate platelet activation requires knowledge at several levels. First, it is necessary to identify the SFKs that are involved in important platelet activation pathways. Second, for each of these pathways, the types of inter-molecular interactions that support binding of the SFKs to the receptors that initiate these pathways must be characterized. For those pathways that involve SH2 domain-dependent interactions with pY-containing sequences in other proteins, the phosphatases that dephosphorylate those proteins and the adaptor molecules that recruit relevant phosphatases to the site of SFK activation have to be identified. Finally, for those pathways that require re-phosphorylation of the SFK C-terminal inhibitory Y residue, it is necessary to identify the kinase responsible and define how it gets to the site of SFK activation. Platelets express at least six SFKs, including Fgr, Fyn, Lck, Lyn, Src, and Yes [9Pestina T.I. Stenberg P.E. Druker B.J. Steward S.A. Hutson N.K. Barrie R.J. Jackson C.W. Identification of the Src family kinases, Lck and Fgr in platelets. Their tyrosine phosphorylation status and subcellular distribution compared with other Src family members.Arterioscler Thromb Vasc Biol. 1997; 17: 3278-85Crossref PubMed Google Scholar, 10Stenberg P.E. Pestina T.I. Barrie R.J. Jackson C.W. The Src family kinases, Fgr, Fyn, Lck, and Lyn, colocalize with coated membranes in platelets.Blood. 1997; 89: 2384-93Crossref PubMed Google Scholar]. Fyn and Lyn are involved in activation of platelets following binding of collagen or laminin to the platelet-specific GPVI/Fc receptor (FcR) γ-chain complex [11Ezumi Y. Shindoh K. Tsuji M. Takayama H. Physical and functional association of the Src family kinases Fyn and Lyn with the collagen receptor glycoprotein VI-Fc receptor g chain complex on human platelets.J Exp Med. 1998; 188: 267-76Crossref PubMed Scopus (0) Google Scholar]. The SH3 domains of Fyn and Lyn both associate with a proline-rich sequence in the cytoplasmic domain of the GPVI subunit [12Suzuki-Inoue K. Tulasne D. Shen Y. Bori-Sanz T. Inoue O. Jung S.M. Moroi M. Andrews R.K. Berndt M.C. Watson S.P. Association of Fyn and Lyn with the proline-rich domain of glycoprotein VI regulates intracellular signaling.J Biol Chem. 2002; 277: 21561-6Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar]. Activation of Fyn and Lyn following ligand binding to GPVI initiates a signal transduction pathway that involves phosphorylation of an immunoreceptor tyrosine-based activation motif (ITAM) in the FcR γ-chain, and recruitment of the tyrosine kinase, Syk, to the phosphorylated ITAM [13Watson S.P. Auger J.M. McCarty O.J. Pearce A.C. GPVI and integrin aIIb b3 signaling in platelets.J Thromb Haemost. 2005; 3: 1752-62Crossref PubMed Scopus (0) Google Scholar, 14Gibbins J.M. Platelet adhesion signalling and the regulation of thrombus formation.J Cell Sci. 2004; 117: 3415-25Crossref PubMed Scopus (236) Google Scholar]. Subsequent phosphorylation of adaptor molecules results in recruitment and activation of other enzymes that ultimately lead to generation of second messengers required for platelet granule release and cytoskeletal rearrangement [15Ragab A. Severin S. Gratacap M.P. Aguado E. Malissen M. Jandrot-Perrus M. Malissen B. Ragab-Thomas J. Payrastre B. Roles of the C-terminal tyrosine residues of LAT in GPVI-induced platelet activation: insights into the mechanism of PLC gamma 2 activation.Blood. 2007; 110: 2466-74Crossref PubMed Scopus (0) Google Scholar]. Fyn and Lyn have been found to co-precipitate with the FcR γ-chain following GPVI-mediated platelet activation [16Briddon S.J. Watson S.P. Evidence for the involvement of p59fyn and p53/56lyn in collagen receptor signalling in human platelets.Biochem J. 1999; 338: 203-9Crossref PubMed Scopus (82) Google Scholar]; however, it is not known whether the SH2 domains of Fyn and Lyn are involved in these interactions and, if so, whether such interactions stabilize the kinases in active conformations. Down-regulation of the GPVI/FcR γ-chain complex has been observed following activation of both mouse and human platelets [17Rabie T. Varga-Szabo D. Bender M. Pozgaj R. Lanza F. Saito T. Watson S.P. Nieswandt B. Diverging signaling events control the pathway of GPVI downregulation in vivo.Blood. 2007; 110: 529-35Crossref PubMed Scopus (0) Google Scholar, 18Boylan B. Berndt M.C. Kahn M.L. Newman P.J. Activation-independent, antibody-mediated removal of GPVI from circulating human platelets: development of a novel NOD/SCID mouse model to evaluate the in vivoeffectiveness of anti-human platelet agents.Blood. 2006; 108: 908-14Crossref PubMed Scopus (0) Google Scholar]. Whether such receptor down-regulation plays a role in inactivation of Fyn and Lyn following GPVI-mediated platelet activation remains to be determined. Another important platelet activation pathway that involves SFKs, specifically Src itself and Fyn, is that of outside-in signaling by the platelet-specific integrin, αIIbβ3[19Obergfell A. Eto K. Mocsai A. Buensuceso C. Moores S.L. Brugge J.S. Lowell C.A. Shattil S.J. Coordinate interactions of Csk, Src, and Syk kinases with aIIbb3 initiate integrin signaling to the cytoskeleton.J Cell Biol. 2002; 157: 265-75Crossref PubMed Scopus (0) Google Scholar, 20Reddy K.B. Smith D.M. Plow E.F. Analysis of Fyn function in hemostasis and aIIbb3-integrin signaling.J Cell Sci. 2008; 121: 1641-8Crossref PubMed Scopus (0) Google Scholar]. Src has been reported to associate with an arginine–glycine–threonine sequence at the extreme C-terminus of the integrin β3subunit [21Arias-Salgado E.G. Lizano S. Shattil S.J. Ginsberg M.H. Specification of the direction of adhesive signaling by the integrin b cytoplasmic domain.J Biol Chem. 2005; 280: 29699-707Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar], whereas Fyn is thought to bind to a sequence in β3that is nearer the inner face of the plasma membrane [20Reddy K.B. Smith D.M. Plow E.F. Analysis of Fyn function in hemostasis and aIIbb3-integrin signaling.J Cell Sci. 2008; 121: 1641-8Crossref PubMed Scopus (0) Google Scholar]. SFK activation following ligation of αIIbβ3on human platelets initiates a signal transduction pathway very similar to that of the GPVI/FcR γ-chain complex [22Shattil S.J. Integrins and Src: dynamic duo of adhesion signaling.Trends Cell Biol. 2005; 15: 399-403Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar], except that it begins with phosphorylation of an ITAM in the cytoplasmic domain of FcγRIIA [23Boylan B. Gao C. Rathore V. Gill J.C. Newman D.K. Newman P.J. Identification of FcgRIIa as the ITAM-bearing receptor mediating aIIbb3 outside-in integrin signaling in human platelets.Blood. 2008; 112: 2780-6Crossref PubMed Scopus (0) Google Scholar]. Additional studies are needed to determine the extent to which the SH3 and/or SH2 domains of Src and Fyn are involved in their interactions with αIIbβ3and components of the αIIbβ3signal transduction pathway, whether such interactions contribute to activation of these SFKs, and how the activity of these enzymes is controlled following αIIbβ3engagement. Finally, SFKs, Lyn in particular [24Yin H. Liu J. Li Z. Berndt M.C. Lowell C.A. Du X. Src family tyrosine kinase Lyn mediates VWF/GPIb-IX-induced platelet activation via the cGMP signaling pathway.Blood. 2008; 112: 1139-46Crossref PubMed Scopus (88) Google Scholar], have also been shown to be involved in activation of platelets by the GPIb/V/IX receptor for von Willebrand Factor [25Du X. Signaling and regulation of the platelet glycoprotein Ib-IX-V complex.Curr Opin Hematol. 2007; 14: 262-9Crossref PubMed Scopus (109) Google Scholar]. 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New roles for an old molecule in platelet and vascular cell biology.Arterioscler Thromb Vasc Biol. 2003; 23: 953-64Crossref PubMed Scopus (0) Google Scholar]. PECAM-1 deficiency in mice results in platelet hyper-responsiveness, especially to GPVI-specific stimuli [27Patil S. Newman D.K. Newman P.J. PECAM-1 serves as an inhibitory receptor that modulates platelet responses to collagen.Blood. 2000; 96: 446aGoogle Scholar, 28Falati S. Patil S. Gross P.L. Stapleton M. Merrill-Skoloff G. Barrett N.E. Pixton K.L. Weiler H. Cooley B. Newman D.K. Newman P.J. Furie B.C. Furie B. Gibbins J.M. Platelet PECAM-1 inhibits thrombus formation in vivo.Blood. 2006; 107: 535-41Crossref PubMed Scopus (161) Google Scholar]. Recently, Carcinoembryonic Antigen-related Cell Adhesion Molecule-1 (CEACAM-1), which possesses cytoplasmic ITIMs that support recruitment and activation of SHP-1, has been shown to behave similarly to PECAM-1 in that its deficiency in mice also results in platelet hyper-responsiveness to GPVI-specific agonists [29Wong C. Liu Y. Yip J. Chand R. Wee J.L. Oates L. Nieswandt B. Reheman A. Ni H. Beauchemin N. Jackson D.E. CEACAM1 negatively regulates platelet-collagen interactions and thrombus growth in vitroand in vivo.Blood. 2009; 113: 1818-28Crossref PubMed Scopus (0) Google Scholar]. Platelets also express additional ITIM-containing molecules, including G6b-B [30Senis Y.A. Tomlinson M.G. Garcia A. Dumon S. Heath V.L. Herbert J. Cobbold S.P. Spalton J.C. Ayman S. Antrobus R. Zitzmann N. Bicknell R. Frampton J. Authi K.S. Martin A. Wakelam M.J. Watson S.P. A comprehensive proteomics and genomics analysis reveals novel transmembrane proteins in human platelets and mouse megakaryocytes including G6b-B, a novel immunoreceptor tyrosine-based inhibitory motif protein.Mol Cell Proteomics. 2007; 6: 548-64Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar] and TREM-like Transcript-1 (TLT-1) [31Washington A.V. Schubert R.L. Quigley L. Disipio T. Feltz R. Cho E.H. McVicar D.W. A TREM family member, TLT-1, is found exclusively in the alpha-granules of megakaryocytes and platelets.Blood. 2004; 104: 1042-7Crossref PubMed Scopus (87) Google Scholar]. Although there is evidence that G6b-B can modulate GPVI signaling [32Newland S.A. Macaulay I.C. Floto A.R. De Vet E.C. Ouwehand W.H. Watkins N.A. Lyons P.A. Campbell D.R. The novel inhibitory receptor G6B is expressed on the surface of platelets and attenuates platelet function in vitro.Blood. 2007; 109: 4806-9Crossref PubMed Scopus (0) Google Scholar, 33Mori J. Pearce A.C. Spalton J.C. 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The steps involved in re-establishment of the intra-molecular interaction between the SFK SH2 domain and its C-terminal inhibitory pY residue have also not been completely delineated for any of the SFK-dependent platelet activation pathways. However, much is known about the kinases responsible for phosphorylation of the C-terminal Y residue, and also about the mechanism by which these kinases are activated at sites of SFK activity. The kinases involved include the aptly named C-terminal Src kinase (Csk) itself and its close relative, Csk-homologous kinase (Chk) [36Chong Y.P. Mulhern T.D. Cheng H.C. C-terminal Src kinase (CSK) and CSK-homologous kinase (CHK) – endogenous negative regulators of Src-family protein kinases.Growth Factors. 2005; 23: 233-44Crossref PubMed Scopus (0) Google Scholar]. Both of these enzymes are known to be expressed in platelets [19Obergfell A. Eto K. Mocsai A. Buensuceso C. Moores S.L. Brugge J.S. Lowell C.A. Shattil S.J. Coordinate interactions of Csk, Src, and Syk kinases with aIIbb3 initiate integrin signaling to the cytoskeleton.J Cell Biol. 2002; 157: 265-75Crossref PubMed Scopus (0) Google Scholar, 37Hirao A. Hamaguchi I. Suda T. Yamaguchi N. Translocation of the Csk homologous kinase (Chk/Hyl) controls activity of CD36-anchored Lyn tyrosine kinase in thrombin-stimulated platelets.EMBO J. 1997; 16: 2342-51Crossref PubMed Scopus (66) Google Scholar]. Csk and Chk have a domain organization similar to that of SFKs, except for the absence of both the N-terminal SH4 domain and a C-terminal inhibitory Y residue [36Chong Y.P. Mulhern T.D. Cheng H.C. C-terminal Src kinase (CSK) and CSK-homologous kinase (CHK) – endogenous negative regulators of Src-family protein kinases.Growth Factors. 2005; 23: 233-44Crossref PubMed Scopus (0) Google Scholar]. Consequently, Csk and Chk are cytoplasmic enzymes that are not regulated directly by tyrosine phosphorylation; instead, they are recruited to the membrane and activated upon binding of their SH2 domains to pY residues within Csk binding proteins, which are themselves phosphorylated by SFKs [36Chong Y.P. Mulhern T.D. Cheng H.C. C-terminal Src kinase (CSK) and CSK-homologous kinase (CHK) – endogenous negative regulators of Src-family protein kinases.Growth Factors. 2005; 23: 233-44Crossref PubMed Scopus (0) Google Scholar]. Although many molecules with Csk or Chk binding functions have been identified [38Chong Y.P. Ia K.K. Mulhern T.D. Cheng H.C. Endogenous and synthetic inhibitors of the Src-family protein tyrosine kinases.Biochim Biophys Acta. 2005; 1754: 210-20Crossref PubMed Scopus (87) Google Scholar], the only protein thus far shown to have such function in platelets is paxillin, which regulates Lyn activity downstream of αIIbβ3engagement [39Rathore V.B. Okada M. Newman P.J. Newman D.K. Paxillin family members function as Csk-binding proteins that regulate Lyn activity in human and murine platelets.Biochem J. 2007; 403: 275-81Crossref PubMed Scopus (0) Google Scholar]. Other well-characterized Csk binding proteins that are expressed in platelets include Csk binding protein (Cbp)/Phosphoprotein Associated with glycosphingolipid-enriched membrane microdomains (PAG) [40Wonerow P. Obergfell A. Wilde J.I. Bobe R. Asazuma N. Brdicka T. Leo A. Schraven B. Horejsi V. Shattil S.J. Watson S.P. Differential role of glycolipid-enriched membrane domains in glycoprotein VI- and integrin-mediated phospholipase Cg2 regulation in platelets.Biochem J. 2002; 364: 755-65Crossref PubMed Scopus (0) Google Scholar], caveolin-1 [41Jayachandran M. Miller V.M. Human platelets contain estrogen receptor alpha, caveolin-1 and estrogen receptor associated proteins.Platelets. 2003; 14: 75-81Crossref PubMed Scopus (0) Google Scholar], and insulin receptor substrate-1[42Ferreira I.A. Eybrechts K.L. Mocking A.I. Kroner C. Akkerman J.W. IRS-1 mediates inhibition of Ca2+ mobilization by insulin via the inhibitory G-protein Gi.J Biol Chem. 2004; 279: 3254-64Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar]. The extent to which these molecules function as Csk binding proteins in platelets and the stages of the platelet activation process at which they regulate SFK activity remains to be determined. In summary, although progress has been made toward developing a complete understanding of how SFK activity is regulated during platelet activation, much remains to be learned. Specifically, whereas the SFKs that are involved in platelet activation by GPVI and αIIbβ3have been identified, the types of inter-molecular interactions that maintain these SFKs in an active conformation downstream of receptor engagement have yet to be fully characterized. Similarly, whereas we have begun to identify the tyrosine phosphatases (and the scaffolding molecules that recruit these phosphatases to sites of SFK activity, where necessary) that are capable of interfering with SH2-dependent, SFK-activating interactions, the substrates that must be dephosphorylated to return SFKs to an inactive conformation remain to be identified. Finally, more information is needed regarding the inventory of molecules that can serve as Csk binding proteins in platelets, the extent to which these molecules recruit Csk vs. Chk to sites of SFK activity, and the identities of the pathways that require re-phosphorylation of the SFK C-terminal inhibitory Y residue to effect SFK inactivation. It is hoped that significant advances will be made in these areas in the coming years. The authors state that they have no conflict of interest. The author apologizes to all those whose contributions were not cited due to space limitations. This work was supported by R01 HL090883 from the Heart, Lung, and Blood Institute of the National Institutes of Health.
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