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alpha-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 alpha-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 alpha-actinin protein containing a Tyr --> 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 alpha-actinin phosphorylation. Purified wild type alpha-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 alpha-actinin that cosedimented with actin filaments. These results establish that alpha-actinin is a direct substrate for FAK and suggest that alpha-actinin mediates FAK-dependent signals that could impact the physical properties of the cytoskeleton.

Platelet cytoskeletons were isolated by extracting these highly contractile cells with a solution containing 1% Triton X-100 and 10 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid as recently described (Rosenberg, S., Stracher, A., and Lucas, R. C. (1981) J. Cell Biol. 91, 201-211). The Triton-insoluble cytoskeleton consists mostly of actin, a high molecular weight actin-binding protein and a previously unidentified protein with an apparent molecular weight on sodium dodecyl sulfate gels of 105,000 (+/- 5,000). We describe the purification of this 105,000-dalton protein from the platelet cytoskeleton using ammonium sulfate fractionation and ion exchange chromatography. This 105,000-dalton protein was found to cross-react with antibodies to beef cardiac alpha-actinin. One-dimensional partial proteolysis maps showed similarity to, but not identity with, the major peptides of the platelet 105,000-dalton protein and skeletal muscle alpha-actinin. The platelet 105,000-dalton cytoskeletal protein binds to and causes the sedimentation of skeletal muscle F-actin under comparatively low centrifugal force. This process, however, is inhibited by calcium ions, unlike the binding of any of the muscle alpha-actinins described to date. Thus, it is likely that the 105,000-dalton protein is the platelet form of alpha-actinin, its different structure accounting for its different actin-binding behavior.