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Phosphatidylinositol 3-Kinase Is Required for Insulin-stimulated Tyrosine Phosphorylation of Shc in 3T3-L1 Adipocytes*

  • Satoshi Ugi
    Affiliations
    Department of Medicine, Division of Endocrinology and Metabolism, University of California, San Diego, La Jolla, California 92093-0673, San Diego Veterans Affairs Hospital, Research Service, San Diego, California 92161, and The Whittier Diabetes Institute, La Jolla, California 92037
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  • Prem M. Sharma
    Affiliations
    Department of Medicine, Division of Endocrinology and Metabolism, University of California, San Diego, La Jolla, California 92093-0673, San Diego Veterans Affairs Hospital, Research Service, San Diego, California 92161, and The Whittier Diabetes Institute, La Jolla, California 92037
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  • William Ricketts
    Footnotes
    Affiliations
    Department of Medicine, Division of Endocrinology and Metabolism, University of California, San Diego, La Jolla, California 92093-0673, San Diego Veterans Affairs Hospital, Research Service, San Diego, California 92161, and The Whittier Diabetes Institute, La Jolla, California 92037
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  • Takeshi Imamura
    Affiliations
    Department of Medicine, Division of Endocrinology and Metabolism, University of California, San Diego, La Jolla, California 92093-0673, San Diego Veterans Affairs Hospital, Research Service, San Diego, California 92161, and The Whittier Diabetes Institute, La Jolla, California 92037
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  • Jerrold M. Olefsky
    Correspondence
    To whom correspondence should be addressed: Dept. of Medicine (0673), University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0673. Tel.: 858-534-6651; Fax: 858-534-6653
    Affiliations
    Department of Medicine, Division of Endocrinology and Metabolism, University of California, San Diego, La Jolla, California 92093-0673, San Diego Veterans Affairs Hospital, Research Service, San Diego, California 92161, and The Whittier Diabetes Institute, La Jolla, California 92037
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  • Author Footnotes
    * This work was supported by National Institutes of Health Research Grant DK 33651, the Veterans Administration San Diego Health Care System, Research Service, and the Whittier Diabetes Institute. 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.
    ‡ Present address: ICN Pharmaceuticals, Inc., 3300 Hyland Ave., Costa Mesa, CA 92626.
Open AccessPublished:March 15, 2002DOI:https://doi.org/10.1074/jbc.M201019200
      The interactions between the phosphatidylinositol 3-kinase (PI 3-kinase) and Ras/MAPK kinase pathways have been the subject of considerable interest. In the current studies, we find that epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) lead to rapid phosphorylation of Shc (maximum at 1–2 min), whereas insulin-mediated Shc phosphorylation is relatively delayed (maximum at 5–10 min), suggesting that an intermediary step may be necessary for insulin stimulation of Shc phosphorylation. The Src homology-2 (SH2) domain of Shc is necessary for PDGF- and EGF-mediated Shc phosphorylation, whereas the phosphotyrosine binding (PTB) domain is critical for the actions of insulin. Because the Shc PTB domain can interact with phospholipids, we postulated that PI 3-kinase might be a necessary intermediary step facilitating insulin-stimulated phosphorylation of Shc. In support of this, we found that the PI 3-kinase inhibitors, wortmannin and LY294002, blocked insulin-stimulated but not EGF- or PDGF-stimulated Shc phosphorylation. Furthermore, overexpression of a dominant negative PI 3-kinase construct (p85N-SH2) blocked insulin, but not EGF- or PDGF-induced Shc phosphorylation. All three growth factors cause localization of Shc to the plasma membrane, but only the effect of insulin was inhibited by wortmannin, supporting the view that PI 3-kinase-generated phospholipids mediate insulin-stimulated Shc phosphorylation. Consistent with this, expression of a constitutively active PI 3-kinase (p110CAAX) increased membrane localization of Shc, and this was completely blocked by wortmannin. A mutant Shc with a disrupted PTB domain (Shc S154) did not localize to the membrane in p110CAAX-expressing cells or after insulin stimulation and was not phosphorylated by insulin. In summary, 1) PI 3-kinase is a necessary early step in insulin-stimulated Shc phosphorylation, whereas the effects of EGF and PDGF on Shc phosphorylation are independent of PI 3-kinase. 2) PI 3-kinase-stimulated generation of membrane phospholipids can localize Shc to the plasma membrane through the Shc PTB domain facilitating phosphorylation by the insulin receptor.
      Growth factor signaling initiates a variety of biologic responses, many of which are mediated through the PI 3-kinase
      The abbreviations used are: PI 3-kinase
      phosphatidylinositol 3-kinase
      MAP kinase
      mitogen-activated protein kinase
      PP2A
      protein phosphatase 2A
      RTK
      receptor tyrosine kinase
      PDGF
      platelet-derived growth factor
      EGF
      epidermal growth factor
      SH2
      Src homology 2
      PTB
      phosphotyrosine binding
      PIP2
      phosphatidylinositol 3,4-bisphosphate
      PIP3
      phosphatidylinositol 3,4,5-trisphosphate
      DMEM
      Dulbecco's modified Eagle's medium
      m.o.i.
      multiplicity of infection
      1The abbreviations used are: PI 3-kinase
      phosphatidylinositol 3-kinase
      MAP kinase
      mitogen-activated protein kinase
      PP2A
      protein phosphatase 2A
      RTK
      receptor tyrosine kinase
      PDGF
      platelet-derived growth factor
      EGF
      epidermal growth factor
      SH2
      Src homology 2
      PTB
      phosphotyrosine binding
      PIP2
      phosphatidylinositol 3,4-bisphosphate
      PIP3
      phosphatidylinositol 3,4,5-trisphosphate
      DMEM
      Dulbecco's modified Eagle's medium
      m.o.i.
      multiplicity of infection
      and the Ras/MAP kinase pathway. PI 3-kinase is a dual protein and lipid kinase composed of an 85-kDa regulatory subunit (p85) and a 110-kDa catalytic subunit (p110). PI 3-kinase phosphorylates phosphoinositides at the 3′-position of the inositol ring to generate phosphorylated lipid products and can also phosphorylate proteins on serine/threonine residues (
      • Dhand R.
      • Hiles I.
      • Panayotou G.
      • Roche S.
      • Fry M.J.
      • Gout I.
      • Totty N.F.
      • Truong O.
      • Vicendo P.
      • Yonezawa K.
      • et al.
      ,
      • Lam K.
      • Carpenter C.L.
      • Ruderman N.B.
      • Friel J.C.
      • Kelly K.L.
      ). PI 3-kinase plays a central role in a diverse range of cellular responses, including cell growth, differentiation, protein synthesis, glucose uptake, lipogenesis, and membrane trafficking (
      • Shepherd P.R.
      • Withers D.J.
      • Siddle K.
      ).
      The Ras/MAP kinase pathway is another key component in the transduction of mitogenic signals. Activation of the insulin receptor or other growth factor receptors results in the tyrosine phosphorylation of Shc, which then interacts with the adapter protein Grb2, which is pre-associated with SOS, a guanine nucleotide exchange factor (
      • Avruch J.
      ,
      • Downward J.
      ). SOS stimulates formation of active GTP-bound Ras, which then initiates a sequence of phosphorylation events, activating a cascade of protein serine/threonine kinases. Ras activates Raf-1 kinase leading to phosphorylation and activation of mitogen-activated/extracellular signal-regulated kinase kinase, which in turn phosphorylates and activates MAP kinase (
      • Ceresa B.P.
      • Pessin J.E.
      ). Thus, in this pathway, Ras functions as a molecular switch converting tyrosine kinase signals into a serine/threonine kinase cascade (
      • Yamauchi K.
      • Holt K.
      • Pessin J.E.
      ).
      Several investigators have demonstrated an interaction between the PI3-kinase and Ras/MAP kinase pathways; however, the results have been somewhat conflicting. PI 3-kinase has been shown to stimulate Ras by some groups (
      • Hu Q.
      • Klippel A.
      • Muslin A.J.
      • Fantl W.J.
      • Williams L.T.
      ,
      • Egawa K.
      • Sharma P.M.
      • Nakashima N.
      • Huang Y.
      • Huver E.
      • Boss G.R.
      • Olefsky J.M.
      ) but to be a target of Ras by others (
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Dhand R.
      • Vanhaesebroeck B.
      • Gout I.
      • Fry M.J.
      • Waterfield M.D.
      • Downward J.
      ,
      • Marte B.M.
      • Rodriguez-Viciana P.
      • Wennstrom S.
      • Warne P.H.
      • Downward J.
      ). Furthermore, inhibition of PI 3-kinase with wortmannin or dominant negative PI 3-kinase can block MAP kinase activation in some, but not, all cells (
      • Hara K.
      • Yonezawa K.
      • Sakaue H.
      • Ando A.
      • Kotani K.
      • Kitamura T.
      • Kitamura Y.
      • Ueda H.
      • Stephens L.
      • Jackson T.R.
      • et al.
      ,
      • Duckworth B.C.
      • Cantley L.C.
      ,
      • DePaolo D.
      • Reusch J.E.
      • Carel K.
      • Bhuripanyo P.
      • Leitner J.W.
      • Draznin B.
      ,
      • Suga J.
      • Yoshimasa Y.
      • Yamada K.
      • Yamamoto Y.
      • Inoue G.
      • Okamoto M.
      • Hayashi T.
      • Shigemoto M.
      • Kosaki A.
      • Kuzuya H.
      • Nakao K.
      ,
      • Sharma P.M.
      • Egawa K.
      • Huang Y.
      • Martin J.L.
      • Huvar I.
      • Boss G.R.
      • Olefsky J.M.
      ).
      To date, most of the attention has been focused on potential direct interactions between PI 3-kinase and Ras, but in this study, we have concentrated on an upstream activator of Ras, and we have explored potential interactions between PI 3-kinase signaling and Shc activation. These studies have shown that PI 3-kinase activity is necessary for insulin-stimulated tyrosine phosphorylation of Shc, whereas other growth factors, such as PDGF and EGF, can efficiently signal to Shc in the absence of the PI 3-kinase requirement. As such, these experiments demonstrate a novel mechanism of cross-talk between the PI 3-kinase and Ras/MAP kinase signaling pathways and demonstrate the specificity of this mechanism for the insulin action cascade.

      DISCUSSION

      Growth factors, such as EGF, PDGF, and insulin bind to their cognate receptor tyrosine kinases (RTKs) leading to rapid tyrosine phosphorylation of Shc with subsequent activation of the Ras/MAP kinase pathway (
      • Ceresa B.P.
      • Pessin J.E.
      ,
      • Pelicci G.
      • Lanfrancone L.
      • Grignani F.
      • McGlade J.
      • Cavallo F.
      • Forni G.
      • Nicoletti I.
      • Pawson T.
      • Pelicci P.G.
      ,
      • Sasaoka T.
      • Rose D.W.
      • Jhun B.H.
      • Saltiel A.R.
      • Draznin B.
      • Olefsky J.M.
      ). Because Shc contains an SH2 and a PTB domain, current thinking is that following ligand-directed tyrosine phosphorylation of RTKs, these receptors directly bind to Shc leading to tyrosine phosphorylation (
      • Isakoff S.J.
      • Yu Y.P.
      • Su Y.C.
      • Blaikie P.
      • Yajnik V.
      • Rose E.
      • Weidner K.M.
      • Sachs M.
      • Margolis B.
      • Skolnik E.Y.
      ,
      • Ricketts W.A.
      • Rose D.W.
      • Shoelson S.
      • Olefsky J.M.
      ,
      • Gustafson T.A., He, W.
      • Craparo A.
      • Schaub C.D.
      • O'Neill T.J.
      ,
      • Yokote K.
      • Mori S.
      • Hansen K.
      • McGlade J.
      • Pawson T.
      • Heldin C.H.
      • Claesson-Welsh L.
      ,
      • Sakaguchi K.
      • Okabayashi Y.
      • Kido Y.
      • Kimura S.
      • Matsumura Y.
      • Inushima K.
      • Kasuga M.
      ,
      • van der Geer P.
      • Wiley S.
      • Lai V.K.
      • Olivier J.P.
      • Gish G.D.
      • Stephens R.
      • Kaplan D.
      • Shoelson S.
      • Pawson T.
      ). The PI 3-kinase and Ras/MAP kinase pathways are clearly interconnected, but there is considerable debate and controversy as to sites and mechanisms of convergence (
      • Hu Q.
      • Klippel A.
      • Muslin A.J.
      • Fantl W.J.
      • Williams L.T.
      ,
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Dhand R.
      • Vanhaesebroeck B.
      • Gout I.
      • Fry M.J.
      • Waterfield M.D.
      • Downward J.
      ,
      • Marte B.M.
      • Rodriguez-Viciana P.
      • Wennstrom S.
      • Warne P.H.
      • Downward J.
      ). In the current studies, we provide evidence for a novel interaction pathway between PI 3-kinase and the Shc/Ras/MAP kinase cascade, with respect to insulin signaling. We find that PI 3-kinase stimulation is a necessary step mediating Shc phosphorylation by the insulin receptor. Our data indicate that PI 3-kinase stimulation leads to generation of plasma membrane lipid products that mediate localization of Shc to the plasma membrane through the Shc PTB domain. This PI 3-kinase-dependent step is necessary for insulin receptor phosphorylation of Shc but not for interactions with the EGF or PDGF receptors.
      RTK activation causes tyrosine phosphorylation of Shc, but the structural basis for Shc tyrosine phosphorylation is different for different growth factors (
      • Isakoff S.J.
      • Yu Y.P.
      • Su Y.C.
      • Blaikie P.
      • Yajnik V.
      • Rose E.
      • Weidner K.M.
      • Sachs M.
      • Margolis B.
      • Skolnik E.Y.
      ,
      • Ricketts W.A.
      • Rose D.W.
      • Shoelson S.
      • Olefsky J.M.
      ,
      • Gustafson T.A., He, W.
      • Craparo A.
      • Schaub C.D.
      • O'Neill T.J.
      ,
      • Yokote K.
      • Mori S.
      • Hansen K.
      • McGlade J.
      • Pawson T.
      • Heldin C.H.
      • Claesson-Welsh L.
      ,
      • Sakaguchi K.
      • Okabayashi Y.
      • Kido Y.
      • Kimura S.
      • Matsumura Y.
      • Inushima K.
      • Kasuga M.
      ). Shc can bind to phosphotyrosine residues through either the Shc PTB or SH2 domains. With respect to the insulin receptor, it is well established that the PTB domain is necessary for interaction and tyrosine phosphorylation (
      • Isakoff S.J.
      • Yu Y.P.
      • Su Y.C.
      • Blaikie P.
      • Yajnik V.
      • Rose E.
      • Weidner K.M.
      • Sachs M.
      • Margolis B.
      • Skolnik E.Y.
      ,
      • Gustafson T.A., He, W.
      • Craparo A.
      • Schaub C.D.
      • O'Neill T.J.
      ). For example, a mutant Shc with a disrupted PTB domain failed to bind to insulin receptors in the two-hybrid system and was not phosphorylated in vivo (
      • Isakoff S.J.
      • Yu Y.P.
      • Su Y.C.
      • Blaikie P.
      • Yajnik V.
      • Rose E.
      • Weidner K.M.
      • Sachs M.
      • Margolis B.
      • Skolnik E.Y.
      ). Conversely, mutant insulin receptors with a disabled PTB domain-binding motif (NP XY domain) cannot bind to or phosphorylate Shc (
      • Gustafson T.A., He, W.
      • Craparo A.
      • Schaub C.D.
      • O'Neill T.J.
      ). In contrast to the insulin receptor, Shc associates with PDGF receptors via its SH2 domain (
      • Yokote K.
      • Mori S.
      • Hansen K.
      • McGlade J.
      • Pawson T.
      • Heldin C.H.
      • Claesson-Welsh L.
      ), whereas both the PTB and the SH2 domain of Shc can bind to EGF receptors (
      • Ricketts W.A.
      • Rose D.W.
      • Shoelson S.
      • Olefsky J.M.
      ,
      • Sakaguchi K.
      • Okabayashi Y.
      • Kido Y.
      • Kimura S.
      • Matsumura Y.
      • Inushima K.
      • Kasuga M.
      ). Membrane localization of Shc is also necessary for growth factor-stimulated phosphorylation (
      • Ravichandran K.S.
      • Zhou M.M.
      • Pratt J.C.
      • Harlan J.E.
      • Walk S.F.
      • Fesik S.W.
      • Burakoff S.J.
      ), and the PTB domain of Shc binds to phospholipids in vitro (
      • Rameh L.E.
      • Arvidsson A.
      • Carraway III, K.L.
      • Couvillon A.D.
      • Rathbun G.
      • Crompton A.
      • VanRenterghem B.
      • Czech M.P.
      • Ravichandran K.S.
      • Burakoff S.J.
      • Wang D.S.
      • Chen C.S.
      • Cantley L.C.
      ,
      • Ravichandran K.S.
      • Zhou M.M.
      • Pratt J.C.
      • Harlan J.E.
      • Walk S.F.
      • Fesik S.W.
      • Burakoff S.J.
      ), potentially mediating membrane localization.
      We found that EGF and PDGF treatment leads to very rapid phosphorylation of Shc, consistent with direct association of the Shc SH2 domain with these RTKs, which then phosphorylate Shc on tyrosine residues (
      • Yokote K.
      • Mori S.
      • Hansen K.
      • McGlade J.
      • Pawson T.
      • Heldin C.H.
      • Claesson-Welsh L.
      ,
      • Sakaguchi K.
      • Okabayashi Y.
      • Kido Y.
      • Kimura S.
      • Matsumura Y.
      • Inushima K.
      • Kasuga M.
      ). In contrast, the time course of insulin-induced Shc phosphorylation is relatively delayed compared with EGF and PDGF, raising the possibility of an intermediary step. This step can be explained by the idea that insulin leads to activation of PI 3-kinase with generation of plasma membrane phospholipids (
      • Shepherd P.R.
      • Withers D.J.
      • Siddle K.
      ,
      • Yang C.
      • Watson R.T.
      • Elmendorf J.S.
      • Sacks D.B.
      • Pessin J.E.
      ), the Shc PTB can bind to PIP2 and PIP3 (
      • Rameh L.E.
      • Arvidsson A.
      • Carraway III, K.L.
      • Couvillon A.D.
      • Rathbun G.
      • Crompton A.
      • VanRenterghem B.
      • Czech M.P.
      • Ravichandran K.S.
      • Burakoff S.J.
      • Wang D.S.
      • Chen C.S.
      • Cantley L.C.
      ), bringing Shc to the plasma membrane, where the Shc PTB can then associate through equilibration to the phosphorylated insulin receptor. In this event, PI 3-kinase activation would facilitate Shc phosphorylation by the insulin receptor, creating a two-step mechanism. Consistent with this concept, we have found that treatment of cells with the PI 3-kinase inhibitors wortmannin and LY294002 impairs insulin but not EGF- or PDGF-induced Shc phosphorylation. We also show that wortmannin interrupts insulin, but not EGF or PDGF, -mediated plasma membrane localization of Shc, as well as Shc association with Grb2. Furthermore, expression of a dominant negative form of PI 3-kinase (p85N-SH2) also inhibits insulin but not EGF- or PDGF-stimulated Shc phosphorylation. Although the suppression of Shc phosphorylation by the dominant negative p85 construct was not 100%, inhibition of Akt phosphorylation was also not complete and was comparable with the magnitude of the inhibition of Shc phosphorylation. Because the inhibition of PI 3-kinase activity by this construct ranges from 70 to 100%, as we reported previously (
      • Sharma P.M.
      • Egawa K.
      • Huang Y.
      • Martin J.L.
      • Huvar I.
      • Boss G.R.
      • Olefsky J.M.
      ), and wortmannin and LY294002 inhibit PI 3-kinase activity to undetectable levels, we cannot definitively rule out the possibility that there is a small component of PI 3-kinase-independent insulin signaling to Shc phosphorylation.
      p110CAAX is a constitutively active, membrane-targeted form of the p110 catalytic subunit of PI 3-kinase (
      • Egawa K.
      • Sharma P.M.
      • Nakashima N.
      • Huang Y.
      • Huver E.
      • Boss G.R.
      • Olefsky J.M.
      ), and our results show that adenoviral mediated expression of this protein results in membrane localization of Shc, consistent with the idea that phospholipids generated by PI 3-kinase recruit Shc to the membrane. Interestingly, insulin-stimulated Shc phosphorylation is reduced in p110CAAX-expressing cells, and we speculate that the large amount of phospholipids generated by p110CAAX can localize Shc to cellular membrane fractions, including the plasma membrane, interfering with Shc-insulin receptor association, and inhibiting insulin mediated Shc phosphorylation. In this way, the excess PI 3-kinase-generated membrane phospholipids effectively compete with the insulin receptor for binding to the Shc PTB domain.
      The S154P Shc contains a disrupted PTB domain (
      • Ricketts W.A.
      • Brown J.H.
      • Olefsky J.M.
      ), and this mutation ablated p110CAAX and insulin-stimulated membrane localization, consistent with the idea that the PTB domain is necessary for this process (
      • Ravichandran K.S.
      • Zhou M.M.
      • Pratt J.C.
      • Harlan J.E.
      • Walk S.F.
      • Fesik S.W.
      • Burakoff S.J.
      ). We also found that insulin failed to phosphorylate this S154P Shc, whereas EGF and PDGF did. Because the Shc SH2 domain can interact with PDGF and EGF receptors (
      • Ricketts W.A.
      • Rose D.W.
      • Shoelson S.
      • Olefsky J.M.
      ,
      • Yokote K.
      • Mori S.
      • Hansen K.
      • McGlade J.
      • Pawson T.
      • Heldin C.H.
      • Claesson-Welsh L.
      ,
      • Sakaguchi K.
      • Okabayashi Y.
      • Kido Y.
      • Kimura S.
      • Matsumura Y.
      • Inushima K.
      • Kasuga M.
      ), these results are consistent with the view that the intact SH2 domain of S154P Shc is sufficient to allow phosphorylation by the EGF and PDGF receptors.
      In summary, these data demonstrate a new insulin-specific mechanism whereby PI 3-kinase stimulation interacts with the Ras/MAP kinase pathway. Thus, PI 3-kinase stimulation is necessary for insulin-induced Shc phosphorylation which then facilitates downstream signaling to Ras. In contrast, the effects of PDGF and EGF on Shc phosphorylation are independent of PI 3-kinase. Our data indicate that the mechanism for this interaction involves PI 3-kinase-induced generation of plasma membrane phospholipid products, which allow targeting of the Shc PTB domain to the cell surface where the Shc PTB domain can then interact with the insulin receptor leading to Shc phosphorylation. In contrast, the Shc SH2 domain is sufficient for interaction with the EGF and PDGF receptors. Taken together, these new data provide a novel mechanism for insulin stimulation of Shc and activation of the Ras/MAP kinase pathway, and point out that the sites of interaction between these two different signaling cascades can be multiple and specific for a particular hormonal input.

      ACKNOWLEDGEMENTS

      We thank Dr. E. Y. Skolnik for pRK5 Shc and Elizabeth Hansen for editorial assistance.

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