Jg. Vostal et Nr. Shulman, VINCULIN IS A MAJOR PLATELET PROTEIN THAT UNDERGOES CA2-DEPENDENT TYROSINE PHOSPHORYLATION(), Biochemical journal, 294, 1993, pp. 675-680
When intracellular Ca2+ pools are released during platelet stimulation
by thrombin, elevation of platelet cytosolic Ca2+ concentration induc
es tyrosine phosphorylation of a 130 kDa protein, and refilling the po
ols mediates dephosphorylation of this protein [Vostal, Jackson and Sh
ulman (1991) J. Biol. Chem. 266, 16911-16916]. In the present work the
130 kDa protein was identified as vinculin by the following criteria.
(1) It is detected on protein immunoblots of thrombin-activated plate
lets by both monoclonal anti-phosphotyrosine and anti-vinculin antibod
ies. (2) Removal of N-linked sugars with peptide-N-glycosidase or redu
ction did not change the molecular mass of vinculin or of the 130 kDa
protein on SDS/PAGE. (3) The 130 kDa tyrosine-phosphorylated protein a
ssociates with Triton-soluble fraction of platelets as does vinculin.
(4) The 130 kDa protein immunoprecipitated by anti-vinculin monoclonal
antibody reacts with anti-phosphotyrosine antibody; when immunoprecip
itated by anti-phosphotyrosine antibody it reacts with anti-vinculin a
ntibody. (5) The 130 kDa tyrosine-phosphorylated protein and vinculin
focus isoelectrically at pI 5.4-5.8. Our finding that vinculin is a ma
jor platelet protein that undergoes Ca2+-dependent tyrosine phosphoryl
ation during platelet activation may provide clues to the function of
this protein.