Identification of p122RhoGAP (deleted in liver cancer-1) Serine 322 as a substrate for protein kinase B and ribosomal S6 kinase in insulin-stimulated cells.
Protein kinase B (PKB or Akt) plays an essential role in the actions of insulin, cytokines, and growth factors, although the substrates for PKB that are relevant to many of its actions require identification. In this study, we have reported the identification of p122RhoGAP, a GTPase-activating protein selective for RhoA and rodent homologue of the tumor suppressor deleted in liver cancer (DLC1) as a novel insulin-stimulated phosphoprotein in primary rat adipocytes. We have demonstrated that Ser-322 is phosphorylated upon insulin stimulation of intact cells and that this site is directly phosphorylated in vitro by PKB and ribosomal S6 kinase, members of the AGC (protein kinases A, G, and C) family of insulin-stimulated protein kinases. Furthermore, expression of constitutively active mutants of PKB or mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) stimulates Ser-322 phosphorylation in intact cells, demonstrating that activation of the PKB or MEK pathway is sufficient for Ser-322 phosphorylation in vivo. Indeed, in primary adipocytes, insulin-stimulated Ser-322 phosphorylation was almost exclusively regulated by the phosphatidylinositol 3-kinase/PKB pathway, whereas in immortalized cells, insulin-stimulated phosphorylation was predominantly regulated by the MEK/extracellular signal-regulated kinase/ribosomal S6 kinase pathway, with the phosphatidylinositol 3-kinase/PKB pathway playing a minor role. These results demonstrate that p122RhoGAP Ser-322 acts as an integrator of signal transduction in a manner dependent on the cellular context.
Hers
Ingeborg
I
Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom. i.hers@bris.ac.uk
Wherlock
Matthew
M
Homma
Yoshimi
Y
Yagisawa
Hitoshi
H
Tavaré
Jeremy M
JM
D017667:Adipocytes / Q000378:metabolism; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D016466:CHO Cells; D002478:Cells, Cultured; D002852:Chromatography, Ion Exchange; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D004822:Epididymis / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000737:chemistry* / Q000502:physiology*; D006801:Humans; D047468:Immunoprecipitation; D007328:Insulin / Q000378:metabolism*; D008297:Male; D009154:Mutation; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D011972:Receptor, Insulin / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D038744:Ribosomal Protein S6 Kinases, 90-kDa / Q000378:metabolism*; D012694:Serine / Q000737:chemistry*; D015398:Signal Transduction; D014162:Transfection; D025521:Tumor Suppressor Proteins / Q000737:chemistry*; D000077191:Wortmannin; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
D000730:Androstadienes; D048049:Extracellular Signal-Regulated MAP Kinases; D020690:GTPase-Activating Proteins; D007328:Insulin; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D011972:Receptor, Insulin; D011994:Recombinant Proteins; D038744:Ribosomal Protein S6 Kinases, 90-kDa; D012694:Serine; D025521:Tumor Suppressor Proteins; D000077191:Wortmannin; D020742:rhoA GTP-Binding Protein; C430864:DLC-1 (deleted in liver cancer) protein, mouse; C509915:Dlc1 protein, rat; C069329:RPS6KA1 protein, human; C512964:Rps6ka1 protein, mouse; C512963:Rps6ka1 protein, rat