The p85 subunit of phosphatidylinositol 3-kinase associates with the Fc receptor gamma-chain and linker for activitor of T cells (LAT) in platelets stimulated by collagen and convulxin.

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 gamma-chain (a component of the collagen receptor complex that includes glycoprotein VI). The associations of the Fc receptor gamma-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 tyrosinephosphorylated 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 ␥-chain (a component of the collagen receptor complex that includes glycoprotein VI). The associations of the Fc receptor ␥-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.
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 ␣ IIb ␤ 3 , and the secretion of various mediators that culminate in the formation of an irreversible platelet aggregate, or hemostatic plug. The integrin ␣ 2 ␤ 1 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 (1,2), which is noncovalently associated with the Fc receptor (FcR) ␥-chain (3). Additional components may also exist. The FcR ␥-chain is recognized for its role in the expression of, and signaling by, the high affinity receptor for IgE (Fc⑀RI) (4 -6) and IgG (Fc␥RI) (7,8) and the low affinity IgG receptor (Fc␥RIII) (8). The FcR ␥-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 where X represents any amino acid (9). 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 (3,4,10). 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 (11,12).
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 ␥-chain, presumably on the ITAM. Tyrosine phosphorylation of the FcR ␥-chain facilitates the recruitment of the tyrosine kinase Syk to the receptor complex, which binds to the FcR ␥-chain via its tandem Src-homology 2 (SH2) domains (3). Using mice that have been engineered to lack either the FcR ␥-chain or Syk, we have demonstrated that both proteins are essential for collagenstimulated activation of platelets and that activation of Syk is essential for phosphorylation and activation of phospholipase C␥2 (PLC␥2) (13). Platelets express a low affinity receptor for IgG, Fc␥RIIA, and this is believed to signal through the same pathway as for GPVI. Cross-linking of Fc␥RIIA results in tyrosine phosphorylation of its intrinsic ITAM sequence, leading to activation of Syk and PLC␥2. Chacko et al. (14) propose that Fc␥RIIA 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 Fc␥RIIA was dependent on platelets being activated by Fc␥RIIA cross-link-ing, 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 ␥-chain ITAM through the SH2 domains of p85. In addition, we show that LAT, recently cloned from T cells (15), becomes tyrosinephosphorylated on stimulation of GPVI and associates with p85.

EXPERIMENTAL PROCEDURES
Materials-Horm-Chemie collagen (collagen fibers from equine tendon) was purchased from Nycomed (Munich, Germany), and venom from the tropical rattlesnake (Crotalus durissus terrificus) and Sephacryl S300HR was supplied by Sigma. Ly294002 was purchased from Calbiochem-Novabiochem. Sulfolink and BCA protein assay were from Pierce and Warriner (Chester, UK). Anti-phosphotyrosine monoclonal antibody 4G10, polyclonal anti-LAT, and anti-p85 antiserum were from Upstate Biotechnology (TCS Biologicals Ltd., Buckinghamshire, UK), and polyclonal anti-Syk N-19 was from Santa Cruz (Autogen Bioclear, Wiltshire, UK). Polyclonal anti-Syk rabbit antiserum, raised against the peptide EPTGGPWGPDGRL corresponding to amino acid residues 318 -330 in murine Syk, was provided by Dr. V. Tybulewicz (National Institute for Medical Research, Mill Hill, London). Rabbit antiserum generated against whole human PLC␥2 was a gift from Dr. Young Han Lee (16) (University of Science and Technology, Pohang, South Korea). GST fusion constructs containing the single and double SH2 domains of p85 were kindly provided by Professor T. Pawson (University of Toronto, Ontario, Canada). Fc receptor ␥-chain-deficient mice were generated as described elsewhere (17,18). Other reagents were from previously described sources (1,3,13).
Preparation of Platelets-Human platelets from drug-free volunteers were prepared on the day of the experiment as described previously (19) and suspended in modified Tyrodes-Hepes buffer (134 mM NaCl, 0.34 mM Na 2 HPO 4 , 2.9 mM KCl, 12 mM NaHCO 3 , 20 mM Hepes, 5 mM glucose, 1 mM MgCl 2 , pH 7.3) to a density of 8 ϫ 10 8 cells/ml. Mouse platelets were prepared as described previously (13). Platelets from each mouse were suspended in 250 l of modified Tyrodes-Hepes buffer, and 40 l of suspension was used for each assay. Stimulation of platelets with collagen (100 g/ml), convulxin (1 g/ml), and thrombin (10 units/ml) was performed for 90 s at 37°C in an aggregometer with continuous stirring (1200 rpm). For protein precipitation experiments, platelets were resuspended in buffer that contained 1 mM EGTA and 10 M indomethacin; a limited number of experiments were performed in the presence of 2 units/ml apyrase.
Preparation of Convulxin-Convulxin was isolated from the venom of the tropical rattlesnake Crotalus durissus terrificus using a method based on the procedure described by Polgár et al. (20). 250 mg of lyophilized snake venom was dissolved in 8 ml of 100 mM ammonium formate buffer, pH 3.5, containing 300 mM NaCl. The solution was mixed for 2 h, and insoluble material removed by centrifugation. The supernatant was loaded onto a Sephacryl S300HR size-exclusion chromatography column (length 100 cm, internal diameter 1.6 cm), which was run in ammonium formate buffer (as above) at a flow rate of 30 ml/h. 5-ml fractions were collected, and protein content was assessed using an in-line UV monitor (280 nm). Fractions were assayed for their ability to stimulate platelet aggregation. The presence of convulxin in the relevant fractions was confirmed by SDS-PAGE and staining with silver to detect a protein of approximately 85 kDa under nonreducing conditions and 14 kDa under reducing conditions. Convulxin-containing fractions were pooled and dialyzed against 1 mM HCl before redissolving in ammonium formate buffer and rechromatography on Sephacryl S300HR. A single peak of protein was detected, and relevant fractions were pooled for dialysis against 150 mM NaCl in 50 mM phosphate buffer, pH 5 and then pH 6.5 and pH 7.4. Protein content was estimated by bicinchoninic acid assay. The convulxin preparation was stored at 4°C.

5-Hydroxytryptamine (5-HT) Secretion
Assay-Mouse platelets were loaded with [ 3 H]5-HT by incubation with 0.2 mCi/ml of platelet-rich plasma for 1 h at 37°C. Platelets were prepared from the platelet-rich plasma as described above. Stimulation of platelets was terminated by microcentrifugation, and the level of [ 3 H]5-HT release into the supernatant was determined by scintillation spectrometry. [ 3 H]5-HT secre-tion was expressed as a percentage of the total tissue content following subtraction of release under basal conditions as described previously (21).
Phosphopeptide Binding Assay-Peptides based on the ITAM sequences of the FcR ␥-chain and the low affinity IgG receptor Fc␥RIIA (see Table I) were synthesized and purified by high performance liquid chromatography to greater than 98%. The mass spectrum of each peptide was obtained to confirm correct molecular masses. An N terminus cysteine residue was included to facilitate covalent coupling to a solidphase support Sulfolink™. Peptides were coupled to the matrix following the manufacturer's instructions at a concentration of 500 g/ml packed gel. After treatment of platelets with buffer, collagen, or convulxin, stimulation was terminated by the addition of an equal volume of lysis buffer (20 mM Tris, 300 mM NaCl, 10 mM EDTA, 2% (v/v) Nonidet P40, 1 mM phenylmethylsulfonyl fluoride, 2 mM Na 3 VO 4 , 10 g/ml leupeptin, 10 g/ml aprotinin, 1 g/ml pepstatin A, pH 7.3). Detergent-insoluble material was removed by centrifugation before the addition of solid-phase peptide (10 g) and incubation at 4°C for 1 h. This high concentration of peptide was used to ensure that levels of tyrosine-phosphorylated peptides were not limiting. Solid-phase peptide pellets were washed in 4 ϫ 1-ml lysis buffer (1 ϫ strength without protease inhibitors) and boiled in SDS-PAGE sample treatment buffer. Precipitated proteins were separated by SDS-PAGE and immunoblotted as described below.
Immunoprecipitation Studies-Platelet stimulation (750-l and 50-l total volumes for human and mouse platelets, respectively) was terminated by the addition of an equal volume of ice-cold lysis buffer (20 mM Tris, 300 mM NaCl, 10 mM EDTA, 2% (v/v) Nonidet P40, 1 mM phenylmethylsulfonyl fluoride, 2 mM Na 3 VO 4 , 10 g/ml leupeptin, 10 g/ml aprotinin, 1 g/ml pepstatin A, pH 7.3). Detergent-insoluble debris was removed, and the lysates were precleared by mixing with protein A-Sepharose for 1 h at 4°C (20 l of a 50% (w/v) suspension of protein A-Sepharose in Tris-buffered saline-Tween 20 (TBS-T: 20 mM Tris, 137 mM NaCl, 0.1% (v/v) Tween 20, pH 7.6)). Protein A-Sepharose was removed from the lysates before the addition of relevant antibodies (1 l of anti-p85 serum, 2 l of anti-murine Syk serum; 2 l of anti-PLC␥2 serum). After rotation at 4°C for 1 h, 25 l of protein A-Sepharose suspension was added to each sample, and mixing was continued for 1 h. The Sepharose pellet was washed in lysis buffer and then TBS-T, before the addition of Laemmli sample treatment buffer. Proteins were separated by SDS-PAGE using 10 -18% gradient slab gels and transferred to polyvinylidene difluoride membranes for immunoblotting.
Immunoblotting-Proteins were separated by SDS-PAGE on 10 -18% gradient slab gels and transferred to polyvinylidene difluoride membranes that were then blocked by incubation in 10% (w/v) bovine serum albumin dissolved in TBS-T. Primary and secondary antibodies were diluted in TBS-T containing 2% (w/v) bovine serum albumin and incubated with polyvinylidene difluoride membranes for 1 h at room temperature. Blots were washed for 2 h in TBS-T after each incubation with antibodies and then developed using an enhanced chemiluminescence detection system. Primary antibodies were used at a concentration of 1 g/ml (anti-phosphotyrosine, -human Syk, -PLC␥2, -LAT, -SLP76, -Vav) or diluted 1:1000 (anti-p85, -murine Syk). Horseradish peroxidase-conjugated secondary antibodies were diluted 1:10000.
Fusion Protein Precipitation Studies-GST fusion proteins containing the single (C terminus, N terminus) and double (N ϩ C termini) SH2 domains were prepared as described elsewhere (22) and bound to glutathione-coated agarose beads. Platelets were stimulated and then lysed as described above for immunoprecipitation. Lysates were precleared using 5 g of GST immobilized on 10 l of glutathione-coated beads before incubation for 2 h at 4°C with 10 g of SH2 domains fusion proteins immobilized on 10-l beads. Proteins isolated with the fusion proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes for immunoblotting as described above.

Mouse Platelets That Lack the FcR ␥-Chain Do Not Respond
to Convulxin-Collagen binds to a number of receptors on the platelet surface including the integrin ␣ 2 ␤ 1 and GPVI. A component of the venom of the tropical rattlesnake (Crotalus durissus terrificus), convulxin, has recently been described to produce its potent platelet activatory activity because of its ability to bind and cross-link GPVI (20,23). To further validate its use as a GPVI-selective agonist, we investigated the effect of the venom on mouse platelets that have been engineered to lack expression of the FcR ␥-chain. Convulxin stimulated secretion of 38% of total [ 3 H]5-HT in control platelets, and this was inhibited completely in FcR ␥-chain-deficient platelets (Fig.  1A). In contrast, the response to thrombin was not altered (Fig.  1A). Syk and PLC␥2 were immunoprecipitated from control mouse platelets or from FcR ␥-chain-deficient platelets under resting conditions or following stimulation with venom or thrombin. Levels of tyrosine phosphorylation were assessed by immunoblotting (Fig. 1B). Stimulation of control platelets with convulxin produced a marked increase in phosphorylation of Syk and PLC␥2, whereas thrombin stimulated a more modest increase. Stimulation of FcR ␥-chain-deficient platelets with venom produced only a small increase in the levels of tyrosine phosphorylation of Syk and PLC␥2, although the response to thrombin was very similar to that observed in controls. A similar residual phosphorylation of Syk and PLC␥2 and abolition of functional response was also seen in FcR ␥-chain-deficient platelets stimulated by collagen (13). These results confirm that convulxin activates platelets through the GPVI-FcR ␥-chain pathway.
The p85 Subunit of PI3-Kinase Associates with the Tyrosinephosphorylated ITAM Sequence of the FcR ␥-Chain-The regulatory subunit of PI3-kinase has been reported to associate indirectly with the phosphorylated ITAM sequence present on the cytoplasmic tail of Fc␥RIIA following cross-linking of the receptor through the tyrosine kinase Syk. We therefore investigated whether the p85 regulatory subunit of PI3-kinase is able to associate with the tyrosine-phosphorylated FcR ␥-chain ITAM sequence. Platelets were lysed and incubated with immobilized peptides that contained either the nonphosphorylated or tyrosine-phosphorylated ITAM sequences derived from the FcR ␥-chain and Fc␥RIIA (see Table I). The identities of proteins and the levels of tyrosine phosphorylation were determined by immunoblot analysis.
Tyrosine-phosphorylated proteins were not detected in precipitation experiments using the nonphosphorylated ITAM peptides. This is in contrast to the tyrosine-phosphorylated peptides, with which a protein of approximately 72 kDa was precipitated (Fig. 2). The level of tyrosine phosphorylation of this protein was increased on stimulation with collagen and convulxin. In addition, a minor band of 75 kDa was observed in stimulated cells, and longer exposures of the anti-phosphotyrosine immunoblots revealed a low level of association with a number of additional proteins, although none had a molecular mass of the 85-kDa regulatory subunit of PI3-kinase (results not shown). The 72-kDa band was shown to contain Syk by reprobing; the identity of the 75-kDa protein is not known and was not recognized by antibodies to SLP76 and Vav. The level of Syk associated with the phosphorylated peptides was similar for the two peptides and was not altered following stimulation with collagen and convulxin.
The peptide blots were further probed for the presence of the p85 regulatory subunit of PI3-kinase. p85 was found to be present in precipitates with both tyrosine-phosphorylated ITAM peptides but did not interact with the nonphosphorylated ITAM peptides (Fig. 2). The level of p85 associated with phosphorylated FcR ␥-chain ITAM sequence was approximately 50% lower than that observed with the phosphorylated Fc␥RIIA peptide. The interaction of p85 with both tyrosinephosphorylated peptides was not dependent on platelet activation. This result contradicts the observations of Chacko et al. (14) who report binding of the p85 subunit of PI3-kinase to a tyrosine-phosphorylated Fc␥RIIA ITAM peptide only under stimulated conditions. Results are representative of four experiments.
Tyrosine-phosphorylated Proteins Associate with the p85 Subunit of PI3-Kinase following Collagen-and Convulxin-Stimulation-Collagen and convulxin stimulate tyrosine phosphorylation of a large number of proteins in platelets. Prominent among these proteins are the FcR ␥-chain (identified previously by immunoprecipitation (3)) and a band of approximately 38 kDa (Fig. 3A). Convulxin is a notably more powerful agonist than collagen, although the profile of phosphorylated proteins is similar to that stimulated by collagen. p85 was immunoprecipitated from the lysates of platelets stimulated with collagen and convulxin and proteins immunoblotted to detect phosphotyrosyl residues (Fig. 3B). Convulxin stimulated a marked increase in tyrosine phosphorylation of a 12-14 kDa protein that comigrates with FcR ␥-chain and a 38 kDa protein.
These two proteins could be seen in immunoprecipitates from collagen-stimulated platelets, albeit at a lower level consistent with the reduced tyrosine phosphorylation seen in whole cell  The peptides below were synthesized to investigate the ability of p85 to interact with the tyrosine-phosphorylated ITAM sequence of the FcR ␥-chain, a component of the activatory collagen receptor. All peptides were produced with an N terminus cysteine residue to facilitate covalent coupling to a solid-phase support.
A recent publication reported the cloning of the adapter protein, LAT, from T cells, which is an essential link between engagement of the T cell receptor and cellular activation (15). LAT, which has a molecular mass of 25 kDa and a putative transmembrane domain, runs on SDS-PAGE as a doublet of 36 -38 kDa and becomes heavily tyrosine-phosphorylated on activation of the T cell receptor. It is a substrate for Syk and ZAP-70 and, when tyrosine-phosphorylated, binds to PLC␥1, the p85 subunit of PI3-kinase and Grb2. We therefore investigated whether the 38-kDa protein observed above is LAT by immunoblotting for its presence in p85 immunoprecipitates. A low level of LAT was associated with p85 from untreated platelets that was enhanced on stimulation with collagen (Fig. 3C). These results are representative of three separate experiments.

The SH2 Domains of p85 Interact with Tyrosine-phosphorylated Fc Receptor ␥-Chain from Collagen-and Convulxin-stimulated Platelets-To investigate the sites of interaction of tyrosine-phosphorylated
proteins with PI3-kinase, we investigated the association of tyrosine-phosphorylated proteins from platelets stimulated with collagen or convulxin with the SH2 domains of p85, expressed as a GST fusion protein.
Lysates obtained from resting platelets or platelets stimulated with collagen or convulxin were precleared by incubation with GST immobilized on agarose and then incubated with immobilized fusion protein containing both of the SH2 domains of p85. Bound proteins were separated by SDS-PAGE and immunoblotted for phosphotyrosine residues. Fig. 4A shows several tyrosine-phosphorylated proteins associating with the p85 dual SH2 domain fusion construct, several of which, particularly those in the lower half of the blot, increased following stimulation with collagen or convulxin. Of note is the presence of the FcR ␥-chain, with its characteristic migration as a tyrosinephosphorylated protein doublet of approximately 12 kDa on SDS-PAGE under reducing conditions. LAT was also detected by immunoblotting samples precipitated with the tandem SH2 domain construct, although Syk was not detected. Fig. 4B shows the association of LAT with the fusion protein through a time course of stimulation with convulxin and also following 90 s of stimulation with collagen. The association was very rapid and detectable at 10 s of stimulation. The association peaked at around 90 s and began to decline by 300 s. This profile was similar to that observed between the dual SH2 domains and the FcR ␥-chain (not shown). An anti-phosphotyrosine reprobe confirmed LAT to be the 38-kDa protein that FIG. 3. Tyrosine-phosphorylated FcR ␥-chain and LAT associate with the p85 subunit of PI3-kinase following collagen and convulxin stimulation. A, whole platelet lysates from resting platelets or following stimulation with collagen (100 g/ml, 90 s) or convulxin (1 g/ml, 90 s) were subjected to SDS-PAGE and immunoblotted to detected phosphotyrosine residues. Arrows highlight the position of the FcR ␥-chain and a protein of approximately 38 kDa. B, the p85 regulatory subunit of PI3-kinase was immunoprecipitated from lysates of human platelets under resting conditions or following stimulation with collagen or convulxin (as above). Precipitated proteins were separated by SDS-PAGE and immunoblotted to detect phosphotyrosine residues. Arrows indicate the positions of the FcR ␥-chain and a protein of approximately 38 kDa, which co-immunoprecipitates with p85. C, the p85 subunit of PI3-kinase was immunoprecipitated from lysates of platelets under basal conditions and following stimulation with collagen (100 g/ml, 90 s). Protein were separated by SDS-PAGE and immunoblotted to detect LAT. The blot was reprobed for p85 to ensure equal sample loading. I.P., immunoprecipitates. associates with the SH2 domains of p85.
These results suggest that the in vivo association of the FcR ␥-chain and LAT with p85 is mediated by the SH2 domains of p85. To define this interaction further, similar precipitation experiments were performed using GST fusion protein that contained the single SH2 domains of p85. Fig. 5A shows the proteins that associated with the C-terminal SH2 domain of p85 from resting platelets and following stimulation with collagen or convulxin. By comparison with the dual SH2 precipitation, the number of associated tyrosine-phosphorylated proteins was low. Of relevance was the presence of LAT but absence of the FcR ␥-chain in these samples. The precipitation experiment was repeated using the N-terminal SH2 domain fusion protein, and the precipitated proteins are shown in 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 (29 -31). 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 (32). 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 myoinositol 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 (33).
There is increasing evidence indicating that PI3-kinase participates in a number of signaling pathways in platelets. PI3kinase is involved in activation of integrin ␣ IIb ␤ 3 (26), thrombin stimulation (34,35), and cytoskeletal rearrangement (25,36,37). Through the use of the structurally distinct PI3-kinase FIG. 4. The SH2 domains of the p85 subunit of PI3-kinase support association with the FcR ␥-chain and LAT. A, the lysates from resting platelets and following stimulation with collagen (100 g/ml, 90 s) and convulxin (1 g/ml, 90 s) were precleared as described under "Experimental Procedures" and incubated with an immobilized GST fusion protein containing the tandem SH2 domains of the p85 subunit of PI3-kinase. Proteins that associated with the fusion protein were separated by SDS-PAGE and immunoblotted for phosphotyrosine residues. Arrows indicate the positions of the FcR ␥-chain and a 38-kDa protein. The 38-kDa protein was identified as LAT (B). Platelets were stimulated with convulxin (Cvx; 1 g/ml) for 10, 20, 45, 90, and 300 s and with collagen (Coll; 100 g/ml for 90 s). Precipitation with the tandem SH2 domain fusion protein was performed as above, and samples were immunoblotted to detect LAT. The blot was reprobed to detect phosphotyrosine residues. inhibitors LY 294002 (38) and wortmannin (39), we have previously shown that this enzyme is required for full activation of PLC␥2 by collagen (40) and convulxin. 2 Several publications have reported the involvement of PI3-kinase in signal transduction generated by Fc receptors (41,42). In a study of the signal transduction generated by the low affinity IgG receptor (Fc␥RIIA) in platelets, Chacko et al. (14) 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 Fc␥RIIA is abrogated by pre-treatment with wortmannin. The authors reported that p85 is capable of binding to Fc␥RIIA-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 ␥-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 (20,23,43). 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 ␥-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 ␥-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 ␥-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 tyrosinephosphorylated ITAM of Fc␥RIIA, and likewise, the levels associated from basal cells are equivalent to those from stimulated platelets. This result contrasts that of Chacko et al. (14), 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 tyrosinephosphorylated 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 ␥-chain both in vitro and in vivo (3). 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 ␥-chain peptide relative to the Fc␥RIIA 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 PI3kinase after immunoprecipitation of either protein (not shown). These results suggest that PI3-kinase associates directly with the phosphorylated ITAMS. The FcR ␥-chain and Fc␥RIIA ITAMs do not conform to the consensus binding motif YXXM that has been defined for p85 SH2 domain binding (32). A similar observation has been previously made for binding to the phosphorylated peptides of the ITAMs present in the T cell receptor -chain complex (44,45). The association of p85 with the tyrosine-phosphorylated ITAM of the FcR ␥-chain provides a potential mechanism that may explain how PI3-kinase is activated on stimulation of the collagen receptor. The FcR ␥-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 PI3kinase 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 (15). 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 ␥-chain and Fc␥RIIA, 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 (15). LAT contains consensus binding motifs for the SH2 domains of PLC␥1 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 (15,46). 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 (15). A 38-kDa protein has previously been described in platelets that associates with Grb2 (47), 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 (15) 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 ␥-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 ␥-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 ␥-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 ␥-chain. A smaller profile of proteins was precipitated with the C-terminal SH2 domain. LAT was present in the adsorbates; however, the FcR ␥-chain was not. This suggests that only the N-terminal SH2 domain of p85 is necessary to support the association with the FcR ␥-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 vivo as reported for the interaction between the T cell -chain and PI3-kinase (45).
Based on the results of this study, we propose a model for signaling by the GPVI-FcR ␥-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 ␥-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 ␥-chain may result in activation of the enzyme leading to the formation of the biologically active intracellular messenger, phosphatidylinositol 3,4,5trisphosphate. On binding the FcR ␥-chain, Syk becomes tyrosine-phosphorylated and activated, which leads to the tyrosine phosphorylation and activation of PLC␥2. Stimulation of the GPVI-FcR ␥-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.