The ubiquitously expressed G-proteins G12 and G13 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 α subunits of both G-proteins can be phosphorylated under physiological conditions. Activation of human platelets by thrombin and the thromboxane A2 receptor agonist U46619 lead to phosphorylation of Gα12 and Gα13. Phosphorylation occurred rapidly after addition of thrombin and was not mediated by glycoprotein IIb/IIIa (integrin αIIbβ3) activation. Phosphorylation of Gα12 and Gα13 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 Gα12/13 phosphorylation induced by thrombin in human platelets. The phosphorylation of both G protein α subunits was reconstituted in COS-7 cells cotransfected with Gα12 or Gα13 and different protein kinase C isoforms. Among the protein knase C isoforms tested, protein kinase C β, δ, and ϵ were most effective in promoting phosphorylation of Gα12 and Gα13 in a phorbol 12-myristate 13-acetate-dependent manner. These data demonstrate that Gα12 and Gα13 are phosphorylated under in vivo conditions and that this phosphorylation involves protein kinase C.
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