Platelet activation triggers integrin αIIbβ 3-dependent signals and the induction of tyrosine phosphorylation of the cytoskeletal protein α-actinin. We have previously reported that α-actinin is phosphorylated by the focal adhesion kinase (FAK) (Izaguirre, G., Aguirre, L., Hu, Y.-P., Lee, H. Y., Schlaepfer, D. D., Aneskievich, B. J., and Haimovich, B. (2001) J. Biol. Chem. 276, 28676-28685). In this study, a phosphatase of 68 kDa that dephosphorylated α-actinin in vitro was isolated from platelet lysates by three sequential chromatography steps. The phosphatase was identified as SHP-1 by electrospray tandem mass spectrometry. α-Actinin was dephosphorylated in vitro by recombinant SHP-1 and by SHP-1 immunoprecipitated from stimulated unstimulated or thrombin-stimulated platelet lysates. SHP-1 immunoprecipitated from lysates of platelets adherent to fibrinogen, however, failed to dephosphorylate α-actinin. In contrast, the activity of SHP-1 against a synthetic substrate was not affected by the mode of platelet activation. The robust and sustained phosphorylation of α-actinin detected in platelets adherent to fibrinogen thus correlates with a decrease in the activity of SHP-1 toward it. Tyrosine phosphorylation of α-actinin is seen in vanadate-treated COS-7 cells that are co-transfected with α-actinin and wild type FAK. Triple transfection of the cells with cDNAs encoding for α-actinin, FAK, and wild type SHP-1 abolished the phosphorylation of α-actinin. The phosphorylation of FAK, however, was barely affected by the expression of wild type SHP-1. Both α-actinin and FAK were phosphorylated in cells co-expressing α-actinin, FAK, and a catalytic domain mutant (C453S) of SHP-1. These findings establish that SHP-1 can dephosphorylate α-actinin in vitro and in vivo and suggest that SHP-1 may regulate the tethering of receptors to the cytoskeleton and/or the extent of cross-linking of actin filaments in cells such as platelets.
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology