Tyrosine Phosphorylation of the beta 3 Cytoplasmic Domain Mediates Integrin-Cytoskeletal Interactions*

Alison L. JenkinsDagger §, Lisa Nannizzi-AlaimoDagger §, Debra Silver, James R. Sellers, Mark H. Ginsbergparallel , Debbie A. LawDagger , and David R. PhillipsDagger **

From Dagger  COR Therapeutics, Inc., South San Francisco, California 94080, the  Section of Cellular and Molecular Motility, Laboratory of Molecular Cardiology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-1762, and the parallel  Scripps Research Institute, La Jolla, California 92037

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Tyrosine phosphorylation of the beta 3 subunit of the major platelet integrin alpha IIbbeta 3 has been shown to occur during thrombin-induced platelet aggregation (1). We now show that a wide variety of platelet stimuli induced beta 3 tyrosine phosphorylation, but that this phosphorylation occurred only following platelet aggregation. Several lines of evidence suggest that the beta 3 cytoplasmic domain tyrosine residues and/or their phosphorylation function to mediate interactions between beta 3 integrins and cytoskeletal proteins. First, phospho-beta 3 was retained preferentially in a Triton X-100 insoluble cytoskeletal fraction of thrombin-aggregated platelets. Second, in vitro experiments show that the cytoskeletal protein, myosin, associated in a phosphotyrosine-dependent manner with a diphosphorylated peptide corresponding to residues 740-762 of beta 3. Third, mutation of both tyrosines in the beta 3 cytoplasmic domain to phenylalanines markedly reduced beta 3-dependent fibrin clot retraction. Thus, our data indicate that platelet aggregation is both necessary and sufficient for beta 3 tyrosine phosphorylation, and this phosphorylation results in the physical linkage of alpha IIbbeta 3 to the cytoskeleton. We hypothesize that this linkage may involve direct binding of the phosphorylated integrin to the contractile protein myosin in order to mediate transmission of force to the fibrin clot during the process of clot retraction.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Integrins are a family of heterodimeric transmembrane proteins that link the extracellular matrix to the cytoskeletal/contractile apparatus within a cell (2). These cytoskeletal linkages are characteristically induced by integrin clustering that can occur by the binding of multivalent or immobilized extracellular ligands, often resulting in the assembly of "focal contacts" in cultured cells. Several cytoskeletal proteins (e.g. talin, actin binding protein, alpha -actinin) directly bind to integrin cytoplasmic domains (3-6), indicating that integrins may interact by multiple mechanisms for focal contact assembly. Focal contact assembly is often followed by signal transduction events such as induction of gene transcription (7) and prevention of apoptosis (8, 9), regulating a diversity of cellular functions from embryonic development to hemostasis (10). Although cytoskeletal and signaling proteins have been identified which bind integrin cytoplasmic domains, major unsolved questions persist. For example, what is the identity of the proteins responsible for the initial interactions of integrins with the cytoskeletal structures? How are these interactions regulated? In this regard, it is relevant to the present study that focal contacts are major sites for protein tyrosine phosphorylation, one of the earliest signaling events observed upon integrin ligation (11).

On platelets, the interaction of the integrin alpha IIbbeta 3 with its adhesive ligands, fibrinogen, or von Willebrand factor leads to platelet aggregation and association of alpha IIbbeta 3 with the cytoskeleton (12, 13). Under normal conditions, platelet aggregation is the desired response to external trauma, allowing for hemostasis. However, inappropriate platelet aggregation does occur, as in ruptured artherosclerotic plaques, resulting in the formation of occlusive thrombi leading to myocardial infarction or thrombolytic stroke (14). The importance of alpha IIbbeta 3-mediated events in both hemostasis and thrombosis is underscored in two ways. First, patients who lack, or have mutated, alpha IIbbeta 3, a condition known as Glanzmann's thrombasthenia, have a bleeding disorder that arises from the failure of the platelets to aggregate (15). Second, clinical trials have shown that antagonists for alpha IIbbeta 3 ligand binding are effective antithrombotics (16).

alpha IIbbeta 3 is involved in both "inside-out" and "outside-in" signaling pathways during platelet aggregation (12). In order to bind soluble forms of its adhesive protein ligands, alpha IIbbeta 3 on resting platelets has to undergo a conformational change. This process, the consequence of "inside-out" alpha IIbbeta 3 signaling, occurs when agonists such as ADP or thrombin activate platelets. Binding of fibrinogen and von Willebrand factor to alpha IIbbeta 3 induces platelet aggregation and alpha IIbbeta 3 clustering: the signals transduced by this process are referred to as "outside-in" signaling events. The cytoplasmic domains of the integrin are thought to play a critical role in these signaling events (17-20). In addition, platelet aggregation induces the direct interaction of alpha IIbbeta 3 with the cytoskeleton (21, 22). The cytoskeletal proteins talin and alpha -actinin have been found to act directly with alpha IIbbeta 3 (4, 6). Along with alpha IIbbeta 3, many other intracellular proteins, including Src and FAK, redistribute to the cytoskeleton of aggregated platelets (22, 23). In these ways, the integrin may play a direct role not only in organizing the cytoskeleton but also in transducing signals to elicit cellular responses. Although it is clear that the cytoplasmic domains of alpha IIbbeta 3 are involved in signal transduction and cytoskeletal reorganization events, the precise mechanisms regulating these processes remain to be discovered.

Previously, we showed that tyrosine phosporylation of beta 3 occurs upon thrombin-induced platelet aggregation, indicating a potential role for integrin cytoplasmic tyrosine residues in outside-in alpha IIbbeta 3 signaling (1). In support of this hypothesis, we observed that the signaling adaptor proteins SHC and Grb2 interacted with peptides corresponding to the tyrosine phosphorylated cytoplasmic domain of beta 3 (1). The present study shows that tyrosine phosphorylation of beta 3 is a unifying event of platelet aggregation and provides in vitro evidence that tyrosine phosphorylation of this integrin subunit may direct its binding to myosin, a specific element contained within the platelet cytoskeleton.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Reagents-- Aprotinin, apyrase, aspirin, prostaglandin I2, phenylmethylsulfonyl fluoride, leupeptin, diisopropyl fluorophosphate, sodium orthovanadate, sodium pyrophosphate, the thromboxane A2 analog 9,11-dideoxy-11a,9a-epoxymethanoprostaglandin F2a (U46619), control mouse IgG, and Sigmacote were all purchased from Sigma. The platelet aggregation reagents ADP, epinephrine, and collagen were purchased from Sigma Diagnostics. Thrombin receptor activating peptide SFLLRN-NH2 (TRAP; Ref. 24) and the cyclic peptide alpha IIbbeta 3 antagonist Mpr-RGDWP-Pen-NH2 (25) were provided by COR Therapeutics, Inc., Medicinal Chemistry Department. Human alpha -thrombin was purchased from Hematologic Technologies, Inc., and human fibrinogen was from Chromogenix. The alpha vbeta 3 antibody LM609 was generously provided by David Cheresh (Scripps Research Institute) (26). The anti-LIBS6 monoclonal antibody has been described (27). Anti-human beta 3 monoclonal antibody C3a.19.5, which recognizes the cytoplasmic tail of the beta 3 subunit, was described previously (1). Anti-myosin monoclonal antibody was from Immunotech, Inc. The anti-phosphotyrosine antibodies PY-20 and 4G10 were from Transduction Laboratories and Upstate Biotechnology, Inc., respectively. The horseradish peroxidase-conjugated secondary reagent sheep anti-mouse Ig and horseradish peroxidase-conjugated streptavidin were from Amersham Pharmacia Biotech. Hank's buffered saline solution and Dulbecco's modified Eagle's medium were from Life Technologies, Inc. Goat anti-mouse fluorescein isothiocyanate-conjugated F(ab')2 was from Jackson and Gamimmune N was from Miles. Biotinylated integrin cytoplasmic domain peptides, synthesized by SynPep Corporation using solid phase Fmoc (N-(9-fluorenyl) methoxycarbonyl) chemistry, were dissolved at a concentration of 2 mg/ml in water and diluted as needed. Chymotrypsin and papain were from Boehringer Mannheim. 4-20% gradient SDS-PAGE 1 gels were from Bio-Rad, and ECL Hybond nitrocellulose was from Amersham Pharmacia Biotech. See-blue molecular mass standards were purchased from Novex.

Platelet Preparation and Aggregation-- Blood from healthy volunteers was drawn on the day of use, and washed platelets were prepared as described previously (28) except 0.6 units/ml apyrase and 50 ng/ml prostaglandin I2 (final concentrations) were present in the collecting solution. Before stimulation, the platelets (~4-8 × 108/ml) were incubated for 1 h at 37 °C in Tyrodes-HEPES buffer (12 mM NaHCO3, 138 mM NaCl, 5.5 mM glucose, 2.9 mM KCl, 10 mM HEPES, pH 7.4, 1 mM CaCl2, 0.5 mM MgCl2) unless otherwise stated. Platelet samples of 0.5 ml were then stirred at 37 °C in a whole blood lumiaggregometer, and various agonists and conditions were examined. When platelet lysates were not prepared, 4× nonreducing Laemmli sample buffer containing vanadate (37 mM Tris, pH 6.8, 11.8% (v/v) glycerol, 2.36% (w/v) SDS, 2 mM sodium orthovanadate, and 0.002% (w/v) bromphenol blue (final concentration)) was added immediately after aggregation, and samples were boiled for 5 min.

Platelet Lysate Preparation-- For two-dimensional gel analysis of detergent-soluble and cytoskeletal fractions, platelets were lysed immediately after aggregation by the addition of an equal volume of ice-cold 2× Triton X-100 lysis buffer (1% (v/v) Triton X-100, 100 mM NaCl, 20 mM Tris, pH 7.0, 2 mM EDTA, 2 mM ethyleneglycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid, 20 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 200 µM leupeptin, 4 mM sodium orthovanadate, 2 mM benzamidine, 0.27 mM diisopropyl fluorophosphate, 5 mM sodium pyrophosphate (final concentrations)). The lysate was then centrifuged for 6 min at 15,000 × g to remove the Triton X-100 insoluble cytoskeletons formed during aggregation (22). The supernatant was reserved and 100 µl of 2× RIPA buffer (see below) was added to the pellet and sonicated for 20 min at room temperature in a Branson 5120 Sonicator to resolubilize the pellet. Nonreducing sample buffer (as described above) was added to each of the samples (supernatant and resolubilized pellet) and boiled for 5 min.

For ligand blot analysis, platelets were lysed in RIPA buffer (1% (w/v) Triton X-100, 1% (w/v) deoxycholic acid, 0.1% sodium dodecyl sulfate, 5 mM ethylenediamine-tetraacetic acid, 20 mM Tris pH 7.5, 5 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 75 µg/ml leupeptin, 20 µg/ml aprotinin (final concentrations)). For resolution of detergent soluble from cytoskeletal fractions for ligand blots, platelets were lysed in Triton X-100 buffer (1% (v/v) Triton X-100, 137 mM NaCl, 2 mM ethylenediamine-tetraacetic acid, 20 mM Tris pH 8, 5 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 75 µg/ml leupeptin, 20 µg/ml aprotinin (final concentrations)) and cytoplasmic actin filaments were sedimented by centrifugation at 15 600 × g for 15 min at 4 °C (22).

Determination of beta 3 Phosphorylation Level-- Nonreduced-reduced two-dimensional gel electrophoresis was performed to visualize the characteristic migration of beta 3 and assess its phosphorylation state as described previously (1, 29). The two-dimensional gels were transferred to nitrocellulose, and blots were probed with anti-phosphotyrosine antibodies PY-20 and 4G10. The blots were washed and incubated with horseradish peroxidase-conjugated sheep anti-mouse Ig and developed using the Enhanced Chemiluminescent (ECL) System. The level of phosphorylation was determined by densitometry using Imagequant software on a Molecular Dynamics densitometer. In each case, three to five different ECL exposures were subjected to densitometry analysis to reduce the risk of erroneous results from nonlinear signals. The blots were then stripped (according to ECL protocol, Amersham Pharmacia Biotech) and reprobed with the beta 3 antibody C3a.19.5 to determine beta 3 protein content for each sample. Phosphorylation results were normalized for the total amount of beta 3 protein present.

Preparation of Proteins-- Myosin was purified from human platelets as described, and purification yielded myosin heavy chain and light chains (30). Controlled proteolytic digests of platelet myosin with papain or chymotrypsin were performed as described (31) except that myosin was not phosphorylated prior to chymotryptic digestion. Papain was activated according to the instructions of the manufacturer. Digests were run on 4-20% SDS-PAGE and subjected to Coomassie Blue staining or transferred to nitrocellulose for ligand blotting.

Ligand Blot Analysis-- Platelet lysates or purified myosin were reduced, separated by SDS-PAGE, and transferred to nitrocellulose. The blots were wet briefly in HEPES blot buffer (HBB) (25 mM HEPES, 25 mM NaCl, 5 mM MgCl2, 1 mM dithiothreitol) at 4 °C. The transferred proteins were denatured by 6 M guanidine HCl in HBB for 10 min at 4 °C and renatured by 2-fold dilution of guanidine HCl (10-min incubations each with 3 M, 1.5 M, 0.75 M, 0.38 M, 0.19 m, and 0 M guanidine HCl in HBB). The blot was blocked in HBB containing 4% bovine serum albumin overnight at 4 °C and probed with 1 µM biotinylated peptide in HBB containing 0.5% bovine serum albumin for 3 h at room temperature. After washing in Tris-buffered saline (20 mM Tris, 150 mM NaCl, pH 7.4)/ 0.01% Nonidet P-40 three times at 4 °C, peptide-reactive bands were visualized by incubating the blots in horseradish peroxidase-conjugated streptavidin and employing ECL detection.

CHO Cell Generation and Flow Cytometry-- beta 3(Y747F, Y759F) was generated and stably transfected into CHO cells as described (32). For flow cytometric analysis, cells were detached with trypsin, washed in Dulbecco's modified Eagle's medium + 25 mM HEPES once and resuspended at 3 × 106 cells/ml in FACS buffer (Hanks buffered saline solution with 3% heat-inactivated fetal bovine serum, 1% bovine serum albumin, 1% normal goat serum, 0.1% Gamimmune N, 0.03% sodium azide). The cells were then seeded at 200 µl/well, pelleted, and incubated with 5 µg/ml primary antibodies LM609 or control mouse IgG for 1 h at 4 °C. After two washes, the cells were incubated with 1:200 goat anti-mouse fluorescein isothiocyanate-conjugated F(ab')2 for 30 min at 4 °C. The cells were washed and resuspended in FACS buffer, and the samples were analyzed by flow cytometry on a FACSort (Becton Dickinson).

Clot Retraction Assays-- Clot retraction experiments were performed as described with minor modifications (33). In brief, cells were trypsinized, washed twice, and resuspended in Dulbecco's modified Eagle's medium + 25 mM HEPES. 0.5 ml of cell suspension containing 5 × 106 cells was mixed with 0.1 ml of fibronectin-depleted plasma in a 12 × 70-mm glass tube treated with Sigmacote. Fibrin clots were formed by adding 1 unit/ml thrombin and allowed to retract at 37 °C over a 2-3-h period. The extent of clot retraction was measured by removing and weighing the clot.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

beta 3 Tyrosine Phosphorylation Is a General Consequence of Platelet Aggregation-- We previously reported that aggregation of platelets by thrombin in a stirred suspension induced a marked increase in the tyrosine phosphorylation of the beta 3 subunit of alpha IIbbeta 3 (1). To determine whether this effect was thrombin-specific, we examined beta 3 tyrosine phosphorylation in response to various agonists. Adding ADP or ADP + epinephrine to a stirred suspension of platelets in the presence of added fibrinogen induced platelet aggregation and an increase in beta 3 phosphorylation, similar to that seen in thrombin-aggregated platelets (Table I). In contrast, when ADP was added in the absence of added fibrinogen or was added without stirring, no platelet aggregation occurred and no increase in beta 3 tyrosine phosphorylation was seen. ADP added in this manner did, however, induce platelet stimulation since other substrates were tyrosine phosphorylated and fibrinogen binding on unstirred preparations occurred (data not shown). An illustration of the increase in beta 3 tyrosine phosphorylation upon thrombin- or ADP-induced platelet aggregation is shown in Fig. 1A. Thus, ADP and ADP + epinephrine induced tyrosine phosphorylation of beta 3 in an aggregation-dependent manner.

                              
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Table I
beta 3 tyrosine phosphorylation is induced by platelet aggregation
Washed platelets were prepared as described under "Experimental Procedures," except that calcium was omitted from the final buffer in the collagen sample and magnesium was omitted and 100 µM aspirin and 0.33 units/ml apyrase were added to the final buffer in the LIBS6 samples. Platelets (approximately 5 × 108/ml) were stimulated at 37 °C and allowed to stir in the aggregometer for 0.5-8 min with the following agonist concentrations: 0.1 units/ml thrombin, 1 µM TRAP, 4-16 µM ADP, 2 µM epinephrine, 0.2 mg/ml collagen, 5 µl LIBS6 ascites, 1 µM U44619. Fibrinogen was added to the indicated samples at a final concentration of 0.25-0.5 mg/ml. In the indicated samples, the cyclic RGD peptide (1 µM) was added to inhibit aggregation. Nonreducing sample buffer containing vanadate was added immediately after aggregation to terminate the reaction. The level of beta 3 phosphorylation was assessed by densitometry after transferring nonreduced-reduced two-dimensional gels to nitrocellulose and probing with anti-phosphotyrosine antibodies. The blots were stripped and reprobed with the anti-beta 3 monoclonal antibody C3a.19.5 to determine protein content. The key to the -fold increase of beta 3 phosphorylation is as follows: 0, no increase in phosphorylation; +, 2-6-fold increase; ++, 7-14-fold increase in phosphorylation as compared with control unstimulated platelets. Densitometry was performed on multiple exposures of the same blot, and similar results were obtained in at least two separate experiments.


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Fig. 1.   beta 3 is dephosphorylated upon reversal of aggregation. Washed platelets were prepared and resuspended in Tyrodes-HEPES buffer containing calcium and 0.33 units/ml apyrase. Aggregation was performed at 37 °C while stirring in the aggregometer, and the reaction was immediately stopped by the addition of 4× nonreducing sample buffer and analyzed by two-dimensional gel electrophoresis as described under "Experimental Procedures." Panel A is the anti-phosphotyrosine immunoblot; panel B shows the identical blot reprobed for beta 3 protein with C3a.19.5 monoclonal antibody; and panel C represents the aggregometer tracing from each sample. The platelet conditions are as follows: lane 1, control unstimulated; lane 2, 3-min aggregation (thrombin 0.1 units/ml); lane 3, 35-s aggregation (16 µM ADP containing 0.4 mg/ml fibrinogen); lane 4, 8-min ADP/fibrinogen aggregation allowing for reversal of aggregation. Duplicate experiments with 10 µM ADP or 8-min thrombin aggregation had similar results (data not shown). Molecular mass standards are indicated to the left.

Threshold concentrations of ADP can result in reversible platelet aggregation (34, 35). We next determined the effect of reversal of ADP-induced aggregation on beta 3 tyrosine phosphorylation. Washed platelets, resuspended in a buffer containing fibrinogen and Ca2+, were stirred with ADP. Platelet aggregation occurred but, as illustrated in Fig. 1C, reversed with time. The reactions were terminated by the addition of SDS-sample buffer to the aggregometer tubes either at maximal ADP-induced aggregation or after aggregation was fully reversed (Fig. 1C). As described above, ADP-induced aggregation led to an increase in beta 3 phosphorylation, as well as the phosphorylation of a number of other substrates, similar to the response induced by thrombin (Fig. 1A). When samples were obtained in which ADP-induced platelet aggregation had reversed, no tyrosine phosphorylation of beta 3 was observed (Fig. 1A). In contrast, when thrombin-induced platelet aggregates were maintained in suspension for up to 10 min prior to addition of SDS-sample buffer, no reversal of beta 3 tyrosine phosphorylation was observed (data not shown).

Platelets aggregated in response to stimulation through the collagen or thromboxane A2 receptors also showed a marked increase in beta 3 tyrosine phosphorylation (Table I). However, if platelet aggregation was prevented by cyclic RGD peptide (a competitive inhibitor of alpha IIbbeta 3), beta 3 phosphorylation was not observed (Table I). Together, these data indicate that many platelet agonists can induce aggregation-dependent tyrosine phosphorylation of beta 3.

In the previous experiments, platelet agonists were used to induce fibrinogen binding and subsequent aggregation. To determine whether beta 3 tyrosine phosphorylation can occur in the absence of an agonist, platelet aggregation was induced by anti-LIBS6, a beta 3-specific antibody that, upon binding to beta 3, activates the receptor such that it becomes competent to bind fibrinogen. This activation occurs even in the absence of detectable platelet stimulation (27). The LIBS6 antibody induced smaller platelet aggregates, and aggregation took slightly longer than that induced by classical platelet agonists. Nevertheless, an average 4-fold increase in the level of beta 3 phosphorylation was still observed (Table I). Again, the addition of cyclic RGD peptide inhibited both platelet aggregation and beta 3 phosphorylation. Thus, alpha IIbbeta 3-dependent platelet aggregation is both necessary and sufficient for tyrosine phosphorylation of beta 3.

Tyrosine Phosphorylated beta 3 Preferentially Redistributes to the Cytoskeleton in Aggregated Platelets-- To gain insight into the functional significance of tyrosine phosphorylation of beta 3, we assessed its effects on the partitioning of the receptor with the cytoskeleton, a process known to occur upon platelet aggregation (21, 22). Platelets were aggregated by the addition of thrombin and lysed with Triton X-100 lysis buffer. Under the solubilization conditions described under "Experimental Procedures," approximately 34% (p = 0.002) of the total beta 3 protein associated with the cytoskeletal fraction upon aggregation, in agreement with earlier studies (21, 22). Notably, previous work has established that only about 5% of the alpha IIbbeta 3 is found in the Triton X-100-insoluble residue of unstimulated platelets (21). Densitometry of anti-phosphotyrosine immunoblots indicated that approximately 72% of the tyrosine-phosphorylated beta 3 redistributes to the cytoskeletal fraction (Fig. 2). Thus, tyrosine-phosphorylated beta 3 is more than twice as likely to become associated with the cytoskeleton (p = 0.018), which may indicate a pivotal role for this beta 3 modification in linking a ligand-occupied receptor on the surface of aggregated platelets to the cytoskeletal/contractile apparatus within.


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Fig. 2.   beta 3 preferentially redistributes to the cytoskeletal fraction following thrombin-induced platelet aggregation. Lysates were prepared from platelets stimulated with 0.1 units/ml thrombin that were allowed to aggregate for 8 min at 37 °C, and Triton X-100-insoluble platelet cytoskeletons were isolated as described under "Experimental Procedures." The supernatant fraction or the insoluble pellet (which was aggressively solubilized) from thrombin-aggregated platelets were subjected to two-dimensional gel analysis as described. This graph depicts the average of the ratio of beta 3 phosphorylation (with basal phosphorylation subtracted)/total amount of beta 3 present in samples as determined by densitometry of five separate experiments. A Student's t test (1-tailed) yielded a p value of 0.018.

Doubly Phosphorylated Cytoplasmic Domain beta 3 Peptide Binds a 200-kDa Protein Identified as Platelet Myosin-- The above observation, that tyrosine-phosphorylated beta 3 preferentially partitions with the cytoskeleton, highlighted the possibility that phosphorylation of beta 3 could be involved in mediating interactions between beta 3 and elements within the cytoskeleton. We have found, as have others (3, 4, 6), that the poor detergent solubility of many of the components of the cytoskeleton makes it technically difficult to observe interactions between cytoskeletal and other proteins in vivo. Thus, to address this issue, we employed an in vitro ligand blotting approach in an attempt to identify candidate proteins that, by binding to tyrosine-phosphorylated cytoplasmic domain of beta 3, could direct association of alpha IIbbeta 3 to the cytoskeleton. Proteins from platelet lysates were separated by SDS-PAGE, transferred to nitrocellulose and renatured on the blot. Synthetic peptides corresponding to the cytoplasmic domain of beta 3, which contained biotin at their amino termini, were used to probe the nitrocellulose blot (Fig. 3A). The direct binding of peptide to renatured proteins was visualized by the addition of streptavidin-horseradish peroxidase and detected by chemiluminescence. As illustrated in Fig. 3B, the beta 3 peptide corresponding to residues 740-762 in the beta 3 cytoplasmic domain bound to a 200-kDa protein in ligand blot analysis of platelet lysates. Binding was detected only when both beta 3 tyrosine residues (Tyr-747 and Tyr-759) were phosphorylated; the nonphosphorylated beta 3 peptide and singly phosphorylated peptides failed to bind the 200-kDa protein under the conditions of this assay (data not shown). To test the specificity of binding of the phosphorylated cytoplasmic domain of beta 3 to this 200-kDa protein, a second, doubly phosphorylated beta 3 peptide containing a naturally occurring single point mutation of beta 3 found in a patient with Glanzmann's thrombasthenia, was used (36). alpha IIbbeta 3 harboring this mutation does not support platelet aggregation (36) and is defective in signaling (37). Despite having two phosphorylated tyrosine residues, the S752P Glanzmann's mutant peptide failed to bind the 200-kDa protein in renatured blots. Furthermore, an unrelated diphosphorylated peptide based on the sequence of the zeta  chain of the T cell receptor complex containing an immune receptor tyrosine-based activation motif (ITAM) domain, was also negative in this assay.


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Fig. 3.   Binding of beta 3 cytoplasmic domain peptides to platelet proteins. A, single letter amino acid sequence of biotinylated peptides used as probes in ligand blots. B, ligand blots of platelet proteins. Platelet lysates were run on 7.5% SDS-PAGE, transferred to nitrocellulose, renatured, and probed with the indicated biotinylated peptides (1 µM) using the ligand blot protocol as described under "Experimental Procedures."

This 200-kDa protein partitioned almost exclusively to the Triton X-100 insoluble cytoskeletal fraction of thrombin-aggregated platelets (data not shown) and appeared, therefore, to be an integral component of the platelet cytoskeleton. Together, its cytoskeletal distribution and apparent molecular mass suggested that the 200-kDa protein was the heavy chain of platelet myosin. To test this hypothesis, myosin was purified from platelets and examined in the ligand blot assay. Myosin heavy chain displayed the same peptide binding specificity as the 200-kDa protein in platelet lysates in that it bound the di-phosphorylated beta 3 peptide but not the nonphosphorylated beta 3 peptide, the diphosphorylated S752P peptide or the diphosphorylated ITAM peptide (Fig. 4B and data not shown). A similar peptide-binding specificity was also observed in solid phase assays, in which platelet myosin was coated on plates and binding of the various biotinylated peptides were detected enzymatically with horseradish peroxidase-conjugated streptavidin (data not shown). Phenylphosphate, a compound known to compete for phosphotyrosine binding sites (38), was used to further establish that the beta 3 phosphotyrosines were required for the interaction of beta 3 with myosin. 10 mM phenylphosphate inhibited completely the binding of the diphosphorylated beta 3 peptide to purified myosin in renatured blots (Fig. 4C), indicating that the beta 3-myosin interaction was phosphotyrosine-dependent under these binding conditions. To further demonstrate the specificity of binding and to rule out nonspecific charge effects, a beta 3 peptide with tyrosines 747 and 759 replaced by glutamates also failed to bind purified myosin (data not shown). Taken together, these observations establish the identity of the 200-kDa protein as myosin heavy chain and demonstrate that its direct interaction with the beta 3 cytoplasmic domain peptide is phosphotyrosine-dependent.


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Fig. 4.   Binding of beta 3 cytoplasmic domain peptides to purified platelet myosin. Myosin was purified from human platelet concentrates as described. Approximately 10 µg of purified platelet myosin (lane 1) and 40 µg of platelet lysate (lane 2) were analyzed by immunoblot with anti-myosin antibodies (panel A; 1 µg/ml) or probed with the diphosphorylated (di- with circled P) beta 3 peptide (panel B; 1 µM). C, phosphotyrosine specificity of diphosphorylated beta 3 peptide binding. Binding of the diphosphorylated beta 3 peptide to purified myosin was inhibited by phenylphosphate (10 mM).

Further ligand blotting experiments were performed using fragments of myosin generated by controlled proteolytic cleavage (31). Cleavage of myosin with papain yields single-headed soluble subfragment-1 (S-1) and an insoluble coiled-coil rod fragment, whereas cleavage with chymotrypsin results in double-headed heavy meromyosin and coiled-coil light meromyosin (31). We found that the doubly phosphorylated beta 3 peptide bound to the rod portion of the papain digest and the light meromyosin fragment in chymotrypsin-digested myosin (not shown). Neither the unphosphorylated beta 3 peptide nor the diphosphorylated S752P beta 3 peptide bound to any of these myosin fragments. Since the chymotryptic light meromyosin fragment and the rod portion generated by papain cleavage both contain overlapping sequences within the tail region of myosin (31), the data indicate that this region is responsible for binding the diphosphorylated beta 3 cytoplasmic domain.

The Tyrosine Residues within the beta 3 Cytoplasmic Domain Are Important for beta 3-dependent Fibrin Clot Retraction-- The results reported above indicate that the phosphorylation of the beta 3 cytoplasmic tyrosine residues might influence integrin-cytoskeletal interactions. This finding predicts that mutating the tyrosine residues of beta 3 should affect cellular functions dependent upon integrin-cytoskeletal interactions. One such function is the beta 3-dependent retraction of fibrin clots, where the integrin is believed to function as a transmembrane linkage between extracellular adhesion proteins and the cytoskeleton. Due to the difficulty of genetically manipulating platelets, we used a CHO cell system that has proven useful in the study of integrin function (19, 39) to directly address this issue. It has previously been shown that CHO cells transfected with wild-type beta 3 will express the beta 3 on the cell surface in conjunction with endogenous alpha v chains (19). In contrast to nontransfected CHO cells, the beta 3-transfected CHO cells gain the ability to retract fibrin clots (Ref. 19, and data not shown). We generated stable CHO cell lines expressing either wild-type beta 3 or beta 3 bearing the conservative Y747F and Y759F mutations. As illustrated in Fig. 5A, FACS analysis with the alpha vbeta 3-specific antibody LM609, confirmed that these transfectants expressed similar levels of alpha vbeta 3 or alpha vbeta 3(Y747F, Y759F) at the cell surface. When the two CHO cell lines were used in the fibrin clot retraction assay, it was found that clot retraction was reduced markedly in the Y747F, Y759F transfectants, as demonstrated by a 50 ± 11.9% increase in clot weight compared with the clot weights obtained with the wild-type beta 3-expressing CHO cells (Fig. 5B).


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Fig. 5.   Flow cytometric analysis and clot retracting ability of cells expressing mutant beta 3. A, CHO cells transfected with wild-type beta 3 or (Y747F and Y759F) beta 3 were incubated with control mouse IgG (control for wild-type beta 3 shown in thin line, median channel 5.47; for mutant beta 3 median channel was 5.86, not shown), or LM609 (thick line for wild-type beta 3, median channel 108.43; dashed line for Y(747,759)F beta 3, median channel 128.64) and analyzed by flow cytometry. B, wild-type beta 3 CHO transfectants and (Y747F and Y759F) beta 3 CHO transfectants were subjected to the fibrin clot retraction assay as described under "Experimental Procedures." The results from five experiments were expressed as clot weight ± S.E.

To assess whether the presence of alpha IIb would alter the effect of the double tyrosine to phenylalanine mutations on beta 3-mediated fibrin clot retraction, similar experiments were performed with CHO cells that had been co-transfected with alpha IIb in addition to the mutant or wild-type beta 3 cDNA. These cells expressed both alpha vbeta 3 and alpha IIbbeta 3 on their surface, and essentially the same results were obtained: the cells bearing Y747F and Y759F beta 3 showed about a 50% decrease in their ability to retract fibrin clots when compared with those cells expressing wild-type beta 3 (data not shown). Thus, the tyrosine residues within beta 3 do indeed play a critical role in beta 3 integrin-mediated fibrin clot retraction.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

A well established function of integrin cytoplasmic domains is as a bridge between extracellular matrix proteins and the cytoskeletal/contractile machinery within a cell. The data presented in this study indicate that the two tyrosine residues within the beta 3 cytoplasmic domain may be important in mediating some of these interactions and suggest a way in which a modification of these residues, namely by phosphorylation, may also be involved in these bridging processes. We have found that the tyrosine phosphorylation of the beta 3 subunit of alpha IIbbeta 3 occurs as a general consequence of platelet aggregation. That this phosphorylation may in turn affect the interaction of the beta 3 integrin with the platelet cytoskeleton is indicated by the following data. First, phosphorylated beta 3 is located preferentially within the detergent-insoluble cytoskeletal fraction of aggregated platelets; and second, the contractile protein myosin can bind directly, in a phosphotyrosine-dependent manner, to peptide corresponding to the cytoplasmic domain of beta 3. Furthermore, functional data obtained by analysis of CHO cells bearing a beta 3 in which both cytoplasmic tyrosine residues were mutated to phenylalanines also indicates the importance of these beta 3 tyrosines in the beta 3-dependent retraction of fibrin clots. We have previously demonstrated the interaction of tyrosine-phosphorylated beta 3 with the signaling proteins SHC and Grb2 (1) and hypothesized that tyrosine phosphorylation of beta 3 allowed for the recruitment of signaling complexes to the membrane. Our new studies indicate that in addition to the role of beta 3 tyrosine phosphorylation in binding signaling proteins, the phosphorylation may also influence the interaction of beta 3 with the myosin-based contractile apparatus and in doing so play an important role in integrin-dependent functions involving cytoskeletal reorganization.

The present data demonstrate that platelet aggregation is both necessary and sufficient to induce tyrosine phosphorylation of beta 3. First, conditions that only induced the active conformation of alpha IIbbeta 3 and did not allow for aggregation to occur, such as ADP stimulation in the absence of fibrinogen or in the presence of an inhibitory RGD peptide, did not induce beta 3 tyrosine phosphorylation. Second, platelet aggregation induced by LIBS6 independent of platelet stimulation also resulted in beta 3 tyrosine phosphorylation. Third, beta 3 tyrosine phosphorylation did not occur under conditions that induced ligand occupancy of alpha IIbbeta 3 but not platelet aggregation, such as the addition of ADP and fibrinogen in the absence of stirring. Last, a reversal of aggregation-induced beta 3 tyrosine phosphorylation was observed upon reversal of platelet aggregation. In all instances, platelet aggregation, with the subsequent platelet-platelet interactions, was absolutely required for beta 3 tyrosine phosphorylation.

Early studies established that a significant portion of alpha IIb and beta 3 could be isolated with cytoskeletal structures in thrombin-aggregated, but not activated, platelets (21), suggesting that platelet aggregation induces the association of alpha IIbbeta 3 with the platelet cytoskeleton. It was proposed that this integrin became Triton X-100 detergent-insoluble because of the macromolecular associations between the platelet membrane surfaces and actin filaments. Morphological studies have also demonstrated that fibrinogen binding to alpha IIbbeta 3 induced its interaction with the cytoskeleton since the membrane-bound integrin appeared to be co-aligned with cytoskeletal structures of the platelet (40). Further, Fox and co-workers demonstrated an aggregation-dependent redistribution of alpha IIbbeta 3 from the membrane skeleton to the Triton X-100-insoluble fraction of platelets (22). Several tyrosine kinases and other tyrosine-phosphorylated proteins also redistributed to the cytoskeleton upon platelet aggregation (22). The present data points to a possible mechanism for the redistribution of alpha IIbbeta 3 to the cytoskeleton in aggregated platelets. Examination of the phospho-beta 3 distribution between Triton X-100 soluble and insoluble fractions of aggregated platelets demonstrated that phosphorylated beta 3 preferentially redistributes to the cytoskeletal fraction. The observations that tyrosine phosphorylation of the beta 3 cytoplasmic domain is a common consequence of aggregation by a wide range of platelet agonists and is a potential player in driving the redistribution of beta 3 to the cytoskeleton prompted us to examine biochemically whether beta 3 phosphorylation plays a part in mediating novel interactions of the receptor with the platelet cytoskeleton.

The binding of integrin cytoplasmic domains to cytoskeletal proteins is not unprecedented, and although little in vivo data exist, due to the technical difficulties of working with poorly soluble cytoskeletal proteins, a variety of in vitro strategies have been used to discover and characterize such interactions. Interactions between talin and beta 1 integrin cytoplasmic domain were first studied using equilibrium gel filtration of purified proteins (3). In solid phase binding assays, talin was found to bind directly with alpha IIbbeta 3 integrin cytoplasmic tail sequences and to purified alpha IIbbeta 3 (4). alpha -Actinin has been shown to bind directly to the cytoplasmic domain of beta 1 as well as to purified alpha IIbbeta 3 (6). In another study, the cytoskeletal protein skelemin interacted with the beta 3 cytoplasmic domain in a yeast two-hybrid screen and with peptides corresponding to the membrane proximal regions of beta 1 and beta 3 (41). Actin binding protein has also been demonstrated to bind directly to the cytoplasmic domain of beta 2 integrin using peptide affinity chromatography (42) and to the dimerized beta 1 cytoplasmic domains using a novel experimental strategy (5). However, the mechanisms that regulate these integrin-cytoskeletal interactions are unknown.

Given that tyrosine phosphorylation of beta 3 is a general consequence of platelet aggregation and appears to direct the redistribution of alpha IIbbeta 3 to the cytoskeleton, we postulate that beta 3 tyrosine phosphorylation could be a general mechanism for regulating integrin-cytoskeletal interactions. Fittingly, members of the Src family of tyrosine kinases are known to selectively redistribute with a subpopulation of alpha IIbbeta 3 to the actin cytoskeleton in aggregated platelets (22, 23). This redistribution is reduced by treatment of platelets with tyrosine kinase inhibitors, suggesting that tyrosine kinases, either directly or through the phosphorylation of other proteins, may regulate the cytoskeletal attachment of alpha IIbbeta 3 (43). Further, alpha IIbbeta 3-mediated clot retraction is inhibited by tyrosine kinase inhibitors (43). Although there is circumstantial evidence that certain integrin-cytoskeletal interactions are phosphotyrosine-dependent, experimental data directly supporting this hypothesis are lacking.

In the present work, we used in vitro ligand binding methodology to observe a novel interaction between myosin and a beta 3 integrin cytoplasmic domain peptide that was regulated by tyrosine phosphorylation. Phosphorylated and nonphosphorylated integrin cytoplasmic domain peptides were synthesized and used to identify a direct and tyrosine phosphorylation-dependent interaction between the beta 3 cytoplasmic domain peptide and platelet myosin heavy chain. Since the doubly phosphorylated beta 3 peptide bound specifically to myosin, this contractile protein may possess tandem phosphotyrosine binding regions analagous to the tandem SH2 domains of the tyrosine kinases ZAP-70 or Syk, which bind ITAM domains in immune receptor complexes (44, 45). However, to our knowledge, classic phosphotyrosine binding motifs in platelet myosin heavy chain have not yet been identified. We further observed that the tail domain of myosin was responsible for its interaction with beta 3. Interestingly, the tail region of myosin serves as an anchor so that it can translocate actin and has been hypothesized to bind certain myosin isoforms to cell or organelle membranes (46). Together these data suggest that the tyrosine phosphorylated beta 3 binding domain of myosin exists on the tail region of myosin heavy chain and that this domain contains previously unrecognized phosphotyrosine binding motifs. These binding motifs may allow for the interaction of phosphorylated beta 3 with the cytoskeletons of aggregated platelets, providing alignment for certain postaggregation contractile events, such as clot retraction, to occur.

Although our in vitro data strongly suggest that tyrosine phosphorylation of the beta 3 cytoplasmic domain can allow for alpha IIbbeta 3 interaction with myosin, this conclusion was not possible to confirm in vivo because of the aforementioned problems associated with working with many cytoskeletal proteins. Indeed, myosin is insoluble at physiological salt concentrations; only highly stringent co-immunoprecipitation conditions could be employed using detergent lysates that are well known to disrupt protein-protein interactions. In this case, the problem is compounded by the fact that the major portion of tyrosine phosphorylated alpha IIbbeta 3 does itself translocate to the insoluble cytoskeleton in aggregated platelets. Also, robust tyrosine dephosphorylation mechanisms are present in platelets (47), which make it difficult to preserve tyrosine phosphorylation of beta 3 except under denaturing conditions (e.g. by the addition of SDS-containing sample buffer) or in rapid postlytic fractionations (e.g. cytoskeleton isolation) hampering immunoprecipitation experiments. Therefore, our data do not preclude other, possibly phosphotyrosine-independent, interactions between the cytoplasmic domains of alpha IIbbeta 3 and myosin. If other such interactions do indeed exist, it is attractive to hypothesize that tyrosine phosphorylation of beta 3 cytoplasmic domain, possibly at only one of the tyrosine residues, could induce a more stable and avid interaction between previously-associated alpha IIbbeta 3 and myosin.

Thus, our data suggest that tyrosine phosphorylation of the beta 3 cytoplasmic tail may regulate a direct association with myosin, providing anchorage of surface beta 3 integrins to the contractile apparatus. A possible consequence of this interaction is to allow for alpha IIbbeta 3-mediated clot retraction in platelets. We addressed the role of the beta 3 cytoplasmic tyrosines in clot retraction using CHO cells transfected with beta 3. Previous studies using such a CHO cell expression system have proven useful for analyzing the role of both alpha IIb and beta 3 integrin cytoplasmic domains in alpha IIbbeta 3 signaling and adhesive functions (17-19, 36). In particular, CHO cells transfected with alpha IIbbeta 3 gain the ability to contract fibrin clots, whereas both untransfected CHO cells and cells expressing the S752P Glanzmann's mutant alpha IIbbeta 3 fail to do so (36). Another expression system, in which CS-1 melanoma cells are transfected with a cDNA encoding the integrin beta 3 subunit, has been used to characterize the roles of the alpha vbeta 3 integrin cytoplasmic domains in adhesion, spreading, and migration on vitronectin (20). Although clot retraction was not addressed in this study, mutating either tyrosine 747 or 759 on beta 3 to phenylalanine had little or no effect on other alpha vbeta 3 adhesive events (20). In the present work, CHO cells bearing the Y747F and Y759F beta 3 cDNA displayed a pronounced defect in fibrin clot retraction: the first demonstration of an effect of beta 3 tyrosine to phenylalanine mutations on a biologically relevant event. Since integrins are believed to support clot retraction by providing the transmembrane linkage between extracellular adhesive proteins and the contractile cytoskeleton (19), it is interesting to hypothesize that, by mutating the tyrosine residues within the beta 3 cytoplasmic domain, we have disrupted the phosphotyrosine-dependent integrin-myosin interaction and that this could account for the defective clot retraction observed in the mutant CHO cell transfectants.

Our working hypothesis of the role of beta 3 cytoplasmic domain tyrosine phosphorylation in platelet function can be summarized as follows: the receptor is phosphorylated as a common consequence of aggregation in response to a number of platelet agonists. Although direct associations of known tyrosine kinases with alpha IIbbeta 3 have not yet been detected, members of the Src family of tyrosine kinases are capable of phosphorylating the receptor in vitro (1) and Src and Lyn can be cross-linked to beta 3 in intact platelets treated with chemical cross-linking agents (48). Once phosphorylated, the beta 3 integrin tails are capable of associating with signaling proteins SHC and Grb2 to potentially initiate outside-in signaling cascades (1). In addition to providing a scaffold for the recruitment of signaling complexes to the membrane, the doubly phosphorylated cytoplasmic domain of beta 3 can also bind to cytoskeletal proteins. In particular, the present work demonstrates direct binding of a doubly tyrosine-phosphorylated beta 3 integrin cytoplasmic domain peptide to myosin and further reveals that replacement of these tyrosine residues with phenylalanines in alpha IIbbeta 3-transfected CHO cells results in defective beta 3 integrin-mediated retraction of fibrin clots. In light of these data, we postulate that phosphorylation of beta 3 integrin cytoplasmic domain may be an important mechanism for regulating a direct myosin-integrin interaction. Inhibition of this interaction may interfere with the transmission of the mechanical forces that regulate processes such as clot retraction and cell motility. Proving or disproving such hypotheses will be the focus of future work.

    ACKNOWLEDGEMENT

The assistance of Liping Gao in tissue culture work is gratefully acknowledged.

    Note Added in Proof

While this manuscript was in review, a manuscript was published which also reported effects of beta 3 tyrosine mutations on clot retraction (Blystone, S. D., Williams, M. D., Slater, S. E., and Brown, E. J. (1997) J. Biol. Chem. 272, 28757-28761).

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant HL 48728 (to M. H. G).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ These authors contributed equally to this work.

** To whom correspondence should be addressed: COR Therapeutics, Inc., 256 E. Grand Ave., South San Francisco, CA 94080. Tel.: 650-244-6884; Fax: 650-244-9270; E-mail: david_phillips{at}corr.com.

1 The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; HBB, HEPES blot buffer; CHO, Chinese hamster ovary; FACS, fluorescence-activated cell sorter; ITAM, immune receptor tyrosine-based activation motif.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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