PRIMARY STRUCTURE OF A GAMMA-SUBUNIT OF G-PROTEIN, GAMMA(12), AND ITS PHOSPHORYLATION BY PROTEIN-KINASE-C

We have determined the primary structure of a novel gamma subunit (gamma(12), previously designated gamma(S1)) of G protein purified from bovine spleen. The mature gamma(12) protein composed of 68 amino acids had acetylated serine at the N terminus and geranylgeranylated/carboxylmethylated cysteine at the C terminus. This was consistent with the C-terminal prenylation signal in the amino acid sequence, which was predicted from gamma(12) cDNA isolated from a bovine spleen cDNA library. Western blots with the specific antibody against gamma(12) showed that gamma(12) is present in all tissues examined. Among various gamma subunits (gamma(1), gamma(2), gamma(3), gamma(7), and gamma(12)), gamma(12) has a unique property to be phosphorylated by protein kinase C. The phosphorylated amino acid residue was Ser(1) (or Ser(2)). The phosphorylated beta gamma(12) associated with G(o) alpha more tightly than the unphosphorylated form. Exposure of Swiss 3T3 and aortic smooth muscle cells to phorbol 12-myristate 13-acetate and NaF induced phosphorylation of gamma(12). Stimulation of aortic smooth muscle cells with natural vasoactive agents such as angiotensin II and vasopressin also induced phosphorylation of gamma(12). The extent of phosphorylation of beta gamma(12) in vitro was suppressed by a complex formation with G(o) alpha, which was relieved by the addition of guanosine 5'-O-(3-thiotriphosphate) or aluminum fluoride. These results strongly suggest that gamma(12) is phosphorylated by protein kinase C during activation of receptor(s) and G protein(s) in living cells.