Primary Structure of a γ Subunit of G Protein, γ12, and Its Phosphorylation by Protein Kinase C

Rika Morishita,Hiroshi Nakayama,Toshiaki Isobe,Takahiko Matsuda,Yuichi Hashimoto,Toshiyuki Okano,Yoshitaka Fukada,Keiko Mizuno,Shigeo Ohno,Osamu Kozawa,Kanefusa Kato,Tomiko Asano

doi:10.1074/jbc.270.49.29469

Abstract

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 Ser1 (or Ser2). The phosphorylated beta gamma 12 associated with Go 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 Go 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.

Highlights

Heterotrimeric G proteins1 play a major role in the transduction of signals from cell surface receptors to cellular effectors [1]
The extent of phosphorylation of ␤␥12 in vitro was suppressed by a complex formation with Go␣, which was relieved by the addition of guanosine 5؅-O-(3-thiotriphosphate) or aluminum fluoride. These results strongly suggest that ␥12 is phosphorylated by protein kinase C during activation of receptor(s) and G protein(s) in living cells
We found that a novel ␥ subunit of G protein, ␥12, was a good substrate for protein kinase C (PKC) among various forms of ␥ examined in vitro

Summary

Introduction

Heterotrimeric G proteins play a major role in the transduction of signals from cell surface receptors to cellular effectors [1]. The ␤␥ complex regulates several effectors, which include Kϩ channels, adenylyl cyclase, phospholipase A2, phospholipase C-␤, receptor kinases, and phosphatidylinositol 3-kinase [1, 2]. C that hydrolyze phosphatidylinositol 4,5-bisphosphate to produce inositol 1,4,5-trisphosphate and diacylglycerol They regulate the release of Ca2ϩ ions from intracellular stores and the activity of protein kinase C (PKC), respectively [18, 19]. The agonist-induced phospholipase C reactions are inhibited by phorbol esters such as PMA, a potent activator of PKC [19]. Such inhibition by PKC activators appears to provide a feedback control on agoniststimulated phospholipase C.

Methods

Results

Conclusion