Galpha 12 and Galpha 13 Are Phosphorylated during Platelet Activation

Abstract

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 alpha 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 Galpha 12 and Galpha 13. Phosphorylation occurred rapidly after addition of thrombin and was not mediated by glycoprotein IIb/IIIa (integrin alpha IIbbeta 3) activation. Phosphorylation of Galpha 12 and Galpha 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 Galpha 12/13 phosphorylation induced by thrombin in human platelets. The phosphorylation of both G protein alpha subunits was reconstituted in COS-7 cells cotransfected with Galpha 12 or Galpha 13 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 Galpha 12 and Galpha 13 in a phorbol 12-myristate 13-acetate-dependent manner. These data demonstrate that Galpha 12 and Galpha 13 are phosphorylated under in vivo conditions and that this phosphorylation involves protein kinase C.