Vinculin is a major platelet protein that undergoes Ca(2+)-dependent tyrosine phosphorylation.
When intracellular Ca2+ pools are released during platelet stimulation by thrombin, elevation of platelet cytosolic Ca2+ concentration induces tyrosine phosphorylation of a 130 kDa protein, and refilling the pools mediates dephosphorylation of this protein [Vostal, Jackson and Shulman (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 platelets by both monoclonal anti-phosphotyrosine and anti-vinculin antibodies. (2) Removal of N-linked sugars with peptide-N-glycosidase or reduction did not change the molecular mass of vinculin or of the 130 kDa protein on SDS/PAGE. (3) The 130 kDa tyrosine-phosphorylated protein associates 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 immunoprecipitated by anti-phosphotyrosine antibody it reacts with anti-vinculin antibody. (5) The 130 kDa tyrosine-phosphorylated protein and vinculin focus isoelectrically at pI 5.4-5.8. Our finding that vinculin is a major platelet protein that undergoes Ca(2+)-dependent tyrosine phosphorylation during platelet activation may provide clues to the function of this protein.
Vostal
J G
JG
Division of Hematology, Food and Drug Administration, Bethesda, MD 20892.
Shulman
N R
NR
D015214:Antigens, Differentiation, Myelomonocytic / Q000378:metabolism; D001792:Blood Platelets / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D010750:Phosphoproteins / Q000378:metabolism; D019000:Phosphotyrosine; D015539:Platelet Activation; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism; D016596:Vinculin / Q000378:metabolism*
D015214:Antigens, Differentiation, Myelomonocytic; D010750:Phosphoproteins; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D010980:Platelet Membrane Glycoproteins; D016596:Vinculin; D019000:Phosphotyrosine; D014443:Tyrosine; D006026:Glycoside Hydrolases; D002118:Calcium