GTP-binding protein gamma12 subunit phosphorylation by protein kinase C--identification of the phosphorylation site and factors involved in cultured cells and rat tissues in vivo.

We have demonstrated previously that the GTP-binding protein gamma12 subunit is a selective substrate for phosphorylation by protein kinase C among various gamma subunits in vitro, and that a serine residue in the N-terminal region is involved. In the present study, we first determined that the site of phosphorylation was Ser1 with antibodies developed against two N-terminal peptides containing phosphorylated Ser1 and Ser2, respectively. Using an antibody recognizing phosphorylated gamma12 and Swiss 3T3 cells rich in this protein, gamma12 was found to be phosphorylated by stimulation of quiescent cells with various reagents, such as phorbol 12-myristate 13-acetate (PMA), NaF, fetal calf serum, lysophosphatidic acid, endothelin, and growth factors. Pertussis toxin completely and partially prevented phosphorylation of gamma12 induced by lysophosphatidic acid and fetal calf serum and by endothelin, respectively, suggesting a contribution of G(i/o). Phosphorylation of gamma12 was limited when cells were stimulated by a single reagent, even with PMA, a strong activator of protein kinase C, whereas simultaneous stimulation with lysophosphatidic acid and either PMA or platelet-derived growth factor induced a synergistic increase of phosphorylation, suggesting physiological roles for GTP-binding proteins and protein kinase C in combination. Phosphorylated gamma12 was also detected in various tissues of untreated rats. Its decrease by pertussis toxin treatment also suggested the involvement of G(i/o) in vivo.