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 Galpha12 and Galpha13. Phosphorylation occurred rapidly after addition of thrombin and was not mediated by glycoprotein IIb/IIIa (integrin alphaIIbbeta3) activation. Phosphorylation of Galpha12 and Galpha13 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 Galpha12/13 phosphorylation induced by thrombin in human platelets. The phosphorylation of both G protein alpha subunits was reconstituted in COS-7 cells cotransfected with Galpha12 or Galpha13 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 Galpha12 and Galpha13 in a phorbol 12-myristate 13-acetate-dependent manner. These data demonstrate that Galpha12 and Galpha13 are phosphorylated under in vivo conditions and that this phosphorylation involves protein kinase C.
Highlights
Units of some members of the Gi/o family can be phosphorylated in vivo and in vitro
Since the effect of thrombin was mimicked by the protein kinase C activating phorbol ester phorbol 12-myristate 13-acetate (PMA) and since platelet activation is known to involve activation of protein kinase C [25,26,27], we investigated the role of PKC in G␣12 and G␣13 phosphorylation
These data demonstrate that the PKC-dependent phosphorylation of G␣12 and G␣13 observed in human platelets can be reconstituted by cotransfection of different PKC isoforms and both G-protein ␣-subunits in COS-7 cells
Summary
G-protein, heterotrimeric guanine nucleotide-binding protein; U46619, 11␣,9␣-epoxymethano-prostaglandin H2; PMA, phorbol 12-myristate 13-acetate; PKC, protein kinase C; PAGE, polyacrylamide gel electrophoresis; DMEM, Dulbecco’s modified Eagle’s medium. Thrombin, thromboxane A2 analogs, and phorbol esters lead to a protein kinase C mediated phosphorylation of G␣z [6, 7]. This phosphorylation blocks interaction of G␣z with G␥ [8]. Several reports have shown that G␣i can be phosphorylated in various systems under conditions leading to protein kinase C and cGMP dependent protein kinase activation (9 –12). Most of these reports suggest that G␣i phosphorylation correlates with inactivation of signaling via this G-protein [9, 11, 12]. Our data show that treatment of human platelets with physiological platelet activators leads to a rapid and sustained phosphorylation of both G-protein ␣-subunits and that this phosphorylation is mediated by protein kinase C
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