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Assessment of the Roles of Mitogen-activated Protein Kinase, Phosphatidylinositol 3-Kinase, Protein Kinase B, and Protein Kinase C in Insulin Inhibition of cAMP-induced Phosphoenolpyruvate Carboxykinase Gene Transcription*

  • Joyce M. Agati
    Affiliations
    Department of Cellular and Molecular Physiology, The Pennsylvania State University, College of Medicine, Hershey, Pennsylvania 17033
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  • David Yeagley
    Affiliations
    Department of Cellular and Molecular Physiology, The Pennsylvania State University, College of Medicine, Hershey, Pennsylvania 17033
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  • Patrick G. Quinn
    Correspondence
    To whom correspondence should be addressed: Dept. of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033. Tel.: 717-531-6182; Fax: 717-531-7667;
    Affiliations
    Department of Cellular and Molecular Physiology, The Pennsylvania State University, College of Medicine, Hershey, Pennsylvania 17033
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  • Author Footnotes
    * This work was supported in part by National Institutes of Health Grant DK49600 and Juvenile Diabetes Foundation International Grant JDFI 195085.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:July 24, 1998DOI:https://doi.org/10.1074/jbc.273.30.18751
      Transcription of the phosphoenolpyruvate carboxykinase (PEPCK) gene is induced by glucagon, acting through cAMP and protein kinase A, and this induction is inhibited by insulin. Conflicting reports have suggested that insulin inhibits induction by cAMP by activating the Ras/mitogen-activated protein kinase (MAPK) pathway or by activating the phosphatidylinositol 3-kinase (PI3-kinase), but not MAPK, pathway. Insulin activated PI3-kinase phosphorylates lipids that activate protein kinase B (PKB) and Ca2+/diacylglycerol-insensitive forms of protein kinase C (PKC). We have assessed the roles of these pathways in insulin inhibition of cAMP/PKA-induced transcription of PEPCK by using dominant negative and dominant active forms of regulatory enzymes in the Ras/MAPK and PKB pathways and chemical inhibitors of PKC isoforms. Three independently acting inhibitory enzymes of the Ras/MAPK pathway, blocking SOS, Ras, and MAPK, had no effect upon insulin inhibition. However, dominant active Ras prevented induction of PEPCK and also stimulated transcription mediated by Elk, a MAPK target. Insulin did not stimulate Elk-mediated transcription, indicating that insulin did not functionally activate the Ras/MAPK pathway. Inhibitors of PI3-kinase, LY294002 and wortmannin, abolished insulin inhibition of PEPCK gene transcription. However, inhibitors of PKC and mutated forms of PKB, both of which are known downstream targets of PI3-kinase, had no effect upon insulin inhibition. Dominant negative forms of PKB did not interfere with insulin inhibition and a dominant active form of PKB did not prevent induction by PKA. Phorbol ester-mediated inhibition of PEPCK transcription was blocked by bisindole maleimide and by staurosporine, but insulin-mediated inhibition was unaffected. Thus, insulin inhibition of PKA-induced PEPCK expression does not require MAPK activation but does require activation of PI3-kinase, although this signal is not transmitted through the PKB or PKC pathways.
      Insulin stimulates a variety of changes in growth and metabolism in different cell types, ranging from the stimulation of replication, translation, and protein synthesis to the covalent modification of enzymes of intermediary metabolism (
      • White M.F.
      • Kahn C.R.
      ). In addition, insulin regulates the transcription of specific genes, whose products catalyze committed reactions in hepatic glucose metabolism (
      • Pilkis S.J.
      • Granner D.K.
      ). In particular, the amounts of the enzymes that catalyze committed steps at either end of the glucose utilization pathway, glucokinase and phosphoenolpyruvate carboxykinase (PEPCK)
      The abbreviations used are: PEPCK, phosphoenolpyruvate carboxykinase; CRE, cAMP-response element; CREB, cAMP-response element binding protein; IRS, insulin receptor substrate; PKAc, protein kinase A catalytic subunit; P-CREB, PKAc-phosphorylated CREB; CBP, CREB-binding protein; CRU, cAMP response unit; MAPK, mitogen-activated protein kinase; PKB, protein kinase B; PKC, protein kinase C; PI3-kinase, phosphatidylinositol 3-kinase; RSV, Rous sarcoma virus; Luc, luciferase; PH, pleckstrin homology; BIM, bisindolyl maleimide I, HCl; PMA, phorbol 12-myristate 13-acetate; da, dominant active; dn, dominant negative; C/EBP, CAAT/enhancer binding protein.
      1The abbreviations used are: PEPCK, phosphoenolpyruvate carboxykinase; CRE, cAMP-response element; CREB, cAMP-response element binding protein; IRS, insulin receptor substrate; PKAc, protein kinase A catalytic subunit; P-CREB, PKAc-phosphorylated CREB; CBP, CREB-binding protein; CRU, cAMP response unit; MAPK, mitogen-activated protein kinase; PKB, protein kinase B; PKC, protein kinase C; PI3-kinase, phosphatidylinositol 3-kinase; RSV, Rous sarcoma virus; Luc, luciferase; PH, pleckstrin homology; BIM, bisindolyl maleimide I, HCl; PMA, phorbol 12-myristate 13-acetate; da, dominant active; dn, dominant negative; C/EBP, CAAT/enhancer binding protein.
      are regulated solely through modulation of gene transcription in an opposite fashion by insulin and glucagon, which acts through cAMP and PKA (
      • Granner D.
      • Pilkis S.
      ). Transcription of the gene encoding glucokinase, which is required for glycolysis, is induced by insulin and inhibited by cAMP. In contrast, transcription of the gene encoding PEPCK, which is required for gluconeogenesis, is induced by cAMP/PKA and inhibited by insulin (
      • Sasaki K.
      • Cripe T.P.
      • Koch S.R.
      • Andreone T.L.
      • Petersen D.D.
      • Beale E.G.
      • Granner D.K.
      ). The mechanism of insulin action has remained elusive.
      The binding of insulin to its cell surface receptors activates their intrinsic tyrosine kinase activity, leading to receptor autophosphorylation and phosphorylation of cytosolic proteins, known as IRSs, which serve as adapters in intracellular signaling (
      • White M.F.
      • Kahn C.R.
      ). IRS-1, the predominant and most thoroughly characterized IRS, binds a variety of signaling molecules when specific tyrosines are phosphorylated, including the regulatory subunit of phosphatidylinositol 3-kinase (PI3-kinase), Shc-1, and Grb 2 (
      • White M.F.
      • Kahn C.R.
      ,
      • Hadari Y.R.
      • Tzahar E.
      • Nadiv O.
      • Rothenberg P.
      • Roberts Jr., C.T.
      • LeRoith D.
      • Yarden Y.
      • Zick Y.
      ,
      • Myers M.J.
      • White M.
      ). Interaction among these IRS-associated molecules initiates signaling cascades leading to the activation of a variety of protein kinases, including MAPK, protein kinase B, protein kinase C, glycogen synthase kinase-3, pp90rsk II, and p70S6 kinase (
      • Rutter G.A.
      • White M.R.H.
      • Tavaré J.M.
      ,
      • Standaert M.L.
      • Bandyopadhyay G.
      • Farese R.V.
      ,
      • Sutherland C.
      • O'Brien R.M.
      • Granner D.K.
      ,
      • Cheatham B.
      • Vlahos C.J.
      • Cheatham L.
      • Wang L.
      • Blenis J.
      • Kahn C.R.
      ,
      • Nakajima T.
      • Fukamizu A.
      • Takahashi J.
      • Gage F.H.
      • Fisher T.
      • Blenis J.
      • Montminy M.R.
      ,
      • Nakanishi H.
      • Brewer K.A.
      • Exton J.H.
      ,
      • Cross D.A.
      • Alessi D.R.
      • Cohen P.
      • Andjelkovich M.
      • Hemmings B.A.
      ,
      • Mendez R.
      • Kollmorgen G.
      • White M.F.
      • Rhoads R.E.
      ). All of these kinases have been implicated in one or more of the growth or metabolic effects attributed to insulin. In some cases, activation of a single pathway may suffice for regulation, whereas in others more than one of these pathways may need to be activated for regulation by insulin. For example, activation of both PI3-kinase and MAPK is required for stimulation of general protein synthesis by insulin, whereas only the PI3-kinase pathway needs to be activated for stimulation of growth-related protein synthesis by insulin (
      • Mendez R.
      • Kollmorgen G.
      • White M.F.
      • Rhoads R.E.
      ). The lipid products of PI3-kinase, which is essential for mediating many of the metabolic effects of insulin, activate PKB (also known as Rac and Akt) (
      • Datta K.
      • Bellacosa A.
      • Chan T.O.
      • Tsichlis P.N.
      ,
      • Klippel A.
      • Kavanaugh W.M.
      • Pot D.
      • Williams L.T.
      ,
      • Burgering B.M.
      • Coffer P.J.
      ) and novel isoforms of PKC not regulated by calcium and diacylglycerol (
      • Nakanishi H.
      • Brewer K.A.
      • Exton J.H.
      ,
      • Toker A.
      • Meyer M.
      • Reddy K.
      • Falck J.
      • Aneja R.
      • Aneja S.
      • Parra A.
      • Burns D.
      • Ballas L.
      • Cantley L.
      ).
      We previously showed that multiple binding sites for CREB-GAL4 ligated to a minimal PEPCK promoter (5XGT) could mediate induction by cAMP/PKA and that this induction was inhibited, at least in part, by insulin in H4IIe hepatoma cells (
      • Quinn P.G.
      ). We proposed that insulin targeted the CREB·CBP·RNA polymerase II complex to inhibit PEPCK gene transcription. However, we recently reexamined this question with a more sensitive luciferase reporter gene and found that insulin inhibition of 5XGT was cAMP-independent, as it was indistinguishable from insulin inhibition of basal PEPCK gene transcription (
      • Yeagley D.
      • Agati J.M.
      • Quinn P.G.
      ). In addition to the CRE, induction of the PEPCK gene by cAMP requires heterologous binding sites located in the (AC) region (−271/−225) (
      • Yeagley D.
      • Agati J.M.
      • Quinn P.G.
      ,
      • Roesler W.J.
      • Graham J.G.
      • Kolen R.
      • Klemm D.J.
      • McFie P.J.
      ,
      • Roesler W.J.
      • Crosson S.M.
      • Vinson C.
      • McFie P.J.
      ,
      • Roesler W.J.
      • McFie P.J.
      • Puttick D.M.
      ). Factors binding to the AC region and CREB, which is targeted by cAMP-activated PKA, form a cAMP response unit (CRU) that mediates both induction by PKA and inhibition by insulin in the presence of the minimal PEPCK promoter (
      • Yeagley D.
      • Agati J.M.
      • Quinn P.G.
      ).
      Blenis and Montminy and colleagues (
      • Nakajima T.
      • Fukamizu A.
      • Takahashi J.
      • Gage F.H.
      • Fisher T.
      • Blenis J.
      • Montminy M.R.
      ) provided evidence that CBP is targeted by insulin to inhibit PEPCK transcription. Their data indicated that activation of the Ras/MAPK pathway by insulin in H4IIe rat hepatoma cells resulted in activation of pp90rskII by MAPK, leading to its binding to CBP and inhibition of cAMP-induced transcription. On the other hand, Gabbay et al. (
      • Gabbay R.A.
      • Sutherland C.
      • Gnudi L.
      • Kahn B.B.
      • O'Brien R.M.
      • Granner D.K.
      • Flier J.S.
      ) demonstrated that PD98059, a potent and specific MEK inhibitor, had no effect upon insulin inhibition of PEPCK gene transcription in H4IIe cells. In addition, Sutherland et al. (
      • Sutherland C.
      • O'Brien R.M.
      • Granner D.K.
      ) provided evidence that inhibition of PI3-kinase activation abrogated insulin regulation of the PEPCK gene. Thus, there is directly conflicting evidence regarding the insulin-stimulated pathway(s) required for inhibition of PEPCK gene transcription.
      The present study was undertaken to examine the role of potential insulin signaling pathways in the inhibition of PKA-induced PEPCK gene transcription. We expressed dominant negative and dominant active forms of regulatory signaling enzymes in the Ras/MAPK and PKB/Akt pathways and utilized chemical inhibitors of PI3-kinase and protein kinase C isoforms to assess the contribution of these different signaling pathways to insulin inhibition of PEPCK expression.

      DISCUSSION

      The results presented here demonstrate that activation of the insulin, Ras/MAPK, or PKC signaling pathways inhibited PKA-induced PEPCK transcription. However, inhibition of either the Ras/MAPK or PKC pathways had no effect upon insulin inhibition. Although dominant active Ras mimicked insulin inhibition of PKA-induced PEPCK transcription, three independently acting dominant negative inhibitors of the Ras/MAPK pathway had no effect upon insulin inhibition. In addition, we showed that insulin does not functionally activate the Ras/MAPK pathway in H4IIe cells. In contrast, inhibition of PI3-kinase activity with either wortmannin or LY294002 abolished inhibition by insulin. Inhibition of PKB/Akt and PKCζ, known downstream targets of PI3-kinase, or of PMA-activated PKC had no effect upon insulin inhibition of PKA-induced PEPCK-Luc activity. Thus, several potential insulin signaling pathways converge on some common transcription factor or complex, but most of them are dispensable for insulin inhibition of PKA-induced PEPCK gene transcription. Overall, our data suggest that an as yet uncharacterized target of PI3-kinase mediates insulin inhibition of cAMP-induced PEPCK gene transcription or that alternate pathways may be utilized. Our data are summarized in Fig. 8, which also specifies the elements determined to be necessary for opposing regulation of PEPCK gene expression by cAMP and insulin (
      • Yeagley D.
      • Agati J.M.
      • Quinn P.G.
      ).
      Figure thumbnail gr8
      Figure 8Role of alternate signaling pathways to insulin inhibition of PKA-induced transcription of the PEPCK gene.Alternate signaling pathways are shown together with the inhibitors (black ovals) that block specific enzymes. A line is drawn through the name of enzymes whose blockade had no effect upon insulin inhibition of PKA-induced PEPCK transcription. Based on this information, insulin likely activates PI3-kinase which signals through an as yet unidentified mediator to modify the function of the CRU-associated factors (CREB, activator protein-1, C/EBP) or cofactors, that mediate induction by PKA. This summary is based on the results of the present study, except for the PD result, which is based on Gabbay et al. (
      • Gabbay R.A.
      • Sutherland C.
      • Gnudi L.
      • Kahn B.B.
      • O'Brien R.M.
      • Granner D.K.
      • Flier J.S.
      ), and the Ly294002/wortmannin (LY/Wort) result, which is also based on Sutherland et al. (
      • Sutherland C.
      • O'Brien R.M.
      • Granner D.K.
      ). The abbreviations are the same as in Fig. .

      Lack of Involvement of the Ras/MAPK Pathway in Insulin Inhibition

      We found that three different inhibitors of the Ras/MAPK pathway, dn-Ras, dn-Raf, and da-PAC-1, had no effect upon insulin inhibition. The fact that these three inhibitors target three distinct steps in the Ras/MAPK pathway strengthens the argument that the Ras/MAPK pathway plays no obligatory role in insulin signaling. In addition, we showed that insulin does not functionally stimulate the Ras/MAPK pathway in H4IIe cells, as indicated by its failure to stimulate G4-PEPCK-Luc expression in the presence of G4-Elk, a classical MAPK target (
      • Roberson M.S.
      • Misra-Press A.
      • Laurance M.E.
      • Stork P.J.
      • Maurer R.A.
      ). In contrast, da-Ras both inhibited PEPCK-Luc and stimulated G4-PEPCK-Luc expression by 20-fold in the presence of G4-Elk. Thus, if the Ras/MAPK pathway had been activated by insulin, it would have prevented induction by PKA.
      Interestingly, all of the Ras/MAPK pathway inhibitors enhanced induction by PKA, suggesting that the Ras/MAPK pathway may exert a constitutive restraining influence on PEPCK gene transcription. This restraint was relieved when the pathway was inhibited. This relief of restraint was consistently seen in all experiments and with all enzyme mutants that interfere with Ras/MAPK signaling. It was most pronounced with lower amounts of expression vector (data not shown), arguing against it being an artifact of the expression vector. Other examples of constitutive restraint are seen with the kinase-defective PKB, K → A mutant and with phosphorylation of Ser-142 in CREB, the mutation of which potentiates CREB-mediated transcription induction (
      • Yeagley D.
      • Agati J.M.
      • Quinn P.G.
      ,
      • Sun P.
      • Enslen H.
      • Myung P.S.
      • Maurer R.A.
      ).
      Recently, a model was proposed for insulin inhibition of PKA-induced PEPCK gene transcription by Nakajima et al. (
      • Nakajima T.
      • Fukamizu A.
      • Takahashi J.
      • Gage F.H.
      • Fisher T.
      • Blenis J.
      • Montminy M.R.
      ). They used an H4IIe cell line stably transfected with a truncated PEPCK promoter (−134/+69) and Ras pathway mutants to argue that insulin activates the Ras/MAPK pathway, culminating in activation of pp90rsk. The binding of pp90rsk to CBP was proposed to disrupt the P-CREB·CBP·RNA polymerase II complex and terminate activation by cAMP.
      However, recent findings are not consistent with this model. First of all, the single CREB binding site in the PEPCK promoter is insufficient for activation of transcription by PKA; the −134/+69 PEPCK promoter can not mediate induction by PKA in H4IIe cells (
      • Yeagley D.
      • Agati J.M.
      • Quinn P.G.
      ) or in HepG2 cells (
      • Roesler W.J.
      • McFie P.J.
      • Puttick D.M.
      ). Both Roesler and colleagues (
      • Roesler W.J.
      • Graham J.G.
      • Kolen R.
      • Klemm D.J.
      • McFie P.J.
      ,
      • Roesler W.J.
      • Crosson S.M.
      • Vinson C.
      • McFie P.J.
      ,
      • Roesler W.J.
      • McFie P.J.
      • Puttick D.M.
      ,
      • Park E.A.
      • Roesler W.J.
      • Liu J.
      • Klemm D.J.
      • Gurney A.L.
      • Thatcher J.D.
      • Shuman J.
      • Friedman A.
      • Hanson R.W.
      ) and we (
      • Yeagley D.
      • Agati J.M.
      • Quinn P.G.
      ) have shown that additional elements from the upstream PEPCK promoter that bind factors other than CREB are absolutely required for induction of PEPCK transcription by PKA and inhibition of PKA-induced transcription (
      • Yeagley D.
      • Agati J.M.
      • Quinn P.G.
      ). When induction is due to CREB alone, as in CREB-GAL4 + PKA-stimulated transcription of 5XGT-Luc (5 GAL4 sites fused to the minimal PEPCK promoter), it is inhibited to no greater extent than basal transcription by insulin (
      • Yeagley D.
      • Agati J.M.
      • Quinn P.G.
      ). Thus, on these grounds alone, more must be involved than simply the P-CREB·CBP·RNA polymerase II complex, or insulin would have effectively inhibited CREB-dependent induction by PKA in 5XGT, as it does with the complete PEPCK promoter.
      Second, we show here that the Ras/MAPK pathway is not activated in response to insulin and that inhibition of the Ras/MAPK at three different and independent steps has no effect upon insulin inhibition of PKA-induced PEPCK gene transcription. Furthermore, insulin did not functionally activate the Ras/MAPK pathway, as evidenced by the lack of induction of G4-PEPCK with G4-Elk. Finally, using an independent approach, Granner and colleagues (
      • Gabbay R.A.
      • Sutherland C.
      • Gnudi L.
      • Kahn B.B.
      • O'Brien R.M.
      • Granner D.K.
      • Flier J.S.
      ) showed that a MEK inhibitor had no effect upon insulin inhibition of PEPCK transcription and recently showed that the effects of dn-Ras at very high concentrations involve inhibition of PI3-kinase activity (
      • Sutherland C.
      • Waltner-Law M.
      • Gnudi L.
      • Kahn B.B.
      • Granner D.K.
      ). Thus, the combined evidence from these studies, utilizing both chemical inhibitors and dominant negative enzymes, argues against a mechanism for insulin inhibition of PEPCK gene transcription action based on activation of the Ras/MAPK pathway.

      Signaling through PI3-Kinase Is Required for Insulin Inhibition

      PI3-kinase activation by insulin is mediated by phosphorylation of specific tyrosines in IRSs in response to insulin activation of its receptor tyrosine kinase and is independent of activation of the Ras/MAPK pathway by insulin (
      • Hadari Y.R.
      • Tzahar E.
      • Nadiv O.
      • Rothenberg P.
      • Roberts Jr., C.T.
      • LeRoith D.
      • Yarden Y.
      • Zick Y.
      ,
      • Withers D.J.
      • Gutierrez J.S.
      • Towery H.
      • Burks D.J.
      • Ren J.M.
      • Previs S.
      • Zhang Y.
      • Bernal D.
      • Pons S.
      • Shulman G.I.
      • Bonner-Weir S.
      • White M.F.
      ,
      • Folli F.
      • Saad M.J.A.
      • Backer J.M.
      • Kahn C.R.
      ). Many of the cellular effects of insulin, including stimulation of protein synthesis, mitogenesis, translocation of GLUT4 transporters, and the regulation of gene expression require activation of PI3-kinase (
      • White M.F.
      • Kahn C.R.
      ,
      • Sutherland C.
      • O'Brien R.M.
      • Granner D.K.
      ,
      • Cheatham B.
      • Vlahos C.J.
      • Cheatham L.
      • Wang L.
      • Blenis J.
      • Kahn C.R.
      ,
      • Mendez R.
      • Kollmorgen G.
      • White M.F.
      • Rhoads R.E.
      ,
      • Okada T.
      • Kawano Y.
      • Sakakibara T.
      • Hazeki O.
      • Ui M.
      ). Treatment of H4IIe cells with LY294002 or wortmannin, chemical inhibitors of PI3-kinase, directly blocked kinase activity as well as the ability of insulin to inhibit PEPCK gene transcription induced by PKA (Fig. 4) or PKA + Dex (
      • Sutherland C.
      • O'Brien R.M.
      • Granner D.K.
      ). Thus, activation of PI3-kinase appears to be an obligatory part of the insulin signaling pathway.
      PI3-kinase is activated by binding to tyrosine phosphorylated IRS (
      • Folli F.
      • Saad M.J.A.
      • Backer J.M.
      • Kahn C.R.
      ) and phosphorylates phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate to produce the physiologically significant regulators, phosphatidylinositol-3,4-diphosphate and phosphatidylinositol-3,4,5-trisphosphate (
      • Carpenter C.
      • Cantley L.
      ). These lipid products activate both PKB and PKC. Phosphatidylinositol-3,4-diphosphate and phosphatidylinositol-3,4,5-trisphosphate directly and indirectly activate protein kinase B (
      • Datta K.
      • Bellacosa A.
      • Chan T.O.
      • Tsichlis P.N.
      ,
      • Burgering B.M.
      • Coffer P.J.
      ,
      • Bos J.L.
      ,
      • Stokoe D.
      • Stephens L.R.
      • Copeland T.
      • Gaffney P.R.
      • Reese C.B.
      • Painter G.F.
      • Holmes A.B.
      • McCormick F.
      • Hawkins P.T.
      ). Binding of phosphatidylinositol-3,4-diphosphate to the pleckstrin homology domain of PKB directly activates the enzyme in vitro (
      • Klippel A.
      • Kavanaugh W.M.
      • Pot D.
      • Williams L.T.
      ,
      • Franke T.F.
      • Kaplan D.R.
      • Cantley L.C.
      • Toker A.
      ). In addition, the products of phosphatidylinositol-3-kinase contribute to a concerted mechanism for activation of PKB (
      • Cohen P.
      • Alessi D.R.
      • Cross D.A.
      ,
      • Franke T.
      • Cantley L.
      ). The protein kinase, PDK-1, binds phosphatidylinositol-3,4,5-trisphosphate and phosphorylates PKB bound to phosphatidylinositol-3,4-diphosphate, resulting in full activation in vivo (
      • Alessi D.
      • Andjelkovic M.
      • Caudwell B.
      • Cron P.
      • Morrice N.
      • Cohen P.
      • Hemmings B.
      ). In addition, binding of phosphatidylinositol-3,4-diphosphate and/or phosphatidylinositol-3,4,5-trisphosphate to novel isoforms of PKC (not regulated by Ca2+ and phospholipids) directly activates these kinases (
      • Nakanishi H.
      • Brewer K.A.
      • Exton J.H.
      ,
      • Toker A.
      • Meyer M.
      • Reddy K.
      • Falck J.
      • Aneja R.
      • Aneja S.
      • Parra A.
      • Burns D.
      • Ballas L.
      • Cantley L.
      ).

      Lack of Involvement of the PKB/Akt Pathway in Insulin Inhibition

      Protein kinase B was first identified as the viral oncogene, Akt, of which a constitutively active form has been isolated, gagPKB (
      • Burgering B.M.
      • Coffer P.J.
      ,
      • Bellacosa A.
      • Franke T.F.
      • Gonzalez-Portal M.E.
      • Datta K.
      • Taguchi T.
      • Gardner J.
      • Cheng J.Q.
      • Testa J.R.
      • Tsichlis P.N.
      ). Insulin-activated PKB phosphorylates and inactivates glycogen synthase kinase-3, mediating insulin stimulation of glycogen synthesis (
      • Cross D.
      • Alessi D.R.
      • Vandenheede J.R.
      • McDowell H.E.
      • Hundal H.S.
      • Cohen P.
      ). Insulin also stimulates GLUT4 glucose transporter translocation through activation of PI3-kinase and PKB (
      • Okada T.
      • Kawano Y.
      • Sakakibara T.
      • Hazeki O.
      • Ui M.
      ,
      • Cong L.
      • Chen H.
      • Li Y.
      • Zhou L.
      • McGibbon M.
      • Taylor S.
      • Quon M.
      ). In addition, inhibition of either PI3-kinase or of PKB prevents activation of a survival factor and results in increased apoptosis (
      • Kauffmann-Zeh A.
      • Rodriguez-Viciana P.
      • Ulrich E.
      • Gilbert C.
      • Coffer P.
      • Downward J.
      • Evan G.
      ,
      • Marte B.M.
      • Downward J.
      ,
      • Dudek H.
      • Datta S.R.
      • Franke T.F.
      • Birnbaum M.J.
      • Yao R.
      • Cooper G.M.
      • Segal R.A.
      • Kaplan D.R.
      • Greenberg M.E.
      ). PKB dimerization through its pleckstrin homology domain is required for activation of the purified enzymein vitro (
      • Franke T.F.
      • Kaplan D.R.
      • Cantley L.C.
      • Toker A.
      ). Mutants of PKB that are defective for dimerization or that contain only the dimerization domain inhibited activation of PKB in vitro (
      • Franke T.F.
      • Kaplan D.R.
      • Cantley L.C.
      • Toker A.
      ), as well as relieving repression of apoptosis in vivo (
      • Kauffmann-Zeh A.
      • Rodriguez-Viciana P.
      • Ulrich E.
      • Gilbert C.
      • Coffer P.
      • Downward J.
      • Evan G.
      ,
      • Marte B.M.
      • Downward J.
      ,
      • Dudek H.
      • Datta S.R.
      • Franke T.F.
      • Birnbaum M.J.
      • Yao R.
      • Cooper G.M.
      • Segal R.A.
      • Kaplan D.R.
      • Greenberg M.E.
      ). In addition, a phosphorylation defective mutant, PKB, K → A, enhanced apoptosis (
      • Dudek H.
      • Datta S.R.
      • Franke T.F.
      • Birnbaum M.J.
      • Yao R.
      • Cooper G.M.
      • Segal R.A.
      • Kaplan D.R.
      • Greenberg M.E.
      ) and blocked insulin-stimulated translocation of the GLUT4 transporter (
      • Cong L.
      • Chen H.
      • Li Y.
      • Zhou L.
      • McGibbon M.
      • Taylor S.
      • Quon M.
      ). On the other hand, expression of the constitutively active form of PKB inhibited apoptosis (
      • Kauffmann-Zeh A.
      • Rodriguez-Viciana P.
      • Ulrich E.
      • Gilbert C.
      • Coffer P.
      • Downward J.
      • Evan G.
      ) and overexpression of wild type PKB enhanced GLUT 4 translocation (
      • Cong L.
      • Chen H.
      • Li Y.
      • Zhou L.
      • McGibbon M.
      • Taylor S.
      • Quon M.
      ).
      In contrast, using these same reagents, we found that overexpression of PKB mutants with defective dimerization or kinase domains had no effect upon insulin inhibition of PKA-induced PEPCK-Luc transcription. Although constitutively active gagPKB depressed transcription, it had no effect upon inhibition by insulin. Thus, our results suggest that PKB is not required as a downstream mediator of PI3-kinase in the insulin signaling pathway utilized for inhibition of PEPCK gene transcription.

      Activation of PKC Is Not Required for Insulin Inhibition

      Previous work demonstrated that either insulin or PMA-activated PKC inhibits PEPCK gene transcription (
      • Chu D.T.
      • Granner D.K.
      ). The mechanism for inhibition by insulin is independent of that used by PKC because insulin can still suppress hormone-induced PEPCK transcription following down-regulation of PKC by PMA (
      • Chu D.T.
      • Granner D.K.
      ). The BIM inhibition data presented here demonstrate in a new way that the mechanisms utilized by PMA and insulin are initially independent, although they likely converge at some mediator (which also can be targeted by da-Ras) that is required for modification of a crucial transcription factor interaction. PKCζ and other diacylglycerol-independent isoforms of PKC are activated by the lipid products of PI3-kinase (
      • Nakanishi H.
      • Brewer K.A.
      • Exton J.H.
      ,
      • Toker A.
      • Meyer M.
      • Reddy K.
      • Falck J.
      • Aneja R.
      • Aneja S.
      • Parra A.
      • Burns D.
      • Ballas L.
      • Cantley L.
      ). Based on inhibition by staurosporine but not phorbol ester analogs, PKCζ was shown to be involved in the stimulation of protein synthesis by insulin (
      • Mendez R.
      • Kollmorgen G.
      • White M.F.
      • Rhoads R.E.
      ). The lack of effect of staurosporine on insulin inhibition seen here suggests that PKCζ is not involved in insulin signaling to the PEPCK gene. Even though our data are complicated by the unexpected observation of staurosporine induction of PEPCK, this effect was inhibited by insulin, as was induction by PKA. Thus, neither PMA-activated PKC isoforms nor novel PI3-kinase-activated PKC isoforms appear to play an obligatory role in insulin inhibition of PKA-induced PEPCK gene transcription.

      Several Pathways Converge on a Common Target or Complex That Mediates Insulin Inhibition

      Our current results show that activation of the Ras/MAPK pathway by da-Ras can inhibit induction of PEPCK-Luc by PKA, as does insulin. Stimulation of the EGF receptor also activates MAPK and results in inhibition of hormone-induced PEPCK gene transcription (
      • Fillat C.
      • Valera A.
      • Bosch F.
      ), as does activation of PKC by phorbol esters (Fig. 7) (
      • Chu D.T.
      • Granner D.K.
      ). Activation of reactivating kinase (or p38) by oxidant stress also mimicked the effect of insulin inhibition of PEPCK expression, but that insulin inhibition was unaffected by an inhibitor of reactivating kinase (
      • Sutherland C.
      • Tebbey P.W.
      • Granner D.K.
      ). Thus, activation of any of several signaling pathways can inhibit gluconeogenic hormone-induced PEPCK gene expression in a manner indistinguishable from insulin. This strongly suggests that all of these pathways converge on a common transcription factor or complex that is targeted by insulin. Our results indicate that activation of the Ras/MAPK, PKB, or PKC pathways either does not occur (Ras/MAPK, PKC) and/or can not account for insulin inhibition of PEPCK transcription (PKB). On the other hand, inhibition of PI3-kinase activity abolished insulin inhibition. It should be stressed that the possibility that insulin works through alternate, parallel paths, either in vivo or in the H4IIe cell model, can not be excluded with the reagents available.
      We suggest that factors bound to the CRU of the PEPCK promoter interact with CBP and/or other integrator complexes in some unique way that permits discrimination of this gene by insulin signals generated through activation of PI3-kinase. The observation that G4-Elk mediated induction by da-Ras, while PKA induction of PEPCK-Luc was inhibited by da-Ras, illustrates how switching a single factor (CREB → Elk) in a complex regulatory array can alter the response to kinase signals,i.e. from inhibition to activation of transcription in this case. In addition, the lack of effect of insulin on Elk-mediated induction is further evidence that insulin specifically targets a factor in the CRU rather than acting through a more general mechanism. The identity of the specific transcription factors within the CRU of the PEPCK gene that are modified by an insulin-generated signal to inhibit transcription induction remain to be identified, as does the mediator activated by PI3-kinase that transmits the signal for modification of these factors. One or more of the CRU factors and/or their coactivators that cooperate in a unique way to confer induction by PKA must be targeted for inhibition by insulin.

      ACKNOWLEDGEMENTS

      We thank Justin Cho for technical assistance. We thank Drs. R. Maurer, S. Cook, F. McCormick, U. Rapp, P. Coffer, and T. Frank for their generous gifts of plasmids, and David Spector for critical reading of the manuscript.

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