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Insulin Receptor Substrate (IRS)-2 Is Dephosphorylated More Rapidly than IRS-1 via Its Association with Phosphatidylinositol 3-Kinase in Skeletal Muscle Cells*

Open AccessPublished:May 09, 1997DOI:https://doi.org/10.1074/jbc.272.19.12868
      Insulin receptor substrate (IRS)-2 is structurally and functionally similar to IRS-1. Indeed, stimulation with insulin or insulin-like growth factor I led to the rapid tyrosine phosphorylation of both IRS-1 and IRS-2, which in turn activated phosphatidylinositol (PI) 3-kinase in L6 cells and rat skeletal muscle. However, IRS-2 was rapidly dephosphorylated (3–10 min after the addition of insulin/insulin-like growth factor I), whereas IRS-1 phosphorylation continued for at least 60 min. The time courses of the PI 3-kinase activity associated with IRS-1 and IRS-2 paralleled the tyrosine phosphorylation of these proteins. Preincubation with sodium orthovanadate, an inhibitor of protein tyrosine phosphatase, blocked the rapid dephosphorylation of IRS-2, suggesting the involvement of tyrosine phosphatase. The activation of PI 3-kinase apparently plays an important role in the rapid dephosphorylation of IRS-2, as IRS-2 dephosphorylation was inhibited markedly by suppressing PI 3-kinase activity with wortmannin or overexpression of the dominant negative p85 subunit of PI 3-kinase, which cannot bind the p110 catalytic subunit. In addition, platelet-derived growth factor stimulation prior to insulin stimulation decreased IRS-associated PI 3-kinase and significantly inhibited the dephosphorylation of IRS-2. Taken together, these observations suggest that IRS-2 plays a unique role in mediating the signals from the insulin receptor to downstream molecules and that this effect is more transient than that of IRS-1. Tyrosine phosphatase and IRS-associated PI 3-kinase activity thus contribute to the rapid dephosphorylation of IRS-2.
      Insulin binding to its receptor induces activation of the receptor tyrosine kinase followed by the phosphorylation of several cytosolic substrates. A major substrate of the insulin receptor is a 165–185-kDa protein termed insulin receptor substrate (IRS)
      The abbreviations used are; IRS, insulin receptor substrate; PI, phosphatidylinositol; αPY, anti-phosphotyrosine; Δp85, dominant negative p85 mutant; Na3VO4, sodium orthovanadate; IGF-I, insulin-like growth factor I; PDGF, platelet-derived growth factor.
      1The abbreviations used are; IRS, insulin receptor substrate; PI, phosphatidylinositol; αPY, anti-phosphotyrosine; Δp85, dominant negative p85 mutant; Na3VO4, sodium orthovanadate; IGF-I, insulin-like growth factor I; PDGF, platelet-derived growth factor.
      -1 (
      • Sun X.J.
      • Rothenberg P.
      • Kahn C.R.
      • Backer J.M.
      • Araki E.
      • Wilden P.A.
      • Cahill D.A.
      • Goldstein B.J.
      • White M.F.
      ). After insulin stimulation, IRS-1 is rapidly phosphorylated on multiple tyrosine residues and binds to several Src-homology 2 domains containing proteins (SH2 proteins) (
      • Sun X.J.
      • Crimmins D.L.
      • Myers Jr., M.G.
      • Miralpeix M.
      • White M.F.
      ), which include regulatory subunits for phosphatidylinositol (PI) 3-kinase (p85 (
      • Backer J.M.
      • Myers Jr., M.G.
      • Shoelson S.E.
      • Chin D.J.
      • Sun X.J.
      • Miralpeix M.
      • Hu P.
      • Margolis B.
      • Skolnik E.Y.
      • Schlessinger J.
      • White M.F.
      ), p55γ (
      • Pons S.
      • Asano T.
      • Glasheen E.
      • Miralpeix M.
      • Zhang Y.
      • Fisher T.L.
      • Myers Jr., M.G.
      • Sun X.J.
      • White M.F.
      ,
      • Inukai K.
      • Anai M.
      • Van Breda E.
      • Hosaka T.
      • Katagiri H.
      • Funaki M.
      • Fukushima Y.
      • Ogihara T.
      • Yazaki Y.
      • Kikuchi M.
      • Oka Y.
      • Asano T.
      ), and p55α (
      • Inukai K.
      • Anai M.
      • Van Breda E.
      • Hosaka T.
      • Katagiri H.
      • Funaki M.
      • Fukushima Y.
      • Ogihara T.
      • Yazaki Y.
      • Kikuchi M.
      • Oka Y.
      • Asano T.
      )), Grb2 (
      • Lowenstein E.J.
      • Daly R.J.
      • Batzer A.G.
      • Li W.
      • Margolis B.
      • Lammers R.
      • Ullrich A.
      • Skolnik E.Y.
      • Bar-Sagi D.
      • Schlessinger J.
      ), SHPTP2 (
      • Feener E.P.
      • Plutzky J.
      • Neel B.G.
      ), and Nck (
      • Lee C.-H.
      • Li W.
      • Nishimura R.
      • Zhou M.
      • Batzer A.
      • Myers Jr., M.G.
      • White M.F.
      • Schlessinger J.
      • Skolnik E.Y.
      ). Consequently, IRS-1 mediates activation of PI 3-kinase, p21ras and mitogen-activated protein kinase (
      • Skolnik E.Y.
      • Lee C.-H.
      • Batzer A.
      • Vicentini L.M.
      • Zhou M.
      • Daly R.
      • Myers Jr, M.J.
      • Backer J.M.
      • Ullrich A.
      • White M.F.
      • Schlessinger J.
      ), resulting in the promotion of glucose uptake, glycogen synthesis, mitogenesis, or gene expression. Therefore, IRS-1 was originally thought to play important roles in insulin signaling.
      However, studies using IRS-1-deficient mice, derived from targeted gene disruption, have demonstrated that a 180–190-kDa protein functions as an alternative substrate for the insulin receptor (
      • Tamemoto H.
      • Kadowaki T.
      • Tobe K.
      • Yagi T.
      • Sakura H.
      • Hayakawa T.
      • Terauchi Y.
      • Ueki K.
      • Kaburagi Y.
      • Satoh S.
      • Sekihara H.
      • Yoshioka S.
      • Horikoshi H.
      • Furuta Y.
      • Ikawa Y.
      • Kasuga M.
      • Yazaki Y.
      • Aizawa S.
      ,
      • Araki E.
      • Lipes M.A.
      • Patti M.-E.
      • Brüning J.C.
      • Haag III, B.
      • Johnson R.S.
      • Kahn R.C.
      ). Cloning of this protein revealed a structure similar to that of IRS-1, which led this protein to be designated IRS-2 (
      • Sun X.J.
      • Wang L.-M.
      • Zhang Y.
      • Yenush L.
      • Myers Jr., M.G.
      • Glasheen E.
      • Lane W.S.
      • Pierce J.H.
      • White M.F.
      ). IRS-2 has multiple conserved tyrosine phosphorylation sites that can bind to various SH2 proteins (
      • Sun X.J.
      • Wang L.-M.
      • Zhang Y.
      • Yenush L.
      • Myers Jr., M.G.
      • Glasheen E.
      • Lane W.S.
      • Pierce J.H.
      • White M.F.
      ), indicating that IRS-2 is functionally similar to IRS-1. Herein, we investigated the functional differences between IRS-1 and IRS-2. We found that IRS-2 is dephosphorylated much more rapidly and activates PI 3-kinase more transiently than IRS-1 in skeletal muscle cells. Furthermore, important roles of its associated PI 3-kinase in the rapid dephosphorylation of IRS-2 are discussed.

      DISCUSSION

      The binding of insulin to its receptor activates receptor tyrosine kinase. The activated insulin receptor phosphorylates several cytosolic substrates, which contribute to the divergent biological effects of insulin. These include a major insulin receptor substrate, IRS-1, and the recently cloned IRS-2. These two proteins had been considered to be structurally and functionally similar (
      • Sun X.J.
      • Wang L.-M.
      • Zhang Y.
      • Yenush L.
      • Myers Jr., M.G.
      • Glasheen E.
      • Lane W.S.
      • Pierce J.H.
      • White M.F.
      ,
      • Patti M.-E.
      • Sun X.-J.
      • Brüning J.C.
      • Araki E.
      • Lipes M.A.
      • White M.F.
      • Kahn C.R.
      ). To date, little information has been available on the different characteristics of IRS-1 and IRS-2. It was reported that IRS-2 binds to the insulin receptor through its newly identified domain, which is absent in IRS-1 (
      • Sawka-Verhelle D.
      • Tartare-Deckert S.
      • White M.F.
      • Van Obberghen E.
      ,
      • He W.
      • Craparo A.
      • Zhu Y.
      • O'Neill T.J.
      • Wang L.-M.
      • Pierce J.H.
      • Gustafson T.A.
      ). A recent report showed that after treatment with tumor necrosis factor-α, IRS-1 inhibits insulin receptor tyrosine kinase activity, whereas IRS-2 does not (
      • Peraldi P.
      • Hotamisligil G.S.
      • Buurman W.A.
      • White M.F.
      • Spiegelman B.M.
      ). In this study, we demonstrated that IRS-2 is dephosphorylated more rapidly and activates PI 3-kinase more transiently than IRS-1 in skeletal muscle cells. This is the first report detailing the different features of IRS-1 and IRS-2 in tyrosine phosphorylation and signal transduction to downstream molecules.
      Next, we attempted to clarify the molecular mechanisms leading to rapid dephosphorylation of IRS-2. When the cells were incubated with Na3VO4 to block tyrosine phosphatase, IRS-2 tyrosine phosphorylation was prolonged. The rapid dephosphorylation of IRS-2 occurs via protein tyrosine phosphatase, as expected. However, when the cells were incubated with 10 nm wortmannin to inhibit PI 3-kinase, the dephosphorylation was also markedly inhibited. In addition, when the Δp85 mutant was overexpressed, IRS-2 dephosphorylation was markedly inhibited, suggesting that PI 3-kinase activation is necessary for rapid dephosphorylation of IRS-2. It is unlikely that Δp85 binds to phosphotyrosine residues of IRS-1 and IRS-2 in a nonspecific manner because the overexpression of Δp85 affected neither the binding of Grb2 to IRS-1 and IRS-2 nor mitogen-activated protein kinase activation. When IRS-1- and IRS-2-associated PI 3-kinase was reduced, despite PI 3-kinase activity with phosphotyrosine being increased, by sequential addition of PDGF and insulin, IRS-2 dephosphorylation was reduced. We have come to the conclusion that IRS-associated PI 3-kinase activity rather than total PI 3-kinase activity is important for rapid dephosphorylation of IRS-2.
      Although the mechanism by which IRS-associated PI 3-kinase triggers the rapid dephosphorylation of IRS-2 remains unknown, the evidence supports two possibilities. First, IRS-1-associated PI 3-kinase leads to phosphorylation of serine residues of IRS-1. PI 3-kinase possesses not only lipid kinase, but also serine kinase activity (
      • Dhand R.
      • Hiles I.
      • Panayotou G.
      • Roche S.
      • Fry M.J.
      • Gout I.
      • Totty N.F.
      • Truong O.
      • Vicendo P.
      • Yonezawa K.
      • Kasuga M.
      • Courtneidge S.A.
      • Waterfield M.D.
      ). In vitro kinase assays of anti-p85 immunoprecipitates have shown that IRS-1 is serine- phosphorylated by IRS-1-associated PI 3-kinase (
      • Lam K.
      • Carpenter C.L.
      • Ruderman N.B.
      • Friel J.C.
      • Kelly K.L.
      ,
      • Tanti J.-F.
      • Gremeaux T.
      • Van Obberghen E.
      • Le Marchand-Brustel Y.
      ). We speculate that IRS-2-associated PI 3-kinase phosphorylates serine residues of IRS-2 as well. Meanwhile, serine/threonine phosphorylation of IRSs affects their tyrosine phosphorylation (
      • Tanti J.-F.
      • Gremeaux T.
      • Van Obberghen E.
      • Le Marchand-Brustel Y.
      ,
      • Kanety H.
      • Feinstein R.
      • Papa M.Z.
      • Hemi R.
      • Karasik A.
      ). When the serine/threonine phosphorylation of IRS-1 is augmented by okadaic acid (
      • Tanti J.-F.
      • Gremeaux T.
      • Van Obberghen E.
      • Le Marchand-Brustel Y.
      ) or calyculin A (
      • Kanety H.
      • Feinstein R.
      • Papa M.Z.
      • Hemi R.
      • Karasik A.
      ), which are serine/threonine phosphatase inhibitors, insulin-induced tyrosine phosphorylation of IRS-1 is impaired. It is possible that serine/threonine phosphorylation of IRSs modulates not only their tyrosine phosphorylation but also dephosphorylation by tyrosine phosphatase. Different serine/threonine phosphorylation sites in IRS-1 and IRS-2 might be responsible for the different degree of accessibility to tyrosine phosphatase. We speculate that IRS-2-associated PI 3-kinase, which phosphorylates serine residues of IRS-2, modulates rapid tyrosine dephosphorylation of IRS-2.
      Second, a role for PI 3-kinase in protein sorting has been suggested by the finding that a yeast PI 3-kinase homolog, VSP34, is involved in sorting proteins to vacuoles (
      • Schu P.V.
      • Takegawa K.
      • Fry M.J.
      • Stack J.H.
      • Waterfield M.D.
      • Emr S.D.
      ). PI 3-kinase binding to the PDGF receptor is reportedly essential for the postendocytic sorting of this receptor (
      • Joly M.
      • Kazlauskas A.
      • Fay F.S.
      • Corvela S.
      ). We speculated that PI 3-kinase binding to IRS-1 and IRS-2 is necessary for their intracellular trafficking and that they are sorted and dephosphorylated differently.
      In conclusion, IRS-2 is dephosphorylated more rapidly than IRS-1, and IRS-2 dephosphorylation is regulated to some extent by IRS-associated PI 3-kinase activity. Based on the results of this study, we have devised a model for the roles of IRS-1 and IRS-2. We propose that IRS-1 transmits continuous signals from the insulin receptor, whereas IRS-2 mediates transient signals regulated by its associated PI 3-kinase activity. The continuous IRS-1 pathway and the duration-regulated IRS-2 pathway might operate in combination to fine tune the acute and chronic actions of insulin.

      Acknowledgments

      We are grateful to Dr. Izumu Saito (Institute of Medical Science, University of Tokyo) for the generous gift of the recombinant Adex1CALacZ and the cassette cosmid for constructing recombinant adenovirus.

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