Abstract. We have investigated mechanisms involved in integrin-mediated signal transduction in platelets by examining integrin-dependent phosphorylation and activation of a newly identified protein tyrosine kinase, pp125 F~ (FAK, focal adhesion kinase). This kinase was previously shown to be localized in focal adhesions in fibroblasts, and to be phosphorylated on tyrosine in normal and Src-transformed fibroblasts. We show that thrombin and collagen activation of platelets causes an induction of tyrosine phosphorylation of pp125 F~ and that pp125 F~ molecules isolated from activated platelets display enhanced levels of phosphorylation in immune-complex kinase assays, pp125 F~ was not phosphorylated on tyrosine after thrombin or collagen treatment of Glanzmann's thrombasthenic platelets deficient in the fibrinogen receptor GPIIbIIIa, or of platelets pretreated with an inhibitory monoclonal antibody to GP lib-Ilia. Fibrinogen binding to GP Ub-HIa was not sufficient to induce pp125 F~x phosphorylation because pp125 F~ was not phosphorylated on tyrosine in thrombin-treated platelets that were not allowed to aggregate. These results indicate that tyrosine phosphorylation of pp125 s~ is dependent on platelet aggregation mediated by fibrinogen binding to the integrin receptor GP lib-Ilia. The induction of tyrosine phosphorylation of pp125 F~ was inhibited in thrombin-and collagen-treated platelets preincubated with cytochalasin D, which prevents actin polymerization following activation. Under all of these conditions, there was a strong correlation between the induction of tyrosine phosphorylation of pp125 r~ in vivo and stimulation of the phosphorylation of pp125 FA~ in vitro in immune-complex kinase assays. This study provides the first genetic evidence that tyrosine phosphorylation of pp125 F~ is dependent on integrin-mediated events, and demonstrates that there is a strong correlation between tyrosine phosphorylation of pp125 FAK in platelets, and the activation of pp125FAK-associated phosphorylating activity in vitro.T HE interactions of cells with extracellular adhesion molecules play critical roles in regulating the morphology, proliferation, migration, and differentiation of cells. One family of receptors for extracellular adhesion molecules, called integrins, is comprised of heterodimeric r transmembrane proteins (1,17,19,20). Although the extracellular interactions between adhesion molecules and their integrin receptors have been well characterized, the interactions of integrins with cytoplasmic targets and their role in signaling pathways that are responsible for adhesion-induced changes in cell behavior are poorly understood (5).Platelets provide a very useful model system for investigating the mechanisms involved in integrin-induced events. The best characterized platelet integrin receptor, GP Hb-HIa, is required for two essential functions of platelets in hemostasis-platelet-to-platelet aggregation and the spreading of J. S. Brugge's present address is Ariad Pharmaceuticals, Inc.,

As cells encounter new regions of the substrate, they develop bonds with new matrix molecules for migration, matrix remodeling and force generation. How cells orchestrate the assembly of adhesion sites is only partially understood. Here we show that fibroblasts deficient in the SH2 domain containing protein tyrosine phosphatase 2 (Shp2) have an increased number of immature focal complexes deficient in alpha-actinin. Inhibition of FAK restored alpha-actinin to focal complexes, whereas inhibition of RhoA did not. In correlation, adhesion site dynamics, measured by fluorescence recovery after photobleaching (FRAP) of GFP-paxillin and GFP-vinculin were dramatically increased in Shp2(-/-) cells and restored to normal by FAK inhibition. Shp2(-/-) cells failed to strengthen initial integrin-cytoskeleton linkages, as measured by optical tweezers and large bead assays, and were rescued by inhibition of FAK. In contrast, affinity modulation of adhesion receptors was unaffected. In addition, reinforcement correlated with alpha-actinin assembly through decreased dynamics. This shows for the first time that adhesion site dynamics are regulated during adhesion formation and that force-dependent strengthening of integrin-cytoskeleton linkages is correlated with alpha-actinin assembly and decreased adhesion site dynamics.

Abstract. Chicken integrin B1 eDNA and its sitedirected mutants were cloned into a mammalian expression vector and introduced into mouse NIH 3T3 cells. Stable transfectants expressing the chicken/31 subunit or its site-directed mutants were identified by immunostaining with antibodies specific for the chicken integrin/31 subunit. The chicken/~1 proteins were expressed predominately in the endoplasmic reticulum of transfectants and to a lesser degree in the plasma membrane. Immunoblots and immunoprecipitations, using anti-chicken integrin antibodies, revealed three different sizes of the chicken subunit (90, 95, and 120 kD) and a mouse 140-kD cz subunit. Immunoprecipitations of the cell surface receptors showed only two peptides, an 120-kD/31 and an 140-kD ot subunit. Antibodies perturbing mouse and chicken integrin-specific cell adhesions were used to demonstrate that the chimeric receptors functioned in adhesion to both laminin and fibronectin. Immunofluorescent staining with antibodies specific for either the chicken or mouse receptors showed that both the wild type and the chimeric receptors localized in focal contacts. Several mutations in the cytoplasmic domain were synthesized and used in the transfection experiments. In one mutant the tyrosine (Tyr 788) in the consensus sequence for phosphorylation was replaced by a phenylalanine. In another the lysine (Lys 757) at the end of the membrane spanning region was replaced by a leucine. Both of these mutants formed dimers with mouse ~ subunits, participated in adhesion, localized in focal contacts, and displayed biological properties indistinguishable from the wild-type transfection. In contrast, mutants containing deletions greater than 5-15 amino acids nearest the carboxyl end in the cytoplasmic domain neither promoted adhesion nor localized in focal contacts. They did, however, form heterodimers that were expressed on the cell surface.

␣-Actinin is tyrosine-phosphorylated in activated human platelets (Izaguirre, G., Aguirre, L., Ji, P., Aneskievich, B., and Haimovich, B. (1999) J. Biol. Chem. 274, 37012-37020). Analysis of platelet RNA by reverse transcription-polymerase chain reaction revealed that ␣-actinin expressed in platelets is identical to the cytoskeletal/non-muscle isoform. A construct of this isoform containing a His 6 tag at the amino terminus was generated. Robust tyrosine phosphorylation of the recombinant protein was detected in cells treated with the tyrosine phosphatase inhibitor vanadate. The tyrosine phosphorylation site was localized to the amino-terminal domain by proteolytic digestion. A recombinant ␣-actinin protein containing a Tyr 3 Phe mutation at position 12 (Y12F) was no longer phosphorylated when expressed in vanadate-treated cells, indicating that tyrosine 12 is the site of phosphorylation. The wild type recombinant protein was not phosphorylated in cells lacking the focal adhesion kinase (FAK). Re-expression of FAK in these cells restored ␣-actinin phosphorylation. Purified wild type ␣-actinin, but not the Y12F mutant, was phosphorylated in vitro by wild type as well as a Phe-397 mutant of FAK. In contrast, no phosphorylation was detected in the presence of a kinase-dead FAK. Tyrosine phosphorylation reduced the amount of ␣-actinin that cosedimented with actin filaments. These results establish that ␣-actinin is a direct substrate for FAK and suggest that ␣-actinin mediates FAK-dependent signals that could impact the physical properties of the cytoskeleton.