Identification of Shc as the Primary Protein Binding to the Tyrosine-phosphorylated β3 Subunit of αIIbβ3 during Outside-in Integrin Platelet Signaling*

Outside-in signaling mediated by the integrin αIIbβ3 (GPIIbIIIa) is critical to platelet function and has been shown to involve the phosphorylation of tyrosine residues on the cytoplasmic tail of β3. To identify proteins that bind directly to phosphorylated β3, we utilized an affinity column consisting of a peptide modeled on the tyrosine-phosphorylated cytoplasmic domain of β3. Tandem mass spectrometric sequencing and immunoblotting demonstrated that Shc was the primary protein binding to phosphorylated β3. To determine the involvement of Shc in outside-in αIIbβ3 signaling, the phosphorylation of Shc during platelet aggregation was examined; transient Shc phosphorylation was observed when thrombin-stimulated platelets were allowed to aggregate or when aggregation was induced by an LIBS (ligand-induced binding site) antibody, D3. Moreover, Shc was co-immunoprecipitated with tyrosine-phosphorylated β3 in detergent lysates of aggregated platelets. Using purified, recombinant protein, it was found that the binding of Shc to monophosphorylated (C-terminal tyrosine) and diphosphorylated β3 peptides was direct, demonstrating Shc recognition motifs on phospho-β3. Aggregation-induced Shc phosphorylation was also observed to be robust in platelets from wild-type mice, but not in those from mice expressing (Y747F,Y759F) β3, which are defective in outside-in αIIbβ3 signaling. Thus, Shc is the primary downstream signaling partner of β3 in its tyrosine phosphorylation outside-in signaling pathway.

Integrins are a homologous family of receptors that function to mediate cell adhesions and to signal cell activities such as differentiation, proliferation, and migration (1,2). ␣ IIb ␤ 3 is the most prominent platelet integrin, and is capable of binding several adhesive proteins, including fibrinogen and von Willebrand factor, the binding of which mediates platelet aggregation (3). During platelet aggregation, ␣ IIb ␤ 3 is also involved in transmitting signals, so-called "outside-in" ␣ IIb ␤ 3 signaling. Outside-in ␣ IIb ␤ 3 signaling initiates subsequent platelet responses, including various signaling reactions, changes in the platelet cytoskeleton, and platelet secretion (4 -6). The high degree of homology within the integrin family, for example between ␤ 3 and ␤ 1 , and the presence of the ␤ 3 subunit in the widely distributed ␣ v ␤ 3 integrin, suggest that ␣ IIb ␤ 3 signaling serves as a prototype for the elucidation of signaling pathways within the integrin family of adhesion receptors.
Outside-in ␣ IIb ␤ 3 signaling in platelets has been shown to involve the integrin cytoplasmic tyrosine (ICY) 1 domain of the cytoplasmic tail of ␤ 3 (7). The ␤ 3 ICY domain contains two tyrosine sequences, each in an NXXY motif, separated by 11 amino acids. Phosphorylated NXXY motifs in other receptors are known to be recognition sequences for proteins containing phosphotyrosine binding (PTB) domains (8). Phosphorylation of the ICY domain of ␤ 3 occurs during outside-in signaling induced by platelet aggregation, independent of the platelet agonist (9,10). The importance of phosphorylation of ICY domain tyrosines has been established through the analysis of platelet function in the diYF mouse, a mutant strain in which the endogenous ␤ 3 gene was replaced by one in which the two cytoplasmic tyrosines in ␤ 3 (Tyr-747 and Tyr-759) were mutated to phenylalanines. Platelets from the diYF mouse aggregate poorly to low concentrations of thrombin, aggregate reversibly to ADP, and are defective in retracting clots. DiYF mice demonstrate unstable hemostasis, perhaps because of the reversible nature of their platelet aggregates. Given the importance of tyrosine phosphorylation to outside-in ␣ IIb ␤ 3 signaling, characterization of the binding partner(s) of tyrosine-phosphorylated ␤ 3 is essential for characterizing the molecular features of this ␣ IIb ␤ 3 -mediated signaling event. Moreover, because conserved ICY domain sequences are found in the ␤ subunits of several integrins, including ␤ 1 , ␤ 3 , ␤ 6 , and ␤ 7 , and because tyrosine phosphorylation appears to be involved in the migration of cells on ␤ 1 integrins, it is anticipated that information concerning the mechanisms of signaling through ␣ IIb ␤ 3 might be applicable to other integrins.
Previous studies have identified proteins that bind the cytoplasmic tails of ␣ IIb ␤ 3 . These proteins include: ␤ 3 endonexin (11), which specifically interacts with the NITY motif at residues 756 -759 on the distal end of the ␤ 3 cytoplasmic tail (12), in a phosphorylation-independent manner; calcium-and integrin-binding protein (13), which binds in a calcium-dependent manner to the cytoplasmic tail of the ␣ IIb subunit (14); talin (15), a cytoskeletal protein that binds directly with the cytoplasmic tail of both ␣ IIb ␤ 3 subunits, integrin-associated protein (16), a protein that laterally associates with ␣ IIb ␤ 3 and activates this integrin in response to thrombospondin (17); and myosin, Shc, and Grb2, which have previously been shown to bind tyrosine-phosphorylated ␤ 3 peptides (9,10).
In the present study we used affinity chromatography and * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18  tandem mass spectrometric sequencing to identify the major soluble platelet protein binding to a diphosphorylated ␤ 3 ICY domain cytoplasmic tail peptide. Biochemical and genetic strategies were then used to determine whether the protein identified was involved in outside-in signaling through ␣ IIb ␤ 3 . We discovered that Shc was the primary protein associating with the phosphorylated ␤ 3 peptide and showed that Shc becomes transiently phosphorylated during platelet aggregation and can be co-immunoprecipitated with ␤ 3 from lysates in which outside-in ␣ IIb ␤ 3 signaling occurs. We also found that Shc interacts directly with tyrosine-phosphorylated ␤ 3 , because a GST-p52Shc fusion protein binds directly to the diphosphorylated ␤ 3 peptide sequence and to the ␤ 3 peptide sequence monophosphorylated on the C-terminal end. Analysis of platelets from the diYF mouse expressing (Y747F,Y759F) ␤ 3 showed that, in platelets where ␤ 3 tyrosine phosphorylation cannot occur and where outside-in ␣ IIb ␤ 3 signaling is defective, the level of aggregation-induced Shc phosphorylation was abrogated. These data implicate Shc as playing a major role in outside-in ␣ IIb ␤ 3 signaling in platelets by serving as a direct signaling partner for tyrosine-phosphorylated ␤ 3 .

EXPERIMENTAL PROCEDURES
Reagents-Horseradish peroxidase-conjugated Protein A (NA 9120) and ECL Western blotting detection reagents (RPN 2106) were from Amersham Pharmacia Biotech; biotin-conjugated anti-rabbit IgG controls (sc-2040) and horseradish peroxidase-conjugated anti-mouse GST monoclonal antibody (sc-138 HRP) were from Santa Cruz Biotechnologies; anti-rabbit Shc polyclonal antibodies (sc-1695, s14630) were from Santa Cruz Biotechnologies and Transduction Laboratories, respectively; anti-Shc antibody sc-1695 was custom biotinylated at Santa Cruz. The anti-mouse phosphotyrosine antibodies PY-20 (P11120) and 4G10 (05-321) were from Transduction Laboratories and Upstate Biotechnology Inc., respectively. The rabbit polyclonal antibody against ␣ IIb ␤ 3 #4518 was from COR Therapeutics, Inc. The anti-LIBS antibody, D3, was the kind gift of Dr. Lisa K. Jennings (University of Tennessee, Memphis). Peptides were synthesized at SynPep Corp. utilizing solidphase Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry, except for the diphosphorylated, scrambled ␤ 3 peptide, and the ␤ 3 peptide monophosphorylated on the C-terminal tyrosine residue, both synthesized at Research Genetics. Avidin-agarose beads were purchased from Pierce; glutathione-Sepharose 4B beads were from Amersham Pharmacia Biotech. Human ␣-thrombin was from Hemetech. Shc (p52) nucleotide sequence, in-frame with the GST sequence in the pGex-3X bacterial expression vector (for expression of GST-p52Shc fusion protein in Escherichia coli), was a kind gift from Drs. Darren Tyson and Ralph A. Bradshaw, University of California at Irvine. Precast SDS gels were from Bio-Rad.
Affinity Chromatography Procedure-For the column preparation, immobilized avidin on 6% cross-linked beaded agarose (1 mg/ml) was pre-equilibrated in 1:1 TBS/1ϫ lysis buffer A (10 mM Tris base, pH 7.4, 75 mM NaCl, 2 mM EDTA, 1% Nonidet P-40, 2 mM Na 3 VO 4 , 1 mM PMSF, and 10 g/ml of both aprotinin and leupeptin), then incubated 12 h (overnight) with the biotinylated ␤ 3 peptide (biotin-DTANNPLYKEAT-STFTNITYRGT-COOH) or with the diphosphorylated ␤ 3 peptide (biotin-DTANNPLpYKEATSTFTNITpYRGT-COOH). Excess peptide was drained off the column material through 1.5-ϫ 12-cm polypropylene columns (Bio-Rad) by gravity filtration. All three types of columns to be used in the purification process, i.e. 20-ml column beds of avidinagarose, non-phosphorylated, biotin-␤ 3 conjugated to avidin-agarose, or diphosphorylated biotin-␤ 3 conjugated to avidin-agarose, were equilibrated in 1:1 TBS/lysis buffer A. Columns were set up in series, with avidin-agarose as the first, pre-clear column, followed by the nonphosphorylated ␤ 3 column, followed by the diphosphorylated ␤ 3 column. Human platelets were obtained from volunteers subjected to double platelet pheresis, conducted at the Stanford Blood Center, Palo Alto, CA and used within 2 h of collection. Approximately 5 ϫ 10 11 platelets were obtained from each donor. Platelets were washed using CGS buffer (13 mM sodium citrate, 30 mM glucose, 120 mM NaCl, pH 7.0) and resuspended in 210 ml of calcium/magnesium free Tyrode's Hepes buffer (12 mM NaHCO 3 , 138 mM NaCl, 5.5 mM glucose, 2.9 mM KCl, pH 7.4). Platelet suspensions (2.95 Ϯ 0.02 platelets/ml, total volume 170 Ϯ 20 ml) were lysed on ice for 30 min with a 1:1 volume of 2ϫ lysis buffer A. Lysates were centrifuged at 100,000 ϫ g for 1 h at 4°C to remove the majority of cytoskeletal proteins. The supernatants were then loaded at a rate of 20 ml/h onto the columns set up in series. Columns were then separated, washed individually with 8 bed volumes of 1:1 TBS/lysis buffer A, pH 7.4, and eluted with 0.8 M phenyl phosphate. Samples (12 l) from fractions were monitored by silver staining of SDS gels and by Western analysis. Fractions containing proteins detected by silver staining and eluting exclusively off the diphosphorylated ␤ 3 column were pooled, concentrated by centrifugal ultrafiltration (Millipore), precipitated (18), and run on SDS gels. Bands of interest were excised from SDS gels and sequenced by tandem mass spectrometry at the Harvard Microchemistry Facility (Dr. William S. Lane).
Preparation of Human Platelet Lysates and Immunoprecipitation of Shc-Blood was drawn, and washed platelets were prepared as in a previous study (19) with the addition of 50 ng/ml prostaglandin I 2 and 0.6 unit/ml apyrase in the collecting solution. The platelet pellet was resuspended in Tyrode's Hepes buffer, pH 7.4, 1 mM CaCl 2 , and 1 mM MgCl 2 , to a concentration of 7 ϫ 10 8 platelets/ml, and the platelet suspension was allowed to rest at 37°C for 45 min. Platelet aggregation was monitored by light transmittance following the addition of agonists with stirring (1200 r.p.m.) to 500-l aliquots of platelet suspensions. For time courses of aggregated platelets, platelet suspensions were activated with an agonist and were allowed to aggregate until specific time points. When aggregation was induced by the LIBS antibody D3, 0.3 g/ml soluble fibrinogen was added 30 s prior to D3. Reactions were then stopped with the addition of 500 l of 2ϫ lysis buffer B (100 mM Tris base, pH 8.0, 250 mM NaCl, 2% Triton X-100, 20% glycerol, 8 mM Na 3 VO 4 , 2 mM PMSF, 20 g/ml aprotinin, 0.2 mg/ml leupeptin), plus 1 mM MgCl 2 and 1 mM CaCl 2 . Samples were left on ice 30 min. Time courses of activated platelets were determined under identical conditions except platelets were treated with 2.5 g/ml eptifibatide prior to stirring to inhibit aggregation. Each sample was then sonicated for 10 s. Lysates were incubated for 45 min at 4°C with 1 g of biotinylated rabbit IgG followed by another 45-min incubation with 70 l of a 50% slurry of avidin-agarose (in PBS, pH 7.4). At the end of the "pre-clear" steps, lysates were centrifuged at 14,000 ϫ g for 10 min at 4°C. Samples were split, and 2 g of either biotinylated anti-Shc or biotinylated rabbit control IgG was added to each 500-l sample. Incubations with the antibodies continued overnight at 4°C. Samples were then incubated with 70 l of 50% avidin-agarose bead slurry for 1 h at 4°C. Immunoprecipitation reactions were spun 20 s at 14,000 ϫ g, the supernatant was aspirated off, and the pellet was washed with 250 l of 0.5 M NaCl in 1ϫ lysis buffer B. Following recentrifugation, the wash steps were repeated twice using 1ϫ lysis buffer B. The remaining pelleted beads were resuspended in 2ϫ reducing sample buffer, boiled, and subjected to SDS-polyacrylamide gel electrophoresis.
Western Blotting Analysis-Samples from immunoprecipitations and from peptide precipitations of purified recombinant Shc were run on gels, which were then transferred to 0.2-m nitrocellulose supports and blotted with specific antibodies. For the immunoprecipitation experiments, blots of samples were cut in half and probed for phosphorylated tyrosine residues (using PY20 and 4G10) and ␣ IIb ␤ 3 (#4518); blots probed with phosphotyrosine antibodies were then stripped and reprobed with Shc antibodies. These blots of samples from immunoprecipitations using biotinylated antibodies were developed with Protein A conjugated to horseradish peroxidase and detected with ECL Western blotting detection reagents. Thus, IgG heavy chain of the immunoprecipitating, biotinylated anti-rabbit Shc polyclonal and anti-rabbit IgG control antibodies was not detected, and the observed band patterns were not obscured. Blots of IgG control immunoprecipitations showed no co-immunoprecipitation of tyrosine-phosphorylated proteins (data not shown).
Expression and Purification of Recombinant Shc-The pGEX-3X bacterial expression plasmid and the pGex-3X-p52Shc construct (20), the latter which contained the p52 Shc sequence inserted in-frame with the pGex-3X glutathione S-transferase (GST) sequence at the unique EcoRI site, were used to transform the protease-deficient BL21(DE3)pLysS E. coli strain of cells (Novagen). For GST and GST-Shc fusion protein expression, bacterial preparations were induced by 1 mM isopropyl-1thio-D-galactopyranoside at 37°C for 2 h. Bacteria were lysed in 2ϫ lysis buffer B (2% Triton X-100, 1 mM dithiothreitol, 8 mM Na 3 VO 4 , 2 mM PMSF, 20 g/ml leupeptin, and 20 g/ml aprotinin). After centrifugation for 20 min at 14,000 ϫ g at 4°C to pellet the cell wall, the expressed GST and GST fusion proteins were isolated from bacterial cell lysates by affinity chromatography using glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech), which were pre-equilibrated in 1ϫ lysis buffer B. GST and GST-Shc fusion proteins were then eluted with reduced glutathione (10 mM).
Analysis of the Interaction of Recombinant Shc with ␤ 3 -To deter-mine whether Shc can bind to phosphorylated ␤ 3 , equal amounts of GST and GST-Shc fusion proteins were mixed with one of non-phosphorylated, monophosphorylated (on the N-terminal tyrosine; biotin-DTAN-NPLpYKEATSTFTNITYRGT-COOH), monophosphorylated (on the Cterminal tyrosine; biotin-DTANNPLYKEATSTFTNITpYRGT-COOH), scrambled diphosphorylated (biotin-TNIEARTpYASKLDPTGTFT-pYTNN), or diphosphorylated ␤ 3 peptide, 50 g of which was bound to avidin-agarose and suspended in 1ϫ lysis buffer B. After incubation for 2 h at 4°C, the avidin-agarose-bound phospho or non-phospho ␤ 3 peptides were washed with 1ϫ lysis buffer B. Beads were resuspended in 2ϫ sample buffer, and samples were separated on a 4 -15% SDSpolyacrylamide gel. Gels were blotted onto nitrocellulose membranes and probed for the presence of Shc and GST with corresponding polyclonal antibodies (Santa Cruz Biotechnologies). Preparation of Mouse Platelet Lysates and Immunoprecipitation of Shc-The Shc immunoprecipitation procedure described above was optimized for mouse blood. First, blood was obtained by cardiac puncture (700 l) and drawn into a mix of 140 l of 2.5% trisodium citrate/2% dextrose/1.5% citric acid monohydrate, 560 l of saline, and prostaglandin E 1 at 50 ng/ml final concentration. Washed platelets were obtained as described (21). Platelets pooled from five to six animals were resuspended in Tyrode's buffer with 1 mM MgCl 2 at 6 ϫ 10 8 /ml. Thrombin-induced platelet aggregation was measured in the aggregometer. Platelets were lysed 15 s after thrombin by the addition of an equal volume of ice-cold 2ϫ lysis buffer B into the aggregometer tube. Samples were then incubated on ice for 20 min prior to sonication (6 ϫ 10 s bursts at amplitude 60). Lysates were precleared with 50 l of avidinagarose beads (Amersham Pharmacia Biotech) for 1 h at 4°C then spun in a microcentrifuge at 16,000 ϫ g for 15 min at 4°C. Supernatants were recovered, and for each immunoprecipitate, 200 l of lysate was incubated with 2 g of either biotinylated rabbit IgG (control) or biotinylated anti-Shc antibody and 50 l of avidin-agarose overnight at 4°C. Beads were washed and resuspended in sample buffer, as with human immunoprecipitates, and loaded onto SDS gels for Western blotting analysis.

Purification and Identification of Proteins that Bind to
Diphosphorylated ␤ 3 Peptides-To identify platelet proteins that selectively bind the phosphorylated tail of ␤ 3 , we designed an affinity procedure that had three columns in series: avidinagarose, the support matrix; a ␤ 3 peptide (␤ 3 740 -762 ); and a diphosphorylated ␤ 3 peptide ((pY747,pY759) ␤ 3 740 -762 ). The peptides were synthesized with a biotin moiety at their N terminus, which was used to conjugate them to avidin-agarose. We determined in preliminary experiments that the yield of phospho-␤ 3 binding proteins would be small, and therefore used a high platelet count to retrieve detectable yields of proteins on the columns. Fig. 1 shows a silver-stained gel of proteins eluting from the three columns. A similar cohort of bands was distributed throughout all columns, most prominently a 210-kDa band corresponding to platelet myosin heavy chain, an 85-kDa band corresponding to ␣-actinin, and a 45-kDa band corresponding to actin. Cytoskeletal proteins are found in abundance in human platelets, and the soluble forms of these proteins apparently were not completely removed by the ultracentrifugation step used in generating the platelet supernatants that were loaded onto the columns. However, one protein, of molecular mass Х 60 kDa, specifically eluted with phenyl phosphate off the diphosphorylated ␤ 3 affinity column and was not found in eluates of either the avidin-agarose precolumn or the non-phosphorylated ␤ 3 column. Direct protein sequencing revealed that this protein was Shc, because tryptic digests of it contained 22 different peptides, each of which had 100% homology to this adapter protein (Fig. 2). Western immunoblotting with Shc antibody (Fig. 3) confirmed that Shc only eluted from the diphosphorylated ␤ 3 column: the apparent molecular mass of this protein (57 kDa) was identical to the only Shcpositive band identified by Western blotting found in platelets. This and the spectrum of peptides identified by sequencing indicate that we isolated the p52 isoform of Shc and that this is the only Shc isoform found in platelets.

Shc is Tyrosine-phosphorylated during Thrombin-induced
Aggregation-To determine whether Shc is involved in outside-in signaling through ␣ IIb ␤ 3 , we determined the time course of Shc tyrosine phosphorylation during platelet aggregation, an event known to induce ␣ IIb ␤ 3 outside-in signaling. Fig. 4 shows Shc phosphorylation during platelet aggregation induced by thrombin, a potent platelet agonist. Shc tyrosine phosphorylation increased dramatically during thrombin-induced aggregation (11.4 Ϯ 0.8-fold), reaching a maximum 20 s after the addition of thrombin. With continued stirring, the level of tyrosine phosphorylation decreased, to 9.1 Ϯ 0.5-fold at 45 s, to 6.8 Ϯ 0.4-fold at 90 s of aggregation, and to 1.4 Ϯ 0.1-fold at 270 s. A reprobe of the immunoprecipitate blot with Shc polyclonal antibodies showed that each lane contained an equal amount of Shc (middle panel). Thrombin signaling alone, in the absence of aggregation, is sufficient to induce tyrosine phosphorylation of several platelet proteins (22,23). To determine the extent of Shc phosphorylation due to thrombin activation as compared with thrombin aggregation, the ␣ ⍜ ⍜ b ␤ 3 -specific antagonist eptifibatide was used to prevent aggregation. Although Shc phosphorylation still occurred in the absence of aggregation, the -fold increase was approximately half of that observed when aggregation also occurred, 6.2 Ϯ 0.3 at 20 s, 4.3 Ϯ 0.4 at 45 s, and to control levels at 270 s. Thus, Shc tyrosine phosphorylation, much like that observed with Syk, appears to be induced by two pathways in platelets: one induced by thrombin stimulation, most likely a pathway initiated by protease-activated receptor activation and involving insideout ␣ IIb ␤ 3 signaling; and the other induced by platelet aggregation, most likely a pathway initiated by ␣ IIb ␤ 3 outside-in signaling.
Previous studies have shown that outside-in signaling in-FIG. 1. Identification of Shc as the major protein specifically eluting from a diphosphorylated ␤ 3 peptide column. Platelets were lysed in 1% Nonidet P-40 lysis buffer, and the majority of cytoskeletal proteins were removed by ultracentrifugation. The supernatant was run over three columns in series; Pre, avidin-agarose precolumn; ␤ 3 , the non-phosphorylated ␤ 3 peptide column; diP, the doubly phosphorylated ␤ 3 peptide column. Columns were washed separately, and proteins were eluted using 0.8 M phenyl phosphate. Samples from fractions eluted off of all three columns were run on 4 -15% SDSpolyacrylamide gels and silver-stained. The gel shown here represents fractions 17 through 20 and is representative of three independent experiments. The arrow indicates the migration position of a band that specifically eluted from the diphosphorylated ␤ 3 column. Fig. 1. Twenty-two different sequences (black boxes) were identified in the tryptic digest of the novel band in Fig. 1, which corresponded, all with 100% identity, to isoforms of Shc. The position of these isolated bands in the protein sequences of the three Shc isoforms is shown. volves, in part, the tyrosine phosphorylation of ␤ 3 . To determine whether outside-in signaling allows for the interaction of Shc with ␤ 3 , we determined whether ␤ 3 could be co-immunoprecipitated with Shc upon aggregation. The lower panel in Fig.  4 shows that ␤ 3 co-immunoprecipitated with Shc during the initial time points of platelet aggregation, at 20 s and at 45 s. In contrast, no ␤ 3 was detected in immunoprecipitates from thrombin-activated platelets (i.e. in the presence of eptifibatide), even though the activation does induce Shc tyrosine phosphorylation. Thus, outside-in signaling through ␣ IIb ␤ 3 induced Shc association with the integrin.

FIG. 2. Direct sequencing of the isolated protein band from
Shc Is Tyrosine-phosphorylated during LIBS Antibody-induced Aggregation-LIBS antibodies recognize determinants on ␣ IIb ␤ 3 , which induce a conformational change in the integrin that permits the binding of fibrinogen and platelet aggregation, without the requirement for platelet stimulation (24). Previous studies have established that ␤ 3 becomes tyrosine-phosphorylated during LIBS antibody-induced platelet aggregation (10). Fig. 5 shows that LIBS antibody-induced platelet aggregation also induces tyrosine phosphorylation of Shc. A significant increase in Shc tyrosine phosphorylation occurred at the first two time points of platelet aggregation in the presence of LIBS antibody, 1.9 Ϯ 0.1-fold 45 s after the addition of D3, 2.6-fold Ϯ 0.1 90 s after, and 2.6-fold Ϯ 0.2 270 s after. A reprobe of this blot with Shc polyclonal antibodies showed that the protein levels of Shc remained constant (middle panel). The same experiment conducted in the presence of eptifibatide or in the absence of either the LIBS antibody or fibrinogen, conditions that prevented platelet aggregation and outside-in signaling, also prevented the tyrosine phosphorylation of Shc. Thus, Shc phosphorylation occurred when platelet aggregation was induced solely by outside-in ␣ IIb ␤ 3 signaling.
We also investigated whether ␤ 3 co-immunoprecipitated with Shc during platelet aggregation without prior activation of the platelet by a strong agonist like thrombin. The lower panel in Fig. 5 also shows that ␤ 3 co-immunoprecipitated with Shc at time points 45, 90, and 270 s during LIBS-induced aggregation. Thus, platelet stimulation by outside-in ␣ IIb ␤ 3 signaling was sufficient to induce ␤ 3 -Shc interactions.
Recombinant Shc Binds to Phospho-␤ 3 Peptides-The coimmunoprecipitation of Shc with ␤ 3 following platelet aggregation suggests that Shc either binds directly to the tyrosinephosphorylated ␤ 3 or to other phospho-␤ 3 binding proteins. To determine whether the tyrosine-phosphorylated ICY domain of ␤ 3 has Shc recognition motifs, a GST-p52Shc fusion protein was expressed, purified, and studied for its ability to interact with the ␤ 3 ICY domain peptides. As depicted in Fig. 6, the GST-p52Shc fusion protein could be detected in peptide precipitates with the diphosphorylated and the C-terminal monophosphorylated ␤ 3 peptide using both anti-GST (Fig. 6A) and anti-Shc (Fig. 6B) antibodies. Little, if any, GST-Shc was present in precipitates obtained using the unphosphorylated ␤ 3 peptide, the ␤ 3 peptide monophosphorylated at the N terminus, and the diphosphorylated ␤ 3 scramble peptide. Fig. 6C depicts the binding capacity of the C-terminal monophosphorylated and the diphosphorylated ␤ 3 peptides for the purified Shc fusion protein. The diphosphorylated ␤ 3 peptide has a significantly greater binding capacity for Shc than the monophosphorylated peptide. Thus, Shc binds the ICY domain of ␤ 3 , but only when this domain is phosphorylated on ␤ 3 tyrosine residue 759.
Tyrosine Phosphorylation of Shc in Murine Platelets-As a genetic test of the hypothesis that phospho-Shc is the downstream component of ␤ 3 tyrosine phosphorylation during outside-in signaling, we assessed the level of Shc phosphorylation in platelets from the diYF mouse, where the native ␤ 3 gene is replaced by one lacking cytoplasmic tyrosines ((Y747F,Y759F) ␤ 3 ). Wild-type and diYF platelets were aggregated using a high FIG. 3. Western blotting analysis of samples eluted from the diphospho-␤ 3 column. Fractions 1 through 39 eluted from the ␤ 3 diP column using phenyl phosphate, were subjected to gel electrophoresis. The separated proteins were then transferred to nitrocellulose and subjected to immunoblotting with an anti-Shc antibody. Shc was detected in most of the fractions with peak elution being observed in fractions 19 through 24.

FIG. 4. Thrombin-induced aggregation and activation of human platelets result in tyrosine phosphorylation of Shc.
Shc immunoprecipitates of total platelet lysates (lysis time points Ϫ t 0 , 20 s, 45 s, 90 s, 270 s after addition of agonist) that were stimulated with 0.2 unit/ml thrombin plus stirring in the absence (aggregation) or presence (activation) of eptifibatide were subjected to gel electrophoresis and transferred to nitrocellulose. The blot was cut into two pieces at approximately 70 kDa, and the bottom part was subjected to immunoblotting with anti-phosphotyrosine antibodies (top panel), whereas the top part of the blot was probed with anti-␤ 3 antibody (lower panel) to demonstrate co-immunoprecipitation of ␤ 3 with Shc during aggregation. The middle panel shows the same blot as in the top panel, which was stripped and reprobed with an anti-Shc antibody to confirm equal levels of Shc protein between samples. dose of thrombin to induce comparable aggregation in both samples (7). As shown in Fig. 7, after 15 s of thrombin-induced aggregation, similar levels of total platelet protein phosphorylation were observed in platelets from both wild-type and diYF mice. However, immunoprecipitation experiments showed that the level of Shc phosphorylation was reduced by 2.8-fold in the aggregated platelet lysates from diYF compared with wild-type mice. These data are comparable to the findings on human platelets in Fig. 4, whereby initial thrombin-induced aggregation resulted in an approximately 2-fold increase in Shc phosphorylation than was observed during thrombin-induced activation.

DISCUSSION
Previous studies have established that tyrosine phosphorylation of the ICY domain of ␤ 3 is involved in outside-in signaling through ␣ IIb ␤ 3 in platelets (7). We hypothesized that protein(s) that bind to the phosphorylated ␤ 3 cytoplasmic tail could be identified by affinity chromatography. Direct protein sequencing of platelet proteins eluting from a diphosphorylated ␤ 3 peptide column identified Shc (25) as the primary protein that associated in a phosphotyrosine-dependent manner with ␤ 3 . A direct role for Shc in outside-in signaling was established by showing that it was transiently phosphorylated during thrombin-and LIBS-induced aggregation. Shc was found to bind directly to ␤ 3 cytoplasmic peptides either phosphorylated at only the C-terminal tyrosine or phosphorylated at both tyrosines, in the absence of other platelet proteins. In platelets from mice expressing a mutant (Y747F,Y759F) ␤ 3 , which could not be tyrosine-phosphorylated, tyrosine phosphorylation of Shc was significantly reduced. The results from these studies identify Shc as a direct signaling partner for ␣ IIb ␤ 3 , and implicate this molecule in downstream signaling events directly involved in human platelet aggregation.
Numerous strategies have been developed to identify signaling partners of membrane receptors (reviewed in Refs. 26 -29), but we elected to use affinity chromatography, because platelets can be isolated in high yield and they contain high concentrations of signaling proteins (3), and we wanted to identify the primary protein(s) that bind tyrosine-phosphorylated ␤ 3 . Previous studies have established that phosphopeptides modeled on the sequence of membrane receptors are useful for identifying signaling partners (9,31,32). The first of three assumptions made in the affinity approach used in the present study was that diphosphorylated ␤ 3 is generated during platelet aggregation. It has been established that ␤ 3 becomes associated with the myosin-based platelet cytoskeleton following platelet aggregation (19). Because only ␤ 3 peptides that are phosphorylated on both tyrosines bind to purified myosin (9), the data suggest that both tyrosines can become phosphorylated upon platelet aggregation. To identify as many tyrosine phosphorylation-dependent signaling partners of ␤ 3 in platelets as possible, we therefore used the diphosphorylated ␤ 3 peptide in the affinity matrix. Our second assumption was that the bound, phosphorylated ␤ 3 signaling partners would be eluted by phenyl phosphate, a reagent previously shown to disrupt interactions of anti-phosphotyrosine antibodies with tyrosine-phosphorylated proteins (33,34). The third assumption was that proteins capable of binding to diphosphorylated ␤ 3 were present in detergent lysates of unstimulated platelets. This assumption is based on findings showing that phosphorylation of ␤ 3 occurs only upon platelet aggregation (10), suggesting that prior activation of phosphotyrosine binding domains of signaling proteins was not required for binding to the phosphorylated integrins.
The affinity chromatography approach described herein identified the p52 isoform of Shc as the primary protein in human platelets that binds to the phosphorylated ICY domain peptide of ␤ 3 . A previous study found that, of the three Shc isoforms, only p52Shc is present in platelets. In support of this finding, our data using Western analysis of platelet lysates and of the isolated protein detected only p52 Shc and not p46 or p66 isoforms. Also, sequence analysis of the tryptic peptides produced from the isolated Shc only revealed peptides contained within the p52 isoform of the protein.
FIG. 6. Direct binding of recombinant GST-p52Shc protein to phospho-␤ 3 cytoplasmic region peptides. A, Western blot with anti-GST antibody; B, Western blot with anti-Shc antibody; recombinant proteins isolated from E. coli extracts were incubated with biotinylated ␤ 3 peptides. The peptides, and any associated protein, were precipitated using avidin-agarose beads. Lanes in A and B: MW: molecular weight marker; 1, purified GST protein; 2, purified GST-p52Shc protein; 3, GST-p52Shc ϩ ␤ 3 peptide; 4, GST-p52-Shc ϩ NP␤ 3 peptide; 5, GST-p52-Shc ϩ CP␤ 3 peptide; 6, GST-p52-Shc ϩ scrambled diP␤ 3 peptide; 7, GST-p52-Shc ϩ diP␤ 3 peptide; 8, GST ϩ ␤ 3 peptide; 9, GST ϩ NP␤ 3 peptide; 10, GST ϩ CP␤ 3 peptide; 11, GST ϩ scrambled diP␤ 3 peptide; 12, GST ϩ diP␤ 3  Previous studies showed that Shc becomes tyrosine-phosphorylated upon thrombin-induced platelet aggregation and that Shc bound to diphosphorylated peptides (9). Three biochemical studies of platelets have now established that Shc is a proximal component of the phosphotyrosine ␤ 3 signaling pathway. First, because one of the hallmarks of Shc involvement in a signaling pathway is that it becomes tyrosine-phosphorylated (reviewed in Ref. 35), our data demonstrating that tyrosine phosphorylation of Shc occurs upon LIBS-induced aggregation places Shc directly on the ␣ IIb ␤ 3 outside-in signaling pathway. LIBS antibodies are known to induce fibrinogen binding to ␣ IIb ␤ 3 by inducing a conformational change in ␣ IIb ␤ 3 without the prior requirement of activation by a platelet agonist. Thus, these data indicate that Shc phosphorylation can be specifically induced by outside-in ␣ IIb ␤ 3 signaling. Shc phosphorylation in response to thrombin differed from that observed in response to the LIBS antibody in that phosphorylation was induced upon thrombin-induced activation, with further phosphorylation occurring upon subsequent platelet aggregation.
A second hallmark of the involvement of Shc in a signaling pathway is its physical association with the cell membrane receptor responsible for initiating the signaling cascade (for example, with platelet-derived growth factor receptor or components of the B cell receptor complex). In the present study, we found that ␤ 3 could be co-immunoprecipitated with Shc from detergent lysates of aggregated platelets but not from lysates of control, unstimulated platelets or stimulated but not aggregated platelets, demonstrating that aggregation induced an association of Shc with this integrin. Third, we have provided genetic evidence implicating Shc in the ␤ 3 tyrosine phosphorylation signaling pathway. Platelets from the diYF mouse are defective in outside-in ␣ IIb ␤ 3 signaling and form unstable platelet aggregates due to the expression of a mutant ((Y747F,Y759F) ␤ 3 ). Because diYF platelets can be induced to aggregate using high doses of potent agonists such as thrombin, even though the diYF ␤ 3 cannot be tyrosine-phosphorylated, the diYF platelet can be used to determine whether Shc phosphorylation is downstream of ␤ 3 tyrosine phosphorylation. In the diYF platelets, Shc did not show the same increase in elevated tyrosine phosphorylation upon platelet aggregation seen in wild-type platelets. Thus, the functional data on human and mouse platelets support the affinity chromatography data showing that Shc is a primary mediator of the tyrosine phosphorylation signaling pathway in platelets.
It was not known whether Shc bound directly to tyrosinephosphorylated ␤ 3 or whether this interaction was bridged by another adaptor protein. The possibility of a bridging protein was raised by previous studies showing that caveolin-1 could function as an adapter protein, linking signaling proteins like Fyn and Shc to integrin ␣ subunits upon integrin ligation (36). However, we were unable to detect caveolin-1 either in the fractions obtained from the affinity columns or in the Shc immunoprecipitates (data not shown), suggesting that caveolin-1 in platelets neither bound to Shc nor to the integrin ␤ 3 cytoplasmic tail. The possibility of a direct interaction between Shc and ␤ 3 was suggested by the fact that Shc contains two phosphotyrosine binding domains, an SH2 and a PTB, the two tyrosines in the ICY domain of ␤ 3 exist in NXXY motifs, well known to be recognized by PTB domains when phosphorylated (37)(38)(39)(40)(41)(42). We tested the hypothesis that Shc binds directly to tyrosine-phosphorylated ␤ 3 by using recombinant Shc protein and tyrosine-phosphorylated ␤ 3 ICY domain peptides. Our experiments demonstrated a direct interaction between Shc and phospho-␤ 3 . Thus, because Shc was isolated by affinity chromatography without other detectable proteins, direct binding of Shc to ␤ 3 can be demonstrated, and phospho-␤ 3 has motifs that are predicted to be recognized by the PTB domain of Shc, it appears that Shc is a direct signaling partner of tyrosinephosphorylated ␤ 3 .