Primary structure of a γ subunit of G protein, γ₁₂, and its phosphorylation by protein kinase C

We have determined the primary structure of a novel γ subunit (γ₁₂, previously designated γ(S1)) of G protein purified from bovine spleen. The mature γ₁₂ 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 γ₁₂ cDNA isolated from a bovine spleen cDNA library. Western blots with the specific antibody against γ₁₂ showed that γ₁₂ is present in all tissues examined. Among various γ subunits (γ₁, γ₂, γ₃, γ₇, and γ₁₂), γ₁₂ has a unique property to be phosphorylated by protein kinase C. The phosphorylated amino acid residue was Ser¹ (or Ser²). The phosphorylated βγ₁₂ associated with G(o)α 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 γ₁₂. Stimulation of aortic smooth muscle cells with natural vasoactive agents such as angiotensin II and vasopressin also induced phosphorylation of γ₁₂. The extent of phosphorylation of βγ₁₂ in vitro was suppressed by a complex formation with G(o)α, which was relieved by the addition of guanosine 5'-O-(3-thiotriphosphate) or aluminum fluoride. These results strongly suggest that γγ₁₂ is phosphorylated by protein kinase C during activation of receptor(s) and G protein(s) in living cells.