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Modulation of Insulin Receptor Substrate-1 Tyrosine Phosphorylation by an Akt/Phosphatidylinositol 3-Kinase Pathway*

  • Jinping Li
    Affiliations
    From the Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, California 94305
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  • Kathryn DeFea
    Affiliations
    From the Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, California 94305
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  • Richard A. Roth
    Correspondence
    To whom correspondence should be addressed: Dept. of Molecular Pharmacology, Stanford Medical Center, Stanford, CA 94305. Tel.: 650-723-5933; Fax: 650-725-2952;
    Affiliations
    From the Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, California 94305
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  • Author Footnotes
    * This work was supported by National Institutes of Health Grant DK 34926 and by a grant from Metabolex.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:April 02, 1999DOI:https://doi.org/10.1074/jbc.274.14.9351
      Serine/threonine phosphorylation of insulin receptor substrate 1 (IRS-1) has been implicated as a negative regulator of insulin signaling. Prior studies have indicated that this negative regulation by protein kinase C involves the mitogen-activated protein kinase and phosphorylation of serine 612 in IRS-1. In the present studies, the negative regulation by platelet-derived growth factor (PDGF) was compared with that induced by endothelin-1, an activator of protein kinase C. In contrast to endothelin-1, the inhibitory effects of PDGF did not require mitogen-activated protein kinase or the phosphorylation of serine 612. Instead, three other serines in the phosphorylation domain of IRS-1 (serines 632, 662, and 731) were required for the negative regulation by PDGF. In addition, the PDGF-activated serine/threonine kinase called Akt was found to inhibit insulin signaling. Moreover, this inhibition required the same IRS-1 serine residues as the inhibition by PDGF. Finally, the negative regulatory effects of PDGF and Akt were inhibited by rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), one of the downstream targets of Akt. These studies implicate the phosphatidylinositol 3-kinase/Akt kinase cascade as an additional negative regulatory pathway for the insulin signaling cascade.
      SH
      Src homology
      mTOR
      mammalian target of rapamycin
      IRS
      insulin receptor substrate
      MAP
      mitogen-activated protein
      PDGF
      platelet-derived growth factor
      FRAP
      FKBP-rapamycin-associate protein
      PI
      phosphatidylinositol
      HT
      hydroxytamoxifen
      HA
      hemagglutinin
      PCR
      polymerase chain reaction
      After insulin binds to its receptor, it activates an intrinsic tyrosine kinase activity that mediates the tyrosine phosphorylation of a variety of endogenous substrates including insulin receptor substrates 1–4 (
      • White M.F.
      • Yenush L.
      ). Binding of these tyrosine-phosphorylated substrates to the Src homology (SH)1 domain-2 of the regulatory subunit of the heterodimeric p85/p110 phosphatidylinositol (PI) 3-kinase leads to a 3–5-fold stimulation in its enzymatic activity and an increase in the PI 3,4-bisphosphate and 3,4,5-trisphosphate in the cell (
      • Rordorf-Nikolic T.
      • Van Horn D.J.
      • Chen D.
      • White M.F.
      • Backer J.M.
      ,
      • Ruderman N.B.
      • Kapeller R.
      • White M.F.
      • Cantley L.C.
      ). A variety of data from several approaches has demonstrated a role for the production of these lipid products in mediating many, if not all, of the subsequent actions of insulin including for example the stimulation of glucose uptake, activation of both the glycogen synthase and the 70-kDa S6 kinase (
      • Shepherd P.R.
      • Withers D.J.
      • Siddle K.
      ). The effects of PI 3,4,5-trisphosphate may in part be mediated through the activation of the Ser/Thr kinases called Akt or one of the atypical protein kinase Cs (
      • Shepherd P.R.
      • Withers D.J.
      • Siddle K.
      ,
      • Kitamura T.
      • Ogawa W.
      • Sakaue H.
      • Hino Y.
      • Kuroda S.
      • Takata M.
      • Matsumoto M.
      • Maeda T.
      • Konishi H.
      • Kikkawa U.
      • Kasuga M.
      ).
      Because of the critical role of the activation of this lipid kinase in inducing subsequent biological responses, increasing interest has focused on the regulation of this process. Indeed, several mechanisms for regulating this process have been identified. First, long term treatment of cells with insulin or the glucocorticoid dexamethasone has been shown to induce the degradation of IRS-1 (
      • Turnbow M.A.
      • Keller S.R.
      • Rice K.M.
      • Garner C.W.
      ,
      • Rice K.M.
      • Turnbow M.A.
      • Garner C.W.
      ). Second, increased serine phosphorylation of IRS-1 has been observed after treatment of cells with either activators of protein kinase C, Ser/Thr phosphatase inhibitors like okadaic acid, platelet-derived growth factor (PDGF), insulin, angiotensin II, or activation of cellular stress pathways by tumor necrosis factor and other cytokines (
      • Tanti J.-F.
      • Gremeaux T.
      • van Obberghen E.
      • Le Marchand-Brustel Y.
      ,
      • Hotamisligil G.S.
      • Peraldi P.
      • Budavari A.
      • Ellis R.
      • White M.F.
      • Spiegelman B.M.
      ,
      • Kanety H.
      • Feinstein R.
      • Papa M.Z.
      • Hemi R.
      • Karasik A.
      ,
      • Haddad T.C.
      • Conover C.A.
      ,
      • DeFea K.
      • Roth R.A.
      ,
      • Folli F.
      • Kahn C.R.
      • Hansen H.
      • Bouchie J.L.
      • Feener E.P.
      ,
      • Ricort J.-M.
      • Tanti J.-F.
      • van Obberghen E.
      • Le Marchand-Brustel Y.
      ,
      • Staubs P.A.
      • Nelson J.G.
      • Reichart D.R.
      • Olefsky J.M.
      ). This increased serine phosphorylation of IRS-1 has been shown to inhibit the subsequent ability of this substrate to be tyrosine-phosphorylated by the insulin receptor and to bind and activate the PI 3-kinase (
      • Tanti J.-F.
      • Gremeaux T.
      • van Obberghen E.
      • Le Marchand-Brustel Y.
      ,
      • Hotamisligil G.S.
      • Peraldi P.
      • Budavari A.
      • Ellis R.
      • White M.F.
      • Spiegelman B.M.
      ,
      • Kanety H.
      • Feinstein R.
      • Papa M.Z.
      • Hemi R.
      • Karasik A.
      ,
      • Haddad T.C.
      • Conover C.A.
      ,
      • DeFea K.
      • Roth R.A.
      ,
      • Folli F.
      • Kahn C.R.
      • Hansen H.
      • Bouchie J.L.
      • Feener E.P.
      ,
      • Ricort J.-M.
      • Tanti J.-F.
      • van Obberghen E.
      • Le Marchand-Brustel Y.
      ,
      • Staubs P.A.
      • Nelson J.G.
      • Reichart D.R.
      • Olefsky J.M.
      ). In addition, serine phosphorylation of IRS-1 has been shown to interfere with the ability of the IRS-1 to interact with the insulin receptor (
      • Paz K.
      • Hemi R.
      • LeRoith D.
      • Karasik A.
      • Elhanany E.
      • Kanety H.
      • Zick Y.
      ).
      In the case of cells treated with an activator of protein kinase C, an increase in the serine phosphorylation of a particular serine, serine 612, in the IRS-1 molecule was found to play a prominent role in this inhibition (
      • DeFea K.
      • Roth R.A.
      ). This residue appeared to be phosphorylated by one of the MAP kinases (also called extracellular signal-regulated kinases), and an increase in MAP kinase activity was shown to inhibit subsequent signaling by the insulin receptor kinase (
      • De Fea K.
      • Roth R.A.
      ). However, as noted above, a number of other stimuli also appear to negatively regulate the ability of the insulin receptor to tyrosine-phosphorylate IRS-1 by serine phosphorylation. In particular, PDGF has recently been shown to stimulate the Ser/Thr phosphorylation of IRS-1 and to inhibit the ability of insulin to stimulate the subsequent tyrosine phosphorylation of IRS-1 and its association with PI 3-kinase (
      • Ricort J.-M.
      • Tanti J.-F.
      • van Obberghen E.
      • Le Marchand-Brustel Y.
      ,
      • Staubs P.A.
      • Nelson J.G.
      • Reichart D.R.
      • Olefsky J.M.
      ). Since PDGF can activate multiple signaling pathways including the MAP kinase cascade (
      • Heldin C.H.
      • Ostman A.
      • Ronnstrand L.
      ), the present studies were therefore designed to examine whether PDGF was affecting IRS-1 via the same pathway as was observed after PKC activation. In particular, we have tested whether the negative regulation of insulin signaling by PDGF was also mediated via MAP kinase and the phosphorylation of Ser-612 in the IRS-1 molecule. We have found that treatment of cells with PDGF inhibits the insulin signaling pathway via a distinct mechanism, possibly by activation of a serine kinase modulated by the Akt pathway, which appears to require the serine phosphorylation of distinct residues on the IRS-1 molecule. In contrast, we find that endothelin-1, an activator of PKC (
      • Clerk A.
      • Bogoyevitch M.A.
      • Anderson M.B.
      • Sugden P.H.
      ), inhibits the insulin signaling pathway via the MAP kinase pathway and the serine phosphorylation of Ser-612 in IRS-1. These results indicate that there are MAP kinase-dependent and -independent pathways that regulate the insulin-stimulated association of IRS-1 with PI 3-kinase.

      DISCUSSION

      Prior studies have demonstrated that increased Ser/Thr phosphorylation of IRS-1 can be stimulated by a number of factors including activators of protein kinase C, Ser/Thr phosphatase inhibitors like okadaic acid, PDGF, insulin, or activation of cellular stress pathways by tumor necrosis factor and other cytokines (
      • Tanti J.-F.
      • Gremeaux T.
      • van Obberghen E.
      • Le Marchand-Brustel Y.
      ,
      • Hotamisligil G.S.
      • Peraldi P.
      • Budavari A.
      • Ellis R.
      • White M.F.
      • Spiegelman B.M.
      ,
      • Kanety H.
      • Feinstein R.
      • Papa M.Z.
      • Hemi R.
      • Karasik A.
      ,
      • Haddad T.C.
      • Conover C.A.
      ,
      • DeFea K.
      • Roth R.A.
      ,
      • Folli F.
      • Kahn C.R.
      • Hansen H.
      • Bouchie J.L.
      • Feener E.P.
      ,
      • Ricort J.-M.
      • Tanti J.-F.
      • van Obberghen E.
      • Le Marchand-Brustel Y.
      ,
      • Staubs P.A.
      • Nelson J.G.
      • Reichart D.R.
      • Olefsky J.M.
      ,
      • Paz K.
      • Hemi R.
      • LeRoith D.
      • Karasik A.
      • Elhanany E.
      • Kanety H.
      • Zick Y.
      ,
      • De Fea K.
      • Roth R.A.
      ). This serine phosphorylation may be a contributing factor in the development of insulin resistance since an increase in serine phosphorylation interferes with the tyrosine phosphorylation of IRS-1 by the insulin receptor and its subsequent association with PI 3-kinase. Thus, it is important to identify the kinase cascades responsible for the serine phosphorylation of IRS-1.
      Prior studies have identified several kinases that can phosphorylate IRS-1. These include casein kinase II (
      • Tanasijevic M.J.
      • Myers Jr., M.G.
      • Thoma R.S.
      • Crimmins D.L.
      • White M.F.
      • Sacks D.B.
      ), glycogen synthase kinase 3 (
      • Eldar-Finkelman H.
      • Krebs E.G.
      ), MAP kinase (
      • De Fea K.
      • Roth R.A.
      ), and even a lipid kinase, the phosphatidylinositol 3-kinase (
      • Lam K.
      • Carpenter C.L.
      • Ruderman N.B.
      • Friel J.C.
      • Kelly K.L.
      ,
      • Li P.M.
      • Goldstein B.J.
      ). In the case of MAP kinase, a particular serine (serine 612) has been identified in the IRS-1 molecule as being responsible for causing the subsequent inhibition of insulin-stimulated association with PI 3-kinase (
      • De Fea K.
      • Roth R.A.
      ). In this prior work, the MAP kinase was activated by phorbol esters, a potent but non-physiological activator of protein kinase C. In the present work, endothelin-1, a normal regulator of protein kinase C (
      • Clerk A.
      • Bogoyevitch M.A.
      • Anderson M.B.
      • Sugden P.H.
      ), was also found to inhibit the ability of insulin to stimulate the IRS-1 association with PI 3-kinase. This inhibition also appeared to be mediated via the activation of MAP kinase since the MEK inhibitor PD98059 completely blocked the response to endothelin-1. Moreover, this inhibition required the phosphorylation of Ser-612 since endothelin-1 did not inhibit the insulin-stimulated association of a Ser-612 mutant of IRS-1 with the PI 3-kinase (data not shown). Since endothelin-1 can inhibit insulin-stimulated glucose uptake in vivo as well asin vitro (
      • Chou Y.C.
      • Perng J.C.
      • Huan C.C.
      • Jang S.Y.
      • Kwok C.F.
      • Chen W.L.
      • Fong J.C.
      • Ho L.T.
      ,
      • Juan C.C.
      • Fang V.S.
      • Huang Y.J.
      • Kwok C.F.
      • Hsu Y.P.
      • Ho L.T.
      ) and the levels of endothelin-1 are elevated in obesity and diabetes mellitus (
      • Sarman B.
      • Toth M.
      • Somogyi A.
      ,
      • Ferri C.
      • Bellini C.
      • Desideri G.
      • Baldoncini R.
      • Properzi G.
      • Santucci A.
      • DeMattia G.
      ), this pathway may contribute to insulin resistance in these conditions.
      In the present studies, we have also examined the pathway that is involved in the PDGF-induced negative regulation of the insulin signaling cascade. Although PDGF did induce the activation of MAP kinase in the 3T3-L1 cells, this pathway did not appear to be required for the negative regulation of the insulin signaling cascade. In contrast to the results with endothelin-1, the MEK inhibitor had essentially no effect on the PDGF-induced inhibition in IRS-1-associated PI 3-kinase, although it completely inhibited the ability of PDGF to stimulate MAP kinase in these cells. This finding of a lack of inhibition by the MEK inhibitor of the PDGF response was also recently reported by Staubs et al. (
      • Staubs P.A.
      • Nelson J.G.
      • Reichart D.R.
      • Olefsky J.M.
      ).
      These results indicate that PDGF is most likely regulating the insulin-induced increase in IRS-1-associated PI 3-kinase by a pathway independent of the MAP kinase cascade. Further evidence to support this hypothesis is provided by the finding that the IRS-1 mutant lacking Ser-612 is still negatively regulated by PDGF. However, a mutant IRS-1 lacking three other serines in the same domain (Ser-632, -662, and -731) was found to lose its ability to be negatively regulated by PDGF treatment of cells. These results are consistent with the hypothesis that the negative regulation occurs via the serine phosphorylation of IRS-1; however, the sites required for this effect appear to differ from that required for the negative regulation by MAP kinase.
      In addition to activating the MAP kinase cascade, PDGF also stimulates the PI 3-kinase/Akt/70-kDa S6 kinase cascade in cells (
      • Heldin C.H.
      • Ostman A.
      • Ronnstrand L.
      ). Prior studies have shown that the activation of the PI 3-kinase is important in mediating the negative regulation of the insulin response (
      • Ricort J.-M.
      • Tanti J.-F.
      • van Obberghen E.
      • Le Marchand-Brustel Y.
      ,
      • Staubs P.A.
      • Nelson J.G.
      • Reichart D.R.
      • Olefsky J.M.
      ). Consistent with this prior work, wortmannin, an inhibitor of the PI 3-kinase, did block the PDGF inhibitory effect on the insulin-stimulated association of IRS-1 with PI 3-kinase. However, wortmannin had no effect on the endothelin response, further indicating that these two hormones exert their effects through distinct signaling cascades. To determine whether molecules further downstream in this kinase cascade could also induce a negative regulation of the insulin-signaling pathway, we have utilized a conditionally active form of Akt (
      • Kohn A.D.
      • Barthel A.
      • Kovacina K.S.
      • Boge A.
      • Wallach B.
      • Summers S.A.
      • Birnbaum M.J.
      • Scott P.H.
      • Lawrence Jr., J.C.
      • Roth R.A.
      ). This kinase can be turned on independently of other signals by treatment of cells with the estrogen antagonist, hydroxytamoxifen. Activation of this chimeric Akt by treatment of cells with hydroxytamoxifen was found to inhibit the insulin-stimulated increase in IRS-1-associated PI 3-kinase. Control experiments verified that no significant inhibition was induced by hydroxytamoxifen in cells not expressing this chimeric Akt. The inhibition induced by hydroxytamoxifen in MER-Akt-expressing cells paralleled the increase in Akt enzymatic activity of this chimera. This inhibition by Akt also appeared to require the same serines as those required for the negative regulation by PDGF since activation of Akt did not inhibit the insulin-induced association of PI 3-kinase and a mutant IRS-1 lacking these serines.
      The finding that Akt can negatively regulate the insulin-induced increase in IRS-1-associated PI 3-kinase is important since it suggests that, during the insulin-stimulated activation of Akt, there is also a feedback inhibition. This would also explain the insulin resistance that occurs during periods of hyperinsulinemia (
      • Olefsky J.M.
      • Nolan J.J.
      ). Finally, it would explain the decrease in insulin-stimulated biological responses that is observed in cells overexpressing various Akt constructs (
      • Kohn A.D.
      • Barthel A.
      • Kovacina K.S.
      • Boge A.
      • Wallach B.
      • Summers S.A.
      • Birnbaum M.J.
      • Scott P.H.
      • Lawrence Jr., J.C.
      • Roth R.A.
      ,
      • Scott P.H.
      • Brunn G.J.
      • Kohn A.D.
      • Roth R.A.
      • Lawrence Jr., J.C.
      ).
      One explanation of these results is that PDGF activates Akt and Akt itself directly phosphorylates IRS-1, thereby inhibiting its subsequent tyrosine phosphorylation and association with PI 3-kinase. However, the sites identified as the important regulatory ones (serines 612, 632, 662, and 731) do not fit the consensus Akt phosphorylation site (
      • Alessi D.R.
      • Caudwell F.B.
      • Andjelkovic M.
      • Hemmings B.A.
      • Cohen P.
      ). Alternatively, it is possible that a kinase activated by Akt is responsible for this phosphorylation. One such candidate is the enzyme called the mammalian target of rapamycin, mTOR, or FRAP (
      • Abraham R.T.
      ). The kinase activity of this enzyme has been shown to be regulated by Akt, insulin, and PDGF (
      • Scott P.H.
      • Brunn G.J.
      • Kohn A.D.
      • Roth R.A.
      • Lawrence Jr., J.C.
      ,
      • Burnett P.E.
      • Barrow R.K.
      • Cohen N.A.
      • Snyder S.H.
      • Sabatini D.M.
      ). In addition, the sites phosphorylated by this enzyme in one of its substrates, PHAS-I (for properties of heat and acid stability-I), are similar to the regulatory sites identified in IRS-1 in that both have a (Ser/Thr)-Pro motif (
      • Brunn G.J.
      • Fadden P.
      • Haystead T.A.J.
      • Lawrence Jr., J.C.
      ). Finally, the finding that rapamycin at least partly inhibits the negative regulation by both PDGF and Akt is consistent with this hypothesis.

      Acknowledgments

      We thank Dr. Morris White for the IRS-1 cDNA, Dr. Garry Nolan for the Phoenix retroviral packaging cell line and the retroviral vectors, Dr. Alan Saltiel for a gift of PD98059, Dr. Kozui Shii for antibodies to IRS-1 and -2, and Dr. Wei Liu for advice on DNA sequencing.

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