Targeting allosteric protein sites is a promising approach to interfere selectively with cellular signaling cascades. We have discovered a novel class of allosteric insulin-like growth factor-I receptor (IGF-1R) inhibitors. 3-Cyano-1H-indole-7-carboxylic acid {1-[4-(5-cyano-1H-indol-3-yl)butyl]piperidin-4-yl}amide (10) was found with nanomolar biochemical, micromolar, cellular IGF-1R activity and no relevant interference with cellular insulin receptor signaling up to 30 μM. The allosteric binding site was characterized by X-ray crystallographic studies, and the structural information was used to explain the unique mode of action of this new class of inhibitors.
Aberrant signaling through the fibroblast growth factor 19 (FGF19)/fibroblast growth factor receptor 4 (FGFR 4) signaling complex has been shown to cause hepatocellular carcinoma (HCC) in mice and has been implicated to play a similar role in humans. We have developed BLU9931, a potent and irreversible small-molecule inhibitor of FGFR4, as a targeted therapy to treat patients with HCC whose tumors have an activated FGFR4 signaling pathway. BLU9931 is exquisitely selective for FGFR4 versus other FGFR family members and all other kinases. BLU9931 shows remarkable antitumor activity in mice bearing an HCC tumor xenograft that overexpresses FGF19 due to amplification as well as a liver tumor xenograft that overexpresses FGF19 mRNA but lacks FGF19 amplification. Approximately one third of patients with HCC whose tumors express FGF19 together with FGFR4 and its coreceptor klotho β (KLB) could potentially respond to treatment with an FGFR4 inhibitor. These findings are the first demonstration of a therapeutic strategy that targets a subset of patients with HCC.This article documents the discovery of BLU9931, a novel irreversible kinase inhibitor that specifically targets FGFR4 while sparing all other FGFR paralogs and demonstrates exquisite kinome selectivity. BLU9931 is efficacious in tumors with an intact FGFR4 signaling pathway that includes FGF19, FGFR4, and KLB. BLU9931 is the first FGFR4-selective molecule for the treatment of patients with HCC with aberrant FGFR4 signaling.
There is extensive evidence to show that phosphatidylinositol 3-kinase plays an important role in signaling by the immune family of receptors, which has recently been extended to include the platelet collagen receptor, glycoprotein VI. In this report we present two potential mechanisms for the regulation of this enzyme on stimulation of platelets by collagen. We show that on stimulation with collagen, the regulatory subunit of phosphatidylinositol 3-kinase associates with the tyrosine-phosphorylated form of the adapter protein linker for activator of T Cells (LAT) and the tyrosine-phosphorylated immunoreceptor tyrosine-based activation motif of the Fc receptor γ-chain (a component of the collagen receptor complex that includes glycoprotein VI). The associations of the Fc receptor γ-chain and LAT with p85 are rapid and supported by the Src-homology 2 domains of the regulatory subunit. We did not obtain evidence to support previous observations that the regulatory subunit of phosphatidylinositol 3-kinase is regulated through association with the tyrosine kinase Syk. The present results provide a molecular basis for the regulation of the p85/110 form of phosphatidylinositol 3-kinase by GPVI, the collagen receptor that underlies activation. There is extensive evidence to show that phosphatidylinositol 3-kinase plays an important role in signaling by the immune family of receptors, which has recently been extended to include the platelet collagen receptor, glycoprotein VI. In this report we present two potential mechanisms for the regulation of this enzyme on stimulation of platelets by collagen. We show that on stimulation with collagen, the regulatory subunit of phosphatidylinositol 3-kinase associates with the tyrosine-phosphorylated form of the adapter protein linker for activator of T Cells (LAT) and the tyrosine-phosphorylated immunoreceptor tyrosine-based activation motif of the Fc receptor γ-chain (a component of the collagen receptor complex that includes glycoprotein VI). The associations of the Fc receptor γ-chain and LAT with p85 are rapid and supported by the Src-homology 2 domains of the regulatory subunit. We did not obtain evidence to support previous observations that the regulatory subunit of phosphatidylinositol 3-kinase is regulated through association with the tyrosine kinase Syk. The present results provide a molecular basis for the regulation of the p85/110 form of phosphatidylinositol 3-kinase by GPVI, the collagen receptor that underlies activation. platelet glycoprotein VI phosphatidylinositol 3-kinase immunoglobulin Fc receptor immunoreceptor tyrosine-based activation motif Src-homology 2 domain Src-homology 3 domain phospholipase Cγ2 low affinity receptor for immunoglobulin G polyacrylamide gel electrophoresis 5 hydroxytryptamine Tris-buffered saline-Tween 20 glutathione S-transferase linker for avtivator of T cells. Subendothelial collagens are primary platelet agonists and are thereby essential components of the hemostatic system. At sites of vascular damage, platelets adhere to exposed collagen fibers and undergo activation via a tyrosine kinase-dependent signaling pathway. Activation causes an increase in the binding capability of the fibrinogen receptor, integrin αIIbβ3, and the secretion of various mediators that culminate in the formation of an irreversible platelet aggregate, or hemostatic plug. The integrin α2β1 is expressed on the platelet surface and has been shown to support adhesion to collagen, although increasing evidence suggests that a second platelet collagen receptor underlies platelet activation.The collagen receptor that underlies activation comprises the uncharacterized platelet glycoprotein VI (GPVI)1 (1Gibbins J.M. Okuma M. Farndale R. Barnes M. Watson S.P. FEBS Lett. 1997; 413: 255-259Crossref PubMed Scopus (257) Google Scholar, 2Tsuji M. Ezumi Y. Arai M. Takayama H. J. Biol. Chem. 1997; 272: 23528-23531Crossref PubMed Scopus (241) Google Scholar), which is noncovalently associated with the Fc receptor (FcR) γ-chain (3Gibbins J. Asselin J. Farndale R. Barnes M. Law C.L. Watson S.P. J. Biol. Chem. 1996; 271: 18095-18099Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). Additional components may also exist. The FcR γ-chain is recognized for its role in the expression of, and signaling by, the high affinity receptor for IgE (FcεRI) (4Shiue L. Green J. Green O.M. Karas J.L. Morgenstern J.P. Ram M.K. Taylor M.K. Zoller M.J. Zydowsky L.D. Bolen J.B. Brugge J.S. Mol. Cell. Biol. 1995; 15: 272-281Crossref PubMed Google Scholar, 5Paolini R. Renard V. Vivier E. Ochiai K. Jouvin M.H. Malissen B. Kinet J.P. J. Exp. Med. 1995; 181: 247-255Crossref PubMed Scopus (39) Google Scholar, 6Jouvin M.H. Numerof R.P. Kinet J.P. Semin. Immunol. 1995; 7: 29-35Crossref PubMed Scopus (45) Google Scholar) and IgG (FcγRI) (7Duchemin A.-M. Ernst L.K. Anderson C.L. J. Biol. Chem. 1994; 269: 12111-12117Abstract Full Text PDF PubMed Google Scholar,8Greenberg S. Chang P. Silverstein S.C. J. Biol. Chem. 1994; 269: 3897-3902Abstract Full Text PDF PubMed Google Scholar) and the low affinity IgG receptor (FcγRIII) (8Greenberg S. Chang P. Silverstein S.C. J. Biol. Chem. 1994; 269: 3897-3902Abstract Full Text PDF PubMed Google Scholar). The FcR γ-chain is a transmembrane protein that is expressed as a homodimer. The cytoplasmic tail of the protein contains a consensus motif termed an immunoreceptor tyrosine-based activation motif (ITAM),which is defined as YXXLX (6–8)YXXL whereX represents any amino acid (9Reth M. Nature. 1989; 338: 383-384Crossref PubMed Scopus (1154) Google Scholar). This motif, which is also present in subunits of the T and B cell antigen receptors, becomes phosphorylated on the conserved tyrosine residues on ligand binding, enabling association of members of the Syk/Zap70 family of tyrosine kinases (3Gibbins J. Asselin J. Farndale R. Barnes M. Law C.L. Watson S.P. J. Biol. Chem. 1996; 271: 18095-18099Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 4Shiue L. Green J. Green O.M. Karas J.L. Morgenstern J.P. Ram M.K. Taylor M.K. Zoller M.J. Zydowsky L.D. Bolen J.B. Brugge J.S. Mol. Cell. Biol. 1995; 15: 272-281Crossref PubMed Google Scholar, 10Benhamou M. Ryba N.J. Kihara H. Nishikata H. Siraganian R.P. J. Biol. Chem. 1993; 268: 23318-23324Abstract Full Text PDF PubMed Google Scholar). Tyrosine phosphorylation of ITAMs has been demonstrated to involve the activity of Src-family kinases, and two recent reports indicate that this role is performed by Fyn and/or Lyn on stimulation of GPVI in platelets and megakaryocytes (11Melford S.K. Turner M. Briddon S.J. Tybulewicz V.L. Watson S.P. J. Biol. Chem. 1997; 272: 27539-27542Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 12Ezumi Y. Shindoh K. Tsuji M. Takayama H. J. Exp. Med. 1998; 188: 267-276Crossref PubMed Scopus (182) Google Scholar).The binding of collagen to platelets is believed to cause clustering of GPVI, and this is thought to be responsible for initiating tyrosine phosphorylation of the FcR γ-chain, presumably on the ITAM. Tyrosine phosphorylation of the FcR γ-chain facilitates the recruitment of the tyrosine kinase Syk to the receptor complex, which binds to the FcR γ-chain via its tandem Src-homology 2 (SH2) domains (3Gibbins J. Asselin J. Farndale R. Barnes M. Law C.L. Watson S.P. J. Biol. Chem. 1996; 271: 18095-18099Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). Using mice that have been engineered to lack either the FcR γ-chain or Syk, we have demonstrated that both proteins are essential for collagen-stimulated activation of platelets and that activation of Syk is essential for phosphorylation and activation of phospholipase Cγ2 (PLCγ2) (13Poole A. Gibbins J.M. Turner M. van Vugt M.J. van de Winkel J.G.J. Saito T. Tybulewicz V.L.J. Watson S.P. EMBO J. 1997; 16: 2333-2341Crossref PubMed Scopus (386) Google Scholar). Platelets express a low affinity receptor for IgG, FcγRIIA, and this is believed to signal through the same pathway as for GPVI. Cross-linking of FcγRIIA results in tyrosine phosphorylation of its intrinsic ITAM sequence, leading to activation of Syk and PLCγ2. Chacko et al. (14Chacko G.W. Brandt J.T. Coggeshall K.M. Anderson C.L. J. Biol. Chem. 1996; 271: 10775-10781Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar) propose that FcγRIIA becomes coupled to the p85/110 isoform of phosphatidylinositol 3-kinase (PI3-kinase) through Syk, which is recruited to the tyrosine-phosphorylated ITAM sequence of the activated receptor. Association of the p85 regulatory subunit with a phosphopeptide containing the ITAM of FcγRIIA was dependent on platelets being activated by FcγRIIA cross-linking, whereas association with Syk was independent of activation. The authors therefore proposed that Syk may function as an adapter protein, indirectly linking p85 to the phosphorylated ITAM.In this report we present data that suggests two potential pathways of regulation of PI3-kinase by GPVI. We demonstrate that the interaction of p85 with the receptor complex is a direct interaction with the tyrosine-phosphorylated FcR γ-chain ITAM through the SH2 domains of p85. In addition, we show that LAT, recently cloned from T cells (15Zhang W. Sloan-Lancaster J. Kitchen J. Trible R.P. Samelson L.E. Cell. 1998; 92: 83-92Abstract Full Text Full Text PDF PubMed Scopus (1055) Google Scholar), becomes tyrosine-phosphorylated on stimulation of GPVI and associates with p85.DISCUSSIONPhosphatidylinositol 3-kinases are an important class of signaling enzymes that underlie a variety of cellular responses such as cell motility, cytoskeletal rearrangement, regulation of integrin activation, proliferation, and differentiation (24Kimura K. Hattori S. Kabuyama Y. Shizawa Y. Takayanagi J. Nakamura S. Toki S. Matsuda Y. Onodera K. Fukui Y. J. Biol. Chem. 1994; 269: 18961-18967Abstract Full Text PDF PubMed Google Scholar, 25Kovacsovics T.J. Bachelot C. Toker A. Vlahos C.J. Duckworth B. Cantley L.C. Hartwig J.H. J. Biol. Chem. 1995; 270: 11358-11366Crossref PubMed Scopus (196) Google Scholar, 26Zhang J. Zhang J. Shattil S.J. Cunningham M.C. Rittenhouse S.E. J. Biol. Chem. 1996; 271: 6265-6272Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 27Shaw L.M. Rabinovitz I. Wang H.H.F. Toker A. Mercurio A.M. Cell. 1997; 91: 949-960Abstract Full Text Full Text PDF PubMed Scopus (542) Google Scholar, 28Keely P.J. Westwick J.K. Whitehead I.P. Der C.J. Parise L.V. Nature. 1997; 390: 632-636Crossref PubMed Scopus (649) Google Scholar). The family includes the p85/p110 isoform, which comprises two associated proteins, the p85 regulatory subunit and the catalytic subunit p110. p85 is a 724-amino acid protein that contains two SH2 domains and one SH3 domain (29Escodedo J.A. Navankasattusas S. Kavanaugh W.M. Milfay D. Fried V.A. Williams L.T. Cell. 1991; 65: 75-82Abstract Full Text PDF PubMed Scopus (373) Google Scholar, 30Skolnik E.Y. Margolis B. Mohammadi M. Lowenstein E. Fischer R. Drepps A. Ullrich A. Schlessinger J. Cell. 1991; 65: 83-90Abstract Full Text PDF PubMed Scopus (436) Google Scholar, 31Otsu M. Hiles I. Gout I. Fry M.J. Ruiz-Larrea F. Panayotou G. Thompson A. Dhand R. Hsuan J. Totty N. Smith A.D. Morgan S.J. Courtneidge S.A. Parker P.J. Waterfield M.D. Cell. 1991; 65: 91-104Abstract Full Text PDF PubMed Scopus (539) Google Scholar). The SH2 and SH3 domains of p85 allow interactions with other signaling proteins, resulting in translocation and activation of the p110 catalytic subunit. The consensus phosphotyrosine binding motif for the SH2 domains of p85 have been defined as YXXM (32Songyang Z. Shoelson S.E. Chaudhuri M. Gish G. Pawson T. Haser W. King F. Roberts T. Ratnofsky S. Lechleider R.J. Neel B.G. Birge R.B. Fajardo J.E. Chou M.M. Hanafusa H. Schaffhausen B. Cantley L.C. Cell. 1993; 72: 767-778Abstract Full Text PDF PubMed Scopus (2373) Google Scholar). Recruitment of the p85 subunit of PI3-kinase to activated membrane receptors such as the platelet-derived growth factor receptor results in translocation of p110 to the vicinity of substrates. The p110 catalytic subunit is a protein of 1068 amino acids that associates with p85 via region between the two SH2 domains. On activation, this enzyme phosphorylates phosphatidylinositol 4,5-bisphosphate on the 3-position of the myo-inositol ring, resulting in the formation of phosphatidylinositol 3,4,5-trisphosphate. Phosphatidylinositol 3,4,5-trisphosphate is a second messenger which supports membrane binding of proteins with pleckstrin homology domains and the regulation of some protein kinase C isoforms (33Singh S.S. Chauhan A. Brockerhoff H. Chauhan V.P.S. Biochem. Biophys. Res. Commun. 1993; 195: 104-112Crossref PubMed Scopus (68) Google Scholar).There is increasing evidence indicating that PI3-kinase participates in a number of signaling pathways in platelets. PI3-kinase is involved in activation of integrin αIIbβ3 (26Zhang J. Zhang J. Shattil S.J. Cunningham M.C. Rittenhouse S.E. J. Biol. Chem. 1996; 271: 6265-6272Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar), thrombin stimulation (34Mitchell C.A. Jefferson A.B. Bejeck B.E. Brugge J.S. Deuel T.F. Majerus P.W. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9396-9400Crossref PubMed Scopus (22) Google Scholar, 35Zhang J. Fry M.J. Waterfield M.D. Jaken S. Liao L. Fox J.E. Rittenhouse S.E. J. Biol. Chem. 1992; 267: 4686-4692Abstract Full Text PDF PubMed Google Scholar), and cytoskeletal rearrangement (25Kovacsovics T.J. Bachelot C. Toker A. Vlahos C.J. Duckworth B. Cantley L.C. Hartwig J.H. J. Biol. Chem. 1995; 270: 11358-11366Crossref PubMed Scopus (196) Google Scholar, 36Wymann M. Arcaro A. Biochem. J. 1994; 298: 517-520Crossref PubMed Scopus (124) Google Scholar,37Bokoch G.M. Vlahos C.J. Wang Y. Knaus U.G. Traynor-Kaplan A.E. Biochem. J. 1996; 315: 775-779Crossref PubMed Scopus (156) Google Scholar). Through the use of the structurally distinct PI3-kinase inhibitors LY 294002 (38Vlahos C.J. Matter W.F. Hui K.Y. Brown R.F. J. Biol. Chem. 1994; 269: 5241-5248Abstract Full Text PDF PubMed Google Scholar) and wortmannin (39Arcaro A. Wymann M.P. Biochem. J. 1993; 296: 297-301Crossref PubMed Scopus (1045) Google Scholar), we have previously shown that this enzyme is required for full activation of PLCγ2 by collagen (40Mountford J.M. Gross B. Watson S.P. Blood. 1997; 10 Suppl. 2: 2993Google Scholar) and convulxin. 2J. M. Gibbins, P. Thompson, and S. P. Watson, unpublished observation. Several publications have reported the involvement of PI3-kinase in signal transduction generated by Fc receptors (41Ibarrola I. Vossebeld P.J.M. Homburg C.H.E. Thelen M. Roos D. Verhoeven A.J. Biochim. Biophys. Acta. 1997; 1357: 348-358Crossref PubMed Scopus (54) Google Scholar, 42Melendez A.J. Gillooly D.J. Harnett M.M. Aleen J.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2169-2174Crossref PubMed Scopus (26) Google Scholar). In a study of the signal transduction generated by the low affinity IgG receptor (FcγRIIA) in platelets, Chacko et al. (14Chacko G.W. Brandt J.T. Coggeshall K.M. Anderson C.L. J. Biol. Chem. 1996; 271: 10775-10781Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar) show that stimulation of the receptor by cross-linking results in a transient increase in the activity of PI3-kinase, as shown in an in vitro kinase assay. Furthermore, platelet aggregation by stimulation of FcγRIIA is abrogated by pre-treatment with wortmannin. The authors reported that p85 is capable of binding to FcγRIIA-ITAM phosphopeptides in a transient manner and that this was dependent on activation of platelets by cross-linking the receptor. The interaction was therefore proposed to be indirect, possibly mediated via Syk, which also bound to the phosphorylated ITAM peptide.In this study we have investigated mechanisms for the regulation of PI3-kinase following stimulation of the GPVI-FcR γ-chain collagen receptor. Platelets possess several additional proteins or receptors that bind collagen, and their contributions to signal transduction and activation are unclear. We therefore also used a highly potent second platelet agonist convulxin, which selectively binds GPVI (20Polgár J. Clemetson J.M. Kehrel B.E. Wiedemann M. Magnenat E.M. Wells T.N.C. Clemetson K.J. J. Biol. Chem. 1997; 272: 13576-13583Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar, 23Jandrot-Perrus M. Lagrue A.-H. Okuma M. Bon C. J. Biol. Chem. 1997; 272: 27035-27041Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 43Leduc M. Bon C. Biochem. J. 1998; 333: 389-393Crossref PubMed Scopus (66) Google Scholar). The importance of the GPVI pathway in signaling stimulated by convulxin was demonstrated in the present study. Using mouse platelets that have been engineered to lack the FcR γ-chain, which is an essential component of the receptor, we demonstrated that functional and biochemical platelet responses to the venom were absent. This indicates that convulxin stimulates platelet activation through binding to, and activation of, the GPVI-FcR γ-chain collagen receptor.We demonstrate two potential mechanisms for the regulation of PI3-kinase on stimulation of the collagen receptor: through binding to the activated receptor and by binding to tyrosine-phosphorylated LAT. Using peptides based on the ITAM sequence of the FcR γ-chain, we show that when tyrosine-phosphorylated, the motif is able to bind to the p85 subunit of PI3-kinase, and the level of protein associated is not affected by stimulation with collagen or the more powerful agonist convulxin. p85 is also able to bind to the tyrosine-phosphorylated ITAM of FcγRIIA, and likewise, the levels associated from basal cells are equivalent to those from stimulated platelets. This result contrasts that of Chacko et al. (14Chacko G.W. Brandt J.T. Coggeshall K.M. Anderson C.L. J. Biol. Chem. 1996; 271: 10775-10781Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar), who observe association only in stimulated cells and proposed that this was mediated through interaction with Syk. The explanation for this difference is not known. We also show that Syk from resting platelets associates with both of the tyrosine-phosphorylated ITAM peptides at similar levels under resting conditions and following stimulation with collagen or convulxin. The level of tyrosine phosphorylation of Syk bound to the peptides is increased on stimulation with collagen or convulxin. This is consistent with our previous observations of association between the tandem SH2 domains in Syk and the FcR γ-chain both in vitro and in vivo (3Gibbins J. Asselin J. Farndale R. Barnes M. Law C.L. Watson S.P. J. Biol. Chem. 1996; 271: 18095-18099Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). These observations strengthen the conclusion that the interaction of p85 with the tyrosine-phosphorylated ITAMs is not via Syk, as the lower amount of p85 that associates with the FcR γ-chain peptide relative to the FcγRIIA peptide is not accompanied by a similar reduction in the amount of Syk. Furthermore, the increase in tyrosine phosphorylation of Syk that binds to the peptides following stimulation with collagen or convulxin does not result in an increase in the amount of peptide-associated p85. We were also unable to detect coprecipitation of Syk with PI3-kinase after immunoprecipitation of either protein (not shown). These results suggest that PI3-kinase associates directly with the phosphorylated ITAMS. The FcR γ-chain and FcγRIIA ITAMs do not conform to the consensus binding motif YXXM that has been defined for p85 SH2 domain binding (32Songyang Z. Shoelson S.E. Chaudhuri M. Gish G. Pawson T. Haser W. King F. Roberts T. Ratnofsky S. Lechleider R.J. Neel B.G. Birge R.B. Fajardo J.E. Chou M.M. Hanafusa H. Schaffhausen B. Cantley L.C. Cell. 1993; 72: 767-778Abstract Full Text PDF PubMed Scopus (2373) Google Scholar). A similar observation has been previously made for binding to the phosphorylated peptides of the ITAMs present in the T cell receptor ζ-chain complex (44Exley M. Varticovski L. Peter M. Sancho J. Terhorst C. J. Biol. Chem. 1994; 269: 15140-15146Abstract Full Text PDF PubMed Google Scholar, 45Jascur T. Gilman J. Mustelin T. J. Biol. Chem. 1997; 272: 14483-14488Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). The association of p85 with the tyrosine-phosphorylated ITAM of the FcR γ-chain provides a potential mechanism that may explain how PI3-kinase is activated on stimulation of the collagen receptor. The FcR γ-chain was found to be present in p85 immunoprecipitates from platelets stimulated with convulxin (and to a lesser degree, collagen), thus confirming the interaction in vivo.The second potential mechanisms for the regulation of PI3-kinase is through the tyrosine-phosphorylated protein of 38 kDa that was present in p85 immunoprecipitates from platelets stimulated with collagen or convulxin. We have identified this protein to be the recently cloned protein LAT, which is tyrosine-phosphorylated by cross-linking of the T cell receptor (15Zhang W. Sloan-Lancaster J. Kitchen J. Trible R.P. Samelson L.E. Cell. 1998; 92: 83-92Abstract Full Text Full Text PDF PubMed Scopus (1055) Google Scholar). Tyrosine-phosphorylated LAT has been shown to interact with PI3-kinase. LAT was not detected in precipitation experiments with tyrosine-phosphorylated ITAM containing peptides from the FcR γ-chain and FcγRIIA, which suggests that a phosphorylated ITAM does not bind p85 and LAT simultaneously. LAT functions as an essential linker between stimulation of the T cell receptor and T cell activation, whereupon it becomes tyrosine-phosphorylated and binds multiple signaling proteins (15Zhang W. Sloan-Lancaster J. Kitchen J. Trible R.P. Samelson L.E. Cell. 1998; 92: 83-92Abstract Full Text Full Text PDF PubMed Scopus (1055) Google Scholar). LAT contains consensus binding motifs for the SH2 domains of PLCγ1 and Grb2, which support association with the protein when phosphorylated. Although no consensus binding motif for the SH2 domains of p85 is present in the protein, LAT has also been shown to associate with p85 in activated T cells (15Zhang W. Sloan-Lancaster J. Kitchen J. Trible R.P. Samelson L.E. Cell. 1998; 92: 83-92Abstract Full Text Full Text PDF PubMed Scopus (1055) Google Scholar, 46Fukazawa T. Reedquist K.A. Panchamoorthy G. Soltoff S. Trub T. Druker B. Cantley L. Shoelson S. Band H. J. Biol. Chem. 1995; 270: 20177-20182Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Because LAT is believed to be anchored in the plasma membrane through an N-terminal transmembrane domain, the recruitment of PLC and PI3-kinase results in translocation of these proteins to the proximity of lipid substrates. In T cells the Ras activator protein SOS has been shown to co-precipitate with LAT, probably because of its interaction with Grb2 (15Zhang W. Sloan-Lancaster J. Kitchen J. Trible R.P. Samelson L.E. Cell. 1998; 92: 83-92Abstract Full Text Full Text PDF PubMed Scopus (1055) Google Scholar). A 38-kDa protein has previously been described in platelets that associates with Grb2 (47Robinson A. Gibbins J. Rodriguez-Linares B. Finan P.M. Wilson L. Kellie S. Findell P. Watson S.P. Blood. 1996; 88: 522-530Crossref PubMed Google Scholar), and therefore it is possible that LAT may provide a link between the collagen receptor and activation of the mitogen-activated protein kinase pathway through SOS. The mechanism that results in the tyrosine phosphorylation of LAT in platelets has not been determined; however, Zhang (15Zhang W. Sloan-Lancaster J. Kitchen J. Trible R.P. Samelson L.E. Cell. 1998; 92: 83-92Abstract Full Text Full Text PDF PubMed Scopus (1055) Google Scholar) et al. report LAT to be a substrate for Syk and Zap-70. Because Syk is an essential component of the signaling mechanism leading to platelet activation on stimulation with collagen, Syk may also perform this function in collagen-stimulated platelets.We investigated the hypothesis that the interaction of p85 with the FcR γ-chain and LAT is supported by the SH2 domains present in p85. Precipitation experiments were performed using the dual SH2 domains of p85 expressed as GST fusion proteins. Both the FcR γ-chain and LAT were capable of binding to the SH2 domains of p85 following stimulation with collagen or convulxin. Investigation into the kinetics of the interaction following stimulation with convulxin revealed that association of both proteins with the p85 SH2 domain construct was rapid and peaked after 90 s of stimulation, and this was reflected by the level of tyrosine phosphorylation of the proteins. We also examined the contributions of individual p85 SH2 domains to the interaction with the FcR γ-chain and LAT. The profiles of proteins that associated with the individual SH2 domains were quite different. The N-terminal domain was able to bind a large range of proteins with a similar profile to those precipitated with the dual SH2 domains. An increase in the levels of a number of proteins was observed on stimulation, and notably these included the FcR γ-chain. A smaller profile of proteins was precipitated with the C-terminal SH2 domain. LAT was present in the adsorbates; however, the FcR γ-chain was not. This suggests that only the N-terminal SH2 domain of p85 is necessary to support the association with the FcR γ-chain and that the C-terminal SH2 domain is sufficient for LAT binding. However, the tandem SH2 domains of PI3-kinase may support a stronger interaction (increased affinity) with the phosphorylated ITAMs in vivoas reported for the interaction between the T cell ζ-chain and PI3-kinase (45Jascur T. Gilman J. Mustelin T. J. Biol. Chem. 1997; 272: 14483-14488Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar).Based on the results of this study, we propose a model for signaling by the GPVI-FcR γ-chain collagen receptor that allows divergence of signals from the receptor into multiple signaling pathways. Binding of collagen to the receptor results in clustering and the tyrosine phosphorylation of the FcR γ-chain ITAM. This results in the recruitment of PI3-kinase and Syk to tyrosine-phosphorylated ITAM motifs. By analogy with the interaction of PI3-kinase with various growth factor receptors, binding of PI3-kinase to the FcR γ-chain may result in activation of the enzyme leading to the formation of the biologically active intracellular messenger, phosphatidylinositol 3,4,5-trisphosphate. On binding the FcR γ-chain, Syk becomes tyrosine-phosphorylated and activated, which leads to the tyrosine phosphorylation and activation of PLCγ2. Stimulation of the GPVI-FcR γ-chain collagen receptor results in tyrosine phosphorylation of LAT, possibly by receptor-associated and -activated Syk. LAT, which is anchored through the plasma membrane, may then recruit a number of signaling molecules including PI3-kinase, thus providing a second potential mechanism for the regulation of the enzyme on stimulation of platelets with collagen. It is likely that Grb2 also binds to LAT in platelets, and thereby via associated SOS, may feed into the mitogen-activated protein kinase pathway. Subendothelial collagens are primary platelet agonists and are thereby essential components of the hemostatic system. At sites of vascular damage, platelets adhere to exposed collagen fibers and undergo activation via a tyrosine kinase-dependent signaling pathway. Activation causes an increase in the binding capability of the fibrinogen receptor, integrin αIIbβ3, and the secretion of various mediators that culminate in the formation of an irreversible platelet aggregate, or hemostatic plug. The integrin α2β1 is expressed on the platelet surface and has been shown to support adhesion to collagen, although increasing evidence suggests that a second platelet collagen receptor underlies platelet activation. The collagen receptor that underlies activation comprises the uncharacterized platelet glycoprotein VI (GPVI)1 (1Gibbins J.M. Okuma M. Farndale R. Barnes M. Watson S.P. FEBS Lett. 1997; 413: 255-259Crossref PubMed Scopus (257) Google Scholar, 2Tsuji M. Ezumi Y. Arai M. Takayama H. J. Biol. Chem. 1997; 272: 23528-23531Crossref PubMed Scopus (241) Google Scholar), which is noncovalently associated with the Fc receptor (FcR) γ-chain (3Gibbins J. Asselin J. Farndale R. Barnes M. Law C.L. Watson S.P. J. Biol. Chem. 1996; 271: 18095-18099Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). Additional components may also exist. The FcR γ-chain is recognized for its role in the expression of, and signaling by, the high affinity receptor for IgE (FcεRI) (4Shiue L. Green J. Green O.M. Karas J.L. Morgenstern J.P. Ram M.K. Taylor M.K. Zoller M.J. Zydowsky L.D. Bolen J.B. Brugge J.S. Mol. Cell. Biol. 1995; 15: 272-281Crossref PubMed Google Scholar, 5Paolini R. Renard V. Vivier E. Ochiai K. Jouvin M.H. Malissen B. Kinet J.P. J. Exp. Med. 1995; 181: 247-255Crossref PubMed Scopus (39) Google Scholar, 6Jouvin M.H. 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