␣-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.

The integrin ␣ IIb ␤ 3 mediates tyrosine phosphorylation of a 105-kDa protein (pp105) in activated platelets. We have partially purified a 105-kDa tyrosine-phosphorylated protein from platelets stimulated with phorbol 12-myristate 13-acetate and obtained the sequence of an internal 12-mer peptide derived from this protein. The sequence was identical to human ␣-actinin sequences deposited in the Swiss Protein Database. ␣-Actinin, a 105-kDa protein in platelets, was subsequently purified from activated platelets by four sequential chromatographic steps. Fractions were analyzed by Western blotting and probed with ␣-actinin and anti-phosphotyrosine antibodies. The distribution of ␣-actinin and pp105 overlapped throughout the purification. Furthermore, in the course of this purification, a 105-kDa tyrosine-phosphorylated protein was only detected in fractions that contained ␣-actinin. The purified ␣-actinin protein was immunoprecipitated with antibodies to phosphotyrosine in the absence but not in the presence of phenyl phosphate. ␣-Actinin resolved by two-dimensional gel electrophoresis of activated platelet lysates was recognized by the antibodies to phosphotyrosine, whereas pretreatment of the platelets with bisindolylmaleimide, a protein kinase C inhibitor that prevents tyrosine phosphorylation of pp105, inhibited the reactivity of the antibodies to phosphotyrosine with ␣-actinin. Taken together, these data demonstrate that a fraction of ␣-actinin is tyrosine-phosphorylated in activated platelets.Tyrosine phosphorylation of protein substrates is central to all cellular processes that are regulated by the integrin family of extracellular matrix receptors (1) including cell shape change, migration, growth, and survival (for recent reviews see Refs. 2-6). The array of responses regulated by integrins is greatly simplified in platelets that are enucleated, terminally differentiated cells. Platelets, therefore, provide a useful model system to study integrin-mediated protein tyrosine phosphorylation events that are closely linked to cell shape change and the underlying cytoskeleton organization.A rapid increase in protein tyrosine phosphorylation is detected within seconds to minutes of platelet activation by soluble agonists such as thrombin or phorbol 12-myristate 13-acetate (PMA) 1 (7,8). Platelet adhesion to immobilized matrix proteins such as fibrinogen triggers similar tyrosine phosphorylation events (9). Included in the first group of proteins that become tyrosine-phosphorylated are the non-receptor tyrosine kinases pp72syk (10) and RFTK/PYK (11), the guanine nucleotide exchange factor Vav (12), and the mitogen-activated protein (MAP) kinase family members extracellular signal-regulated kinase 2 (ERK2) and p38 (13,14). In platelets stimulated by thrombin, PMA, or adhesion to fibrinogen, a second wave of protein tyrosine phosphorylation that is strictly dependent on both ligand binding to the ␣ IIb ␤ 3 integrin receptor and the cytoskeleton organization is observed (7). As such, tyrosine phosphorylation of this ...