Abstract

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.