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
1998; Elsevier BV; Volume: 273; Issue: 30 Linguagem: Inglês
10.1074/jbc.273.30.18751
ISSN1083-351X
AutoresJoyce M. Agati, David Yeagley, Patrick G. Quinn,
Tópico(s)Pancreatic function and diabetes
ResumoTranscription 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. 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 (1White M.F. Kahn C.R. J. Biol. Chem. 1994; 269: 1-4Abstract Full Text PDF PubMed Google Scholar). In addition, insulin regulates the transcription of specific genes, whose products catalyze committed reactions in hepatic glucose metabolism (2Pilkis S.J. Granner D.K. Annu. Rev. Physiol. 1992; 54: 885-909Crossref PubMed Scopus (691) Google Scholar). In particular, the amounts of the enzymes that catalyze committed steps at either end of the glucose utilization pathway, glucokinase and phosphoenolpyruvate carboxykinase (PEPCK) 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. 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 (3Granner D. Pilkis S. J. Biol. Chem. 1990; 265: 10173-10176Abstract Full Text PDF PubMed Google Scholar). 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 (4Sasaki K. Cripe T.P. Koch S.R. Andreone T.L. Petersen D.D. Beale E.G. Granner D.K. J. Biol. Chem. 1984; 259: 15242-15251Abstract Full Text PDF PubMed Google Scholar). 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 (1White M.F. Kahn C.R. J. Biol. Chem. 1994; 269: 1-4Abstract Full Text PDF PubMed Google Scholar). 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 (1White M.F. Kahn C.R. J. Biol. Chem. 1994; 269: 1-4Abstract Full Text PDF PubMed Google Scholar, 5Hadari Y.R. Tzahar E. Nadiv O. Rothenberg P. Roberts Jr., C.T. LeRoith D. Yarden Y. Zick Y. J. Biol. Chem. 1992; 267: 17483-17486Abstract Full Text PDF PubMed Google Scholar, 6Myers M.J. White M. Diabetes. 1993; 42: 643-650Crossref PubMed Google Scholar). 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 (7Rutter G.A. White M.R.H. Tavaré J.M. Curr. Biol. 1995; 5: 890-899Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 8Standaert M.L. Bandyopadhyay G. Farese R.V. Biochem. Biophys. Res. Commun. 1995; 209: 1082-1088Crossref PubMed Scopus (42) Google Scholar, 9Sutherland C. O'Brien R.M. Granner D.K. J. Biol. Chem. 1995; 270: 15501-15506Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 10Cheatham B. Vlahos C.J. Cheatham L. Wang L. Blenis J. Kahn C.R. Mol. Cell. Biol. 1994; 14: 4902-4911Crossref PubMed Scopus (996) Google Scholar, 11Nakajima T. Fukamizu A. Takahashi J. Gage F.H. Fisher T. Blenis J. Montminy M.R. Cell. 1996; 86: 465-474Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 12Nakanishi H. Brewer K.A. Exton J.H. J. Biol. Chem. 1993; 268: 13-16Abstract Full Text PDF PubMed Google Scholar, 13Cross D.A. Alessi D.R. Cohen P. Andjelkovich M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4283) Google Scholar, 14Mendez R. Kollmorgen G. White M.F. Rhoads R.E. Mol. Cell. Biol. 1997; 17: 5184-5192Crossref PubMed Google Scholar). 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 (14Mendez R. Kollmorgen G. White M.F. Rhoads R.E. Mol. Cell. Biol. 1997; 17: 5184-5192Crossref PubMed Google Scholar). 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) (15Datta K. Bellacosa A. Chan T.O. Tsichlis P.N. J. Biol. Chem. 1996; 271: 30835-30839Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar, 16Klippel A. Kavanaugh W.M. Pot D. Williams L.T. Mol. Cell. Biol. 1997; 17: 338-344Crossref PubMed Scopus (442) Google Scholar, 17Burgering B.M. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1865) Google Scholar) and novel isoforms of PKC not regulated by calcium and diacylglycerol (12Nakanishi H. Brewer K.A. Exton J.H. J. Biol. Chem. 1993; 268: 13-16Abstract Full Text PDF PubMed Google Scholar, 18Toker A. Meyer M. Reddy K. Falck J. Aneja R. Aneja S. Parra A. Burns D. Ballas L. Cantley L. J. Biol. Chem. 1994; 269: 32358-32367Abstract Full Text PDF PubMed Google Scholar). 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 (19Quinn P.G. J. Biol. Chem. 1994; 269: 14375-14378Abstract Full Text PDF PubMed Google Scholar). 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 (20Yeagley D. Agati J.M. Quinn P.G. J. Biol. Chem. 1998; 273: 18743-18750Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). In addition to the CRE, induction of the PEPCK gene by cAMP requires heterologous binding sites located in the (AC) region (−271/−225) (20Yeagley D. Agati J.M. Quinn P.G. J. Biol. Chem. 1998; 273: 18743-18750Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 21Roesler W.J. Graham J.G. Kolen R. Klemm D.J. McFie P.J. J. Biol. Chem. 1995; 270: 8225-8232Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 22Roesler W.J. Crosson S.M. Vinson C. McFie P.J. J. Biol. Chem. 1996; 271: 8068-8074Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 23Roesler W.J. McFie P.J. Puttick D.M. J. Biol. Chem. 1993; 268: 3791-3796Abstract Full Text PDF PubMed Google Scholar). 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 (20Yeagley D. Agati J.M. Quinn P.G. J. Biol. Chem. 1998; 273: 18743-18750Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Blenis and Montminy and colleagues (11Nakajima T. Fukamizu A. Takahashi J. Gage F.H. Fisher T. Blenis J. Montminy M.R. Cell. 1996; 86: 465-474Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar) 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. (24Gabbay R.A. Sutherland C. Gnudi L. Kahn B.B. O'Brien R.M. Granner D.K. Flier J.S. J. Biol. Chem. 1996; 271: 1890-1897Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar) 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. (9Sutherland C. O'Brien R.M. Granner D.K. J. Biol. Chem. 1995; 270: 15501-15506Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar) 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. The PEPCK-Luc plasmid is derived from PEPCK-chloramphenicol acetyltransferase, containing −600/+69 of the PEPCK promoter (25Magnuson M.A. Quinn P.G. Granner D.K. J. Biol. Chem. 1987; 262: 14917-14920Abstract Full Text PDF PubMed Google Scholar). The G4-PEPCK-Luc plasmid contains the entire promoter region (−600/+69) of the PEPCK gene, in which the CRE is replaced by a GAL4 site (26Xing L.P. Quinn P.G. Mol. Endocrinol. 1993; 7: 1484-1494PubMed Google Scholar). The CREB-GAL4 fusion protein expression vector contains the activation domain of CREB (amino acids 1–277) fused to amino acids 4–147 of the GAL4 DNA binding domain and has been described previously (27Quinn P.G. J. Biol. Chem. 1993; 268: 16999-17009Abstract Full Text PDF PubMed Google Scholar, 28Howard P. Day K.H. Kim K.E. Richardson J. Thomas J. Abraham I. Fleischmann R.D. Gottesman M.M. Maurer R.A. J. Biol. Chem. 1991; 266: 10189-10195Abstract Full Text PDF PubMed Google Scholar). The PKA expression vector, RSV-Cα, contains the cDNA for the catalytic subunit of protein kinase A under control of the RSV promoter (28Howard P. Day K.H. Kim K.E. Richardson J. Thomas J. Abraham I. Fleischmann R.D. Gottesman M.M. Maurer R.A. J. Biol. Chem. 1991; 266: 10189-10195Abstract Full Text PDF PubMed Google Scholar). The pRL-SV plasmid is the expression vector for Renilla luciferase, which is used for normalization of the transfection efficiency as assayed by firefly luciferase. The expression vector for dominant-negative Ras, pM2N-RasN17 and for dominant-active Ras, pRSV-Leu61 were provided by S. Cook of Onyx Pharmaceuticals (29Cook S. Rubinfield B. Albert I. McCormick F. EMBO J. 1993; 12: 3475-3485Crossref PubMed Scopus (331) Google Scholar). The expression vector for dominant-negative Raf, RSV-RAF-C4, was from U. Rapp of New York University Medical Center (30Bruder J.T. Heidecker G. Rapp U.R. Genes Dev. 1992; 6: 545-556Crossref PubMed Scopus (396) Google Scholar). The expression vector for dominant-active PAC-1 was provided by K. Kelly, NIH (31Ward Y. Gupta S. Jensen P. Wartman M. Davis R. Kelly K. Nature. 1994; 367: 651-654Crossref PubMed Scopus (295) Google Scholar). The G4-Elk-1 plasmid contains the GAL4 DNA binding domain (amino acids 1–147) fused to the Elk-1 carboxyl-terminal activation domain (307–428) in pcDNA3a with the Neo gene removed and was provided by R. Maurer of Oregon Health Sciences University (32Roberson M.S. Misra-Press A. Laurance M.E. Stork P.J. Maurer R.A. Mol. Cell. Biol. 1995; 15: 3531-3539Crossref PubMed Google Scholar). The HA-Akt, HA-Akt-R25C, and HA-Akt(pleckstrin homology (PH)) plasmids were obtained from T. Franke, Montreal Neurological Institute (33Franke T.F. Kaplan D.R. Cantley L.C. Toker A. Science. 1997; 275: 665-668Crossref PubMed Scopus (1288) Google Scholar). HA-Akt is the expression vector for protein kinase B (PKB/Akt). HA-Akt-R25C is the expression vector for the dimerization-defective PKB. HA-Akt(PH) is the expression vector for a mutant PKB which contains only the pleckstrin homology domain. The pSG5-PKB (wild type), pSG5-PKB, K → A, and pSG5-gagPKB plasmids were obtained from P. Coffer (University Hospital, Utrecht) (34Kauffmann-Zeh A. Rodriguez-Viciana P. Ulrich E. Gilbert C. Coffer P. Downward J. Evan G. Nature. 1997; 385: 544-548Crossref PubMed Scopus (1064) Google Scholar). pSG5-PKB is the expression vector for wild type PKB. pSG5-PKB, K → A is the expression for kinase-defective PKB with a residue change at amino acid 179. The pSG5-gagPKB plasmid is the expression vector for constitutively active PKB. H4IIe cells were cultured and transfected as described previously (19Quinn P.G. J. Biol. Chem. 1994; 269: 14375-14378Abstract Full Text PDF PubMed Google Scholar, 26Xing L.P. Quinn P.G. Mol. Endocrinol. 1993; 7: 1484-1494PubMed Google Scholar). Briefly, 1 ml of calcium phosphate precipitate contained 20 μg of luciferase reporter vector + 2 μg pRL-SV and 2 μg RSV-Cα and/or 2–10 μg expression vector, as indicated in the figure legends. An equal volume of cells was added to the calcium precipitate and incubated for 15 min at room temperature. The cells were plated in replicate dishes, incubated for 4 h at 37 °C, 5% CO2, and treated with 20% Me2SO for 3 min. Where indicated the following were added at the concentration indicated for the last 20 h of the experiment: insulin (Lilly), 10 nm; bisindolyl maleimide I, HCl (BIM) (Calbiochem), 10 μm; and phorbol 12-myristate 13-acetate, (PMA) (Sigma), 1 μm. Following incubation for 20 h, cells were harvested with trypsin treatment and lysed with 1× passive lysis buffer (Promega) and stored at −80 °C for at least 15 min. Lysates (100 μl) were obtained by spinning down the lysed cells, and 10 μl of lysate was used for the luciferase assay. A dual injector Monolight 3010 Luminometer was utilized to measure luciferase activity. 50 μl of each Promega reagent were added to the lysate for the assay. Values were normalized for transfection efficiency and the mean was computed for several experiments. Data shown in figures were obtained from independent transfection experiments performed with different preparations of the various plasmids. H4IIe cells were incubated overnight at 37 °C in serum-free medium containing 0.1% bovine serum albumin, after which they were pretreated with the PI3-kinase inhibitors, wortmannin, or LY294002, for 15 min, prior to adding hormones and incubating with hormone + inhibitor for 3 h. Wortmannin (Sigma) was added to a final concentration of 0.1 mm. LY294002 (Biomole) was added to a final concentration of 10 μm. Insulin (Lilly) was added to a final concentration of 10 nm. 8-(4-Chlorophenylthio)-cAMP (Sigma) was added to a final concentration of 0.1 mm. Cells were harvested and total RNA was isolated as described by Chomczynski and Sacchi (35Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62890) Google Scholar). At the end of the procedure, the RNA was dissolved and reextracted with phenol:chloroform, ethanol-precipitated and dissolved in water. The amount of PEPCK mRNA in 50 μg of total RNA was quantitated by primer extension analysis, as described previously (36Quinn P.G. Granner D.K. Mol. Cell. Biol. 1990; 10: 3357-3364Crossref PubMed Scopus (66) Google Scholar). The molecular pathways proposed to mediate glucagon-stimulated and insulin-inhibited PEPCK gene expression are illustrated in Fig. 1, as are the targets of relevant inhibitors. There is general agreement that glucagon stimulates adenylate cyclase and cAMP activates PKA, a portion of which is translocated to the nucleus where it phosphorylates CREB, leading to CBP binding and activation of gene transcription (37Corbin J.D. Cobb C.E. Beebe S.J. Granner D.K. Koch S.R. Gettys T.W. Blackmore P.F. Francis S.H. Wells J.N. Adv. Second Messenger Phosphoprotein Res. 1988; 21: 75-86PubMed Google Scholar, 38Hoeffler J.P. Meyer T.E. Yun Y. Jameson J.L. Habener J.F. Science. 1988; 242: 1430-1433Crossref PubMed Scopus (522) Google Scholar, 39Gonzalez G.A. Montminy M.R. Cell. 1989; 59: 675-680Abstract Full Text PDF PubMed Scopus (2036) Google Scholar, 40Hagiwara M. Brindle P. Harootunian A. Armstrong R. Rivier J. Vale W. Tsien R. Montminy M.R. Mol. Cell. Biol. 1993; 13: 4852-4859Crossref PubMed Scopus (375) Google Scholar). A region containing putative binding sites for transcription factors of the AP-1 and C/EBP families, as well as the CREB binding site (CRE), are required to form a functional CRU in the PEPCK promoter (20Yeagley D. Agati J.M. Quinn P.G. J. Biol. Chem. 1998; 273: 18743-18750Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 21Roesler W.J. Graham J.G. Kolen R. Klemm D.J. McFie P.J. J. Biol. Chem. 1995; 270: 8225-8232Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 22Roesler W.J. Crosson S.M. Vinson C. McFie P.J. J. Biol. Chem. 1996; 271: 8068-8074Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 23Roesler W.J. McFie P.J. Puttick D.M. J. Biol. Chem. 1993; 268: 3791-3796Abstract Full Text PDF PubMed Google Scholar). This CRU is necessary and sufficient for both induction by PKA and inhibition by insulin of a minimal promoter, whereas CREB alone is not (20Yeagley D. Agati J.M. Quinn P.G. J. Biol. Chem. 1998; 273: 18743-18750Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Thus, more specificity is required for insulin to inhibit PKA-induced PEPCK gene transcription than is provided by the P-CREB/CBP/RNA polymerase II complex alone (20Yeagley D. Agati J.M. Quinn P.G. J. Biol. Chem. 1998; 273: 18743-18750Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). However, it has been suggested that Ras/MAPK-mediated activation of pp90rskII and its binding to CBP is the mechanism utilized by insulin to inhibit PEPCK gene transcription (11Nakajima T. Fukamizu A. Takahashi J. Gage F.H. Fisher T. Blenis J. Montminy M.R. Cell. 1996; 86: 465-474Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). To examine the role of the Ras/MAPK pathway in insulin signaling relevant to inhibition of PEPCK transcription, we determined the effects of expression of dominant negative (dn) and dominant active (da) forms of enzymes involved in transmission and regulation of signals through the Ras/MAPK pathway. In general, dominant negative forms of enzymes interact with the signaling component immediately upstream and prevent further transmission of the signal, whereas dominant active forms of signaling enzymes stimulate all pathway members downstream of that particular enzyme without any requirement for input from receptors or other enzymes upstream. For the experiments shown in Fig. 2, H4IIe cells were cotransfected with PEPCK-Luc and expression vectors for dn-Ras, dn-Raf, da-PAC-1, and da-Ras. Dominant negative Ras interferes with the exchange activity of SOS, preventing exchange of GDP for GTP on endogenous Ras and interfering with downstream signaling (29Cook S. Rubinfield B. Albert I. McCormick F. EMBO J. 1993; 12: 3475-3485Crossref PubMed Scopus (331) Google Scholar, 41Feig L. Cooper G. Mol. Cell. Biol. 1988; 8: 3235-3243Crossref PubMed Scopus (671) Google Scholar). Dominant negative Raf binds to GTP-activated Ras but cannot be activated itself and thus prevents downstream signaling (30Bruder J.T. Heidecker G. Rapp U.R. Genes Dev. 1992; 6: 545-556Crossref PubMed Scopus (396) Google Scholar). Dominant active PAC-1 dephosphorylates and inactivates MAPK, preventing it from activating downstream targets (31Ward Y. Gupta S. Jensen P. Wartman M. Davis R. Kelly K. Nature. 1994; 367: 651-654Crossref PubMed Scopus (295) Google Scholar). As shown in Fig. 2, expression of dn-Ras, dn-Raf, or da-PAC-1 had no affect upon insulin inhibition of PKA-induced PEPCK-Luc. However, all three Ras-pathway inhibitors relieved constitutive restraint of PKA induction through the Ras pathway and enhanced induction. Dominant active Ras (da-Ras) activates downstream components independently of signaling input (42Der C. Finkel T. Cooper G. Cell. 1986; 44: 167-176Abstract Full Text PDF PubMed Scopus (398) Google Scholar). Cotransfection of H4IIe cells with PEPCK-Luc and da-Ras blocked induction by PKA at least as effectively as treatment with insulin. Thus, activation of the Ras/MAPK pathway at the level of Ras is sufficient to inhibit PKA-induced PEPCK expression but not necessary for insulin inhibition. To determine whether insulin functionally activated the Ras/MAPK pathway affecting gene expression, we employed G4-Elk, a MAPK-activated transcription factor. H4IIe cells were cotransfected with G4-PEPCK-Luc and G4-Elk and tested for induction by insulin and inhibition of this induction by the Ras pathway mutant enzymes (Fig. 3). In G4-Elk, the activation domain of the MAPK-activated transcription factor Elk is fused to the GAL4 DNA binding domain. The G4-PEPCK promoter contains a GAL4 site in place of the CRE. Thus, if insulin activated the Ras/MAPK pathway in H4IIe cells, G4-PEPCK-Luc expression should be induced by insulin in the presence of G4-Elk, and this induction should be inhibited by the Ras/MAPK signaling pathway mutants. As illustrated in Fig. 3, insulin did not activate G4-PEPCK-Luc through the G4-Elk factor. However, da-Ras did potently activate transcription mediated by G4-Elk, and this induction was unaffected by insulin. These results demonstrate that insulin stimulation of H4IIe cells did not significantly activate the Ras/MAPK pathway. Together with the results above, these data strongly argue against any role for activation of the Ras/MAPK pathway in insulin-mediated inhibition of PKA-induced PEPCK gene expression. Next, we examined the effects of two different chemical inhibitors of PI3-kinase, wortmannin and LY294002, on endogenous PEPCK mRNA expression (Fig. 4). Total mRNA was prepared from H4IIe cells after 3 h of hormone treatment, a time at which changes in PEPCK mRNA are near maximal (4Sasaki K. Cripe T.P. Koch S.R. Andreone T.L. Petersen D.D. Beale E.G. Granner D.K. J. Biol. Chem. 1984; 259: 15242-15251Abstract Full Text PDF PubMed Google Scholar). PEPCK mRNA was quantitated by primer extension analysis. H4IIe cells were treated with nothing, cAMP alone or cAMP + insulin, in the absence or presence of Me2SO vehicle, 0.1 mm or 0.5 mmwortmannin, as indicated in Fig. 4 A. Wortmannin completely blocked insulin inhibition of PKA-induced PEPCK mRNA accumulation. We also tested another chemical inhibitor of PI3-kinase, LY294002 (10 μm), in the same manner. As shown in Fig. 4 B, LY294002 also completely blocked insulin inhibition of PKA-induced accumulation of PEPCK mRNA. These results confirm those of Granner and colleagues (9Sutherland C. O'Brien R.M. Granner D.K. J. Biol. Chem. 1995; 270: 15501-15506Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar) and indicate that activation of PI3-kinase by insulin is obligatory for insulin inhibition of PKA-induced PEPCK gene expression. Based on other studies of insulin signaling, the most probable downstream targets of PI3-kinase are 1) protein kinase B, also known as Akt (13Cross D.A. Alessi D.R. Cohen P. Andjelkovich M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4283) Google Scholar, 43Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Google Scholar) and 2) nonclassical forms of PKC (12Nakanishi H. Brewer K.A. Exton J.H. J. Biol. Chem. 1993; 268: 13-16Abstract Full Text PDF PubMed Google Scholar, 14Mendez R. Kollmorgen G. White M.F. Rhoads R.E. Mol. Cell. 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A mutant of PKB/Akt that is defective for dimerization, Akt-R25C (dim-def in Fig. 5), cannot be activated in vitro (33Franke T.F. Kaplan D.R. Cantley L.C. Toker A. Science. 1997; 2
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