G alpha(12) and G alpha(13) are phosphorylated during platelet activation

The ubiquitously expressed G-proteins G(12) and G(13) whose function is currently not clear have been shown to be activated in platelet membranes through receptors that stimulate platelet aggregation. We used intact human platelets to determine whether alpha subunits of both G-proteins can be phosphorylated under physiological conditions, Activation of human platelets by thrombin and the thromboxane A(2) receptor agonist U46619 lead to phosphorylation of G alpha(12) and G alpha(13). Phosphorylation occurred rapidly after addition of thrombin and was not mediated by glycoprotein IIb/IIIa (integrin alpha(IIb)beta(3)) activation, Phosphorylation of G alpha(12) and G alpha(13) could be mimicked by phorbol 12-myristate 13-acetate, and thrombin-induced phosphorylation was inhibited by the protein kinase C inhibitor calphostin C indicating an involvement of protein kinase C in G alpha(12/13) phosphorylation in duced by thrombin in human platelets, The phosphorylation of both G protein alpha subunits was reconstituted in COS-7 cells cotransfected with G alpha(12) or G alpha(12) and different protein kinase C isoforms, Among the protein knase C isoforms tested, protein kinase C beta, delta, and epsilon were most effective in promoting phosphorylation of G alpha(12) and G alpha(13) in a phorbol 12-myristate 13-acetate-dependent maimer. These data demonstrate that G alpha(12) and G alpha(13) are phosphorylated under in vivo conditions and that this phosphorylation involves protein kinase C.