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. 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. Protein kinase B (PKB 2The abbreviations used are: PKB, protein kinase B (or Akt); RSK, ribosomal S6 kinase; PAS, phospho-Akt substrate; GAP, GTPase-activating protein; DLC, deleted in liver cancer; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; ERK, extracellular signal-regulated kinase; PI, phosphatidylinositol; mTOR, mammalian target of rapamycin; CHO, Chinese hamster ovary; GFP, green fluorescent protein; myr, myristoylated; GLUT4, glucose transporter 4; PtdIns-3,4,5-P3, phosphatidylinositol 3,4,5-trisphosphate; PtdIns-3-P, phosphatidylinositol-3-phosphate; AGC, protein kinases A, G, and C. 2The abbreviations used are: PKB, protein kinase B (or Akt); RSK, ribosomal S6 kinase; PAS, phospho-Akt substrate; GAP, GTPase-activating protein; DLC, deleted in liver cancer; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; ERK, extracellular signal-regulated kinase; PI, phosphatidylinositol; mTOR, mammalian target of rapamycin; CHO, Chinese hamster ovary; GFP, green fluorescent protein; myr, myristoylated; GLUT4, glucose transporter 4; PtdIns-3,4,5-P3, phosphatidylinositol 3,4,5-trisphosphate; PtdIns-3-P, phosphatidylinositol-3-phosphate; AGC, protein kinases A, G, and C. or Akt) is a protein serine/threonine kinase that plays a key role in intracellular signaling and cellular homeostasis. The kinase is activated by dual phosphorylation on Thr-308 by 3-phosphoinositide-dependent kinase-1 (1Alessi D.R. James S.R. Downes C.P. Holmes A.B. Gaffney P.R. Reese C.B. Cohen P. Curr. Biol. 1997; 7: 261-269Abstract Full Text Full Text PDF PubMed Google Scholar) and on Ser-473 in a hydrophobic motif by a kinase that remains to be identified, although recent evidence suggests that ataxia telangiectasia-mutated, DNA-protein kinase, integrin-linked kinase and/or mTOR/Rictor may be involved (2Sarbassov D.D. Guertin D.A. Ali S.M. Sabatini D.M. Science. 2005; 307: 1098-1101Crossref PubMed Scopus (4981) Google Scholar, 3Viniegra J.G. Martinez N. Modirassari P. Losa J.H. Parada Cobo C. Lobo V.J. Luquero C.I. Alvarez-Vallina L. Ramon y Cajal S. Rojas J.M. Sanchez-Prieto R. J. Biol. Chem. 2005; 280: 4029-4036Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 4Persad S. Attwell S. Gray V. Mawji N. Deng J.T. Leung D. Yan J. Sanghera J. Walsh M.P. Dedhar S. J. Biol. 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Constitutively active PKB mutants induce GLUT4 translocation in the absence of insulin, whereas dominant negative PKB mutants and ablation of PKB using small interfering RNA decrease insulin-stimulated glucose uptake (8Watson R.T. Kanzaki M. Pessin J.E. Endocr. Rev. 2004; 25: 177-204Crossref PubMed Scopus (343) Google Scholar, 9Zhou Q.L. Park J.G. Jiang Z.Y. Holik J.J. Mitra P. Semiz S. Guilherme A. Powelka A.M. Tang X. Virbasius J. Czech M.P. Biochem. Soc. Trans. 2004; 32: 817-821Crossref PubMed Scopus (73) Google Scholar), PKBβ knock-out mice exhibit reduced insulin-stimulated glucose uptake into muscle and adipose tissue (10Cho H. Mu J. Kim J.K. Thorvaldsen J.L. Chu Q. Crenshaw III, E.B. Kaestner K.H. Bartolomei M.S. Shulman G.I. Birnbaum M.J. Science. 2001; 292: 1728-1731Crossref PubMed Scopus (1486) Google Scholar), and deregulation of PKB activation by insulin has been reported to be associated with insulin resistance in type II diabetes (11Karlsson H.K. Zierath J.R. Kane S. Krook A. Lienhard G.E. WallbergHenriksson H. Diabetes. 2005; 54: 1692-1697Crossref PubMed Scopus (211) Google Scholar). PKB also plays a central role in the regulation of many other cellular processes, such as apoptosis and anoikis, neuronal development and degeneration, and the cell cycle (see Ref. 7Brazil D.P. Yang Z.Z. Hemmings B.A. Trends Biochem. Sci. 2004; 29: 233-242Abstract Full Text Full Text PDF PubMed Scopus (702) Google Scholar for a recent review). Despite the central role of PKB in insulin action, many of the substrates that mediate the actions of PKB remain to be identified. Several groups, including our own, have used a commercially available PAS (phospho-Akt substrate) antibody raised against the reported minimal consensus phosphorylation site found in almost all known PKB substrates (RXRXX(pS/pT)) (12Alessi D.R. Caudwell F.B. Andjelkovic M. Hemmings B.A. Cohen P. FEBS Lett. 1996; 399: 333-338Crossref PubMed Scopus (542) Google Scholar, 13Obata T. Yaffe M.B. 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PIKfyve is a PtdIns-3-P 5-kinase, which also appears to play a role in regulating the intracellular trafficking of GLUT4 (17Berwick D.C. Dell G.C. Welsh G.I. Heesom K.J. Hers I. Fletcher L.M. Cooke F.T. Tavare J.M. J. Cell Sci. 2004; 117: 5985-5993Crossref PubMed Scopus (115) Google Scholar). The role of PRAS40 remains unclear, whereas Yes-associated protein attenuates p73-induced apoptosis (18Basu S. Totty N.F. Irwin M.S. Sudol M. Downward J. Mol. Cell. 2003; 11: 11-23Abstract Full Text Full Text PDF PubMed Scopus (606) Google Scholar) and WNK1 negative regulates insulin-stimulated mitogenesis (19Jiang Z.Y. Zhou Q.L. Holik J. Patel S. Leszyk J. Coleman K. Chouinard M. Czech M.P. J. Biol. Chem. 2005; 280: 21622-21628Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Using this approach in the current study, we identified by mass spectrometry an insulin-stimulated phosphoprotein of an apparent molecular weight of 120 kDa in primary as p122RhoGAP, a of the family of GTPase-activating for the and We demonstrated that insulin stimulates p122RhoGAP phosphorylation on Ser-322 in primary and in an cell hamster and that PKB can directly p122RhoGAP on Ser-322 in We also that p122RhoGAP can be phosphorylated in vitro by of the AGC family of protein and that this also in intact cells in an insulin-stimulated manner the signaling Ser-322 phosphorylation of p122RhoGAP acts as an integrator of signal transduction with the of the PKB and MEK in insulin-stimulated p122RhoGAP phosphorylation on the cellular on a and and was The antibody was as K. M. M. H. H. H. Biochem. Soc. Trans. 2004; 32: PubMed Scopus Google Scholar). The antibody and all Cell The antibody was whereas the fluorescent protein antibody all other as I. A.W. Jiang D. Schaefer E. Tavare J.M. Biochem. J. 2002; PubMed Google Scholar). constitutively active PKB and constitutively active of PI by Hemmings and J. Downward and into the The constitutively active was by of The was as M. M. M. K. H. H. H. 2004; PubMed Scopus Google Scholar). using the as a was with the the mutants and of the of as I. A.W. Jiang D. Schaefer E. Tavare J.M. Biochem. J. 2002; PubMed Google Scholar). in and with to the of as (15Berwick D.C. Hers I. Heesom K.J. Moule S.K. Tavare J.M. J. Biol. Chem. 2002; 277: 33895-33900Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar) with a In cells in intracellular and with was used to of cells in the of of was by and a of using a the cells to a and for the with of of and The cells for an and and into to the of into and or with or for to stimulation with insulin for the The was by the cells cell in 120 and of and Cell for and the was for and of cells cells the insulin in and cells in with of or using a of of of to the the cells for with insulin for the in the and in by into of 120 and of and Cell for and the was for p122RhoGAP was by of cell with of antibody and of protein The protein by and in was and by for by using or and to The in in with and with primary and which in The for antibody and using an used a of antibody was for all of was as a with in of cells to an of and protein expression was by the of The cells by and in of and the cells by was by and protein was to protein was on in of p122RhoGAP as an identify novel substrates for PKB, we primary with insulin in the absence and of the PI The cell by and with the PAS in insulin an in phosphorylation of that the and Phosphorylation of the of these was reduced in the of of the of numerous and we this to by phosphoprotein with the PAS and with an apparent molecular weight of kDa was and other upon Hers and J. M. protein was the and with of the using the to the identification of this protein as p122RhoGAP J. PubMed Scopus Google Scholar). Insulin the Phosphorylation of p122RhoGAP Ser-322 in and of the of p122RhoGAP using the the of PKB phosphorylation and of the an phosphorylation site Ser-322 to of the consensus for PKB or of these are phosphorylated upon insulin we primary and cells with p122RhoGAP of and that insulin stimulation to the phosphorylation of p122RhoGAP in primary and cells as by with the PAS of and to on the phosphorylation of p122RhoGAP, of Ser-322 the of p122RhoGAP with the PAS antibody in cell These results demonstrate that Ser-322 is the insulin-stimulated phosphorylation site by the PAS antibody on p122RhoGAP in primary and PKB p122RhoGAP in on p122RhoGAP is a for PKB in we in the and absence of active PKB and Phosphorylation of p122RhoGAP was in the of PKB in PKB also phosphorylated molecular mass of p122RhoGAP in the of The of PKB to p122RhoGAP on the site by the PAS antibody was in the demonstrating that Ser-322 is the phosphorylation site by this antibody p122RhoGAP in a PI in insulin-stimulated p122RhoGAP Ser-322 phosphorylation was regulated of PI and PKB in intact we primary with p122RhoGAP and with or the PI We also the of which the activation of ERK, and which the activation of and the a ribosomal protein S6 that insulin-stimulated p122RhoGAP phosphorylation was in the of whereas and In we the and of the for insulin-stimulated phosphorylation of PKB and These results demonstrate that the insulin-stimulated pathway to p122RhoGAP Ser-322 phosphorylation in primary is PI of Constitutively PI and PKB p122RhoGAP Phosphorylation in of activation of PI and/or PKB is sufficient for phosphorylation of Ser-322 on p122RhoGAP, we primary with constitutively active mutants of PI or PKB in with the p122RhoGAP expression that and an in the phosphorylation of p122RhoGAP on These results demonstrate that activation of PI or PKB is sufficient to Ser-322 phosphorylation of p122RhoGAP in primary adipocytes. Phosphorylation of in on the MEK to the results in primary adipocytes, we found that insulin-stimulated p122RhoGAP Ser-322 phosphorylation in cells was by with insulin-stimulated PKB phosphorylation was In these cells, the MEK reduced p122RhoGAP Ser-322 phosphorylation to of that a kinase of is for the phosphorylation of this site in this cellular The insulin-stimulated p122RhoGAP phosphorylation in the of a was by the of suggests that the pathway plays a dominant role the PI 3-kinase/PKB pathway, with the of the pathway apparent upon of MEK The on p122RhoGAP Ser-322 phosphorylation the of these the These results demonstrate that insulin-stimulated p122RhoGAP Ser-322 phosphorylation in cells is dependent on PI and MEK is of p122RhoGAP Ser-322 Phosphorylation by Constitutively PKB or Constitutively MEK in the MEK pathway appears to play a dominant and role in insulin-stimulated Ser-322 we MEK activation was sufficient to induce Ser-322 phosphorylation in cells using a constitutively active MEK that this was the of with p122RhoGAP in an in the phosphorylation of p122RhoGAP to an that was almost that by insulin the constitutively active although the PI 3-kinase/PKB pathway plays a minor role in insulin-stimulated Ser-322 this pathway is the phosphorylation of this the results demonstrate that activation of PKB or MEK is sufficient to Ser-322 phosphorylation of p122RhoGAP that the MEK pathway in insulin-stimulated Ser-322 phosphorylation in intact p122RhoGAP on activation in cells, MEK and the extracellular signal-regulated kinases and is that Ser-322 is directly phosphorylated by as this site the consensus phosphorylation by these kinases for phosphorylation of Ser-322 on p122RhoGAP of is ribosomal S6 kinase-1 to PKB, is a of the AGC kinase is activated of and the consensus motif as PKB (12Alessi D.R. Caudwell F.B. Andjelkovic M. Hemmings B.A. Cohen P. FEBS Lett. 1996; 399: 333-338Crossref PubMed Scopus (542) Google Scholar, Caudwell F.B. Cohen Cohen P. FEBS Lett. PubMed Scopus Google Scholar). p122RhoGAP on we p122RhoGAP and p122RhoGAP a with active and and p122RhoGAP phosphorylation using the PAS of p122RhoGAP with active to phosphorylation of p122RhoGAP phosphorylation was although in the These results demonstrate that is to p122RhoGAP on Ser-322 in In this study, we have demonstrated that the small GTPase-activating protein p122RhoGAP is a novel insulin-stimulated PKB in cell primary and We that PKB directly p122RhoGAP on Ser-322 in vitro and that expression of active mutants of PI or PKB in primary to Ser-322 the PI insulin-stimulated p122RhoGAP phosphorylation in these cells, we that PKB is and sufficient for the insulin-stimulated phosphorylation of Ser-322 on p122RhoGAP in primary adipocytes. In we found that insulin-stimulated p122RhoGAP phosphorylation in cells, which the insulin on MEK as the MEK reduced p122RhoGAP In these cells, in with of a constitutively active MEK in cells stimulates p122RhoGAP phosphorylation to the as with insulin or a constitutively active These results demonstrate that the pathway is and sufficient for the insulin-stimulated phosphorylation of Ser-322 on p122RhoGAP in In insulin can p122RhoGAP Ser-322 phosphorylation the PI 3-kinase/PKB and with the of pathway dependent on the cellular context. PKB is the kinase to Ser-322 PI we demonstrated that a of the AGC kinase is the to p122RhoGAP of as directly p122RhoGAP in The of phosphorylation by is in the demonstrating that p122RhoGAP the phosphorylation by this kinase other this was phosphorylated by insulin in intact cells as the of p122RhoGAP the PAS antibody was in the for the that and protein and Ser-322 in p122RhoGAP using the mitogen-activated protein kinase in intact cells, in the activation of and protein and kinase M. L.M. Alessi D.R. J. PubMed Scopus Google Scholar). p122RhoGAP phosphorylated in the of protein kinase is I. and J. M. that and protein and kinase are involved in p122RhoGAP The that Ser-322 on p122RhoGAP is a dual PKB and in is of as can be to a small of phosphorylation on For example, of glycogen has been reported to be a for PKB and in cells M. Cohen P. FEBS Lett. PubMed Scopus Google Scholar, D.D. Q. L. FEBS Lett. 2001; PubMed Scopus Google Scholar). PKB and are reported to be to and of B.A. R. J. Sci. 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Ser-322 this its phosphorylation may be involved in regulating the of p122RhoGAP to the to we have been to demonstrate that and mutants to as the Furthermore, which have an to was also found in Phosphorylation of Ser-322 is to of p122RhoGAP to In we have demonstrated that p122RhoGAP Ser-322 is a novel PKB and and insulin-stimulated phosphoprotein in relevant cells as as immortalized cells in site has the to the of signal transduction in a manner dependent on the cellular context. on the of p122RhoGAP phosphorylation in signaling events of PKB, such as GLUT4 the activation of RhoA and cellular We are to of for with mass and of for in the against p122RhoGAP, and Cohen of for