Endogenous Substrates of the Insulin Receptor: Studies with Cells Expressing Wild-Type and Mutant Receptors

  • Kazuyoshi Yonezawa
  • Sarah Pierce
  • Cynthia Stover
  • Martine Aggerbeck
  • William J. Rutter
  • Richard A. Roth
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 293)


The insulin receptor has an intrinsic tyrosine kinase activity which appears to be required for insulin to elicit its various biological responses (1). Identification of endogenous substrates of the insulin receptor kinase and other tyrosine kinases has been greatly advanced in the last few years by the development of high affinity polyclonal and monoclonal antibodies to phosphotyrosine (2–4). These reagents can be used to identify substrates of tyrosine kinases by immunoblotting and immunoprecipitation as well as to purify substrates by utilizing affinity columns composed of anti-phosphotyrosine antibodies. Via these and other techniques, numerous proteins have been identified as becoming phosphorylated on tyrosine residues. These include a number of proteins with unknown functions. One such protein, Mr ~160,000 to 180,000, has been reported to be tyrosine phosphorylated in response to both insulin and insulin-like growth factor I in a variety of cell types (5–6). Other proteins that have been identified as substrates of tyrosine kinases include various enzymes as well as other defined molecules. One such identified molecule is another tyrosine kinase, called pp60C-SRC. Activation of the platelet derived growth factor (PDGF) receptor tyrosine kinase was shown to increase the extent of tyrosine phosphorylation of the SRC protein and increase its enzymatic activity (7, 8). More recently, a phospholipase C (9–11), the type I phosphatidylinositol kinase (12–13), the GTPase activating protein of Ras (called GAP) (14, 15)and several serine/threonine kinases (the MAP2 kinase, the proto-oncogene Raf kinase and a cell cycle dependent kinase, CDC-2) (16–18) have all been shown to be tyrosine phosphorylated. The role of the tyrosine phosphorylation of these different molecules is not always clear. Only the physiological role of the tyrosine phosphorylation of the yeast cell cycle dependent kinase has been directly demonstrated (19).


Insulin Receptor Tyrosine Phosphorylation Chinese Hamster Ovary Cell Thymidine Incorporation Mutant Receptor 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    O.M. Rosen, After insulin binds, Science 237:1452 (1987).PubMedCrossRefGoogle Scholar
  2. 2.
    H.A. Ross, D. Baltimore and H.N. Eisen, Phosphotyrosine-containing proteins isolated by affinity chromatography with antibodies to a synthetic hapten, Nature 294:654 (1981).PubMedCrossRefGoogle Scholar
  3. 3.
    A.R. Frackelton, A.H. Ross and H.N. Eisen, Characterization and use of monoclonal antibodies for isolation of phosphotyrosyl proteins from retrovirus-transformed cells and growth factor-stimulated cells, Mol. Cell. Biol. 3:1343 (1983).PubMedGoogle Scholar
  4. 4.
    J. R. Glenney, L. Zokas and M.J. Kamps, Monoclonal antibodies to phosphotyrosine, J. Immunol. Meth. 109:277 (1988).CrossRefGoogle Scholar
  5. 5.
    M.F. White, R. Maron and C.R. Kahn, Insulin rapidly stimulates tyrosine phosphorylation of a Mr-185,000 protein in intact cells, Nature 318:183 (1985).PubMedCrossRefGoogle Scholar
  6. 6.
    M. Kasuga, T. Izumi, K. Tobe, T. Shiba, K. Momomura, Y. Tashiro-Hashimoto and T. Kadowaki, Substrates for insulin-receptor kinase, Diabetes Care 13:317 (1990).PubMedCrossRefGoogle Scholar
  7. 7.
    R. Ralston and J. M. Bishop, The product of the protooncogene c-src is modified during the cellular response to platelet-derived growth factor, Biochemistry 82:7845 (1985).Google Scholar
  8. 8.
    K.L. Gould and T. Hunter, Platelet-derived growth factor induces multisite phosphorylation of pp60c-src and increases its protein-tyrosine kinase activity, Mol. Cell. Biol. 8:3345 (1988).PubMedGoogle Scholar
  9. 9.
    M.I. Wahl, T.O., Daniel and G. Carpenter, Antiphosphotyrosine recovery of phospholipase C activity after EGF treatment of A-431 cells, Science 241:968 (1988).PubMedCrossRefGoogle Scholar
  10. 10.
    S. Nishibe, M.I. Wahl, S.G. Rhee and G. Carpenter, Tyrosine phosphorylation of phospholipase C-II in vitro by the epidermal growth factor receptor, J. Biol. Chem. 264:10335 (1989).PubMedGoogle Scholar
  11. 11.
    J. Meisenhelder, P.G. Suh, S.G. Rhee and T. Hunter, Phospholipase C-gamma is a substrate for the PDGF and EGF receptor protein-tyrosine kinases in vivo and in vitro, Cell 57:1109 (1989).PubMedCrossRefGoogle Scholar
  12. 12.
    S.A. Courtneidge and A. Heber, An 81 kd protein complexed with middle T antigen and pp 60c-src: A possible phosphatidylinositol kinase, Cell 50:1031 (1987).PubMedCrossRefGoogle Scholar
  13. 13.
    D.R. Kaplan, M. Whitman, B. Schaffhausen, D.C. Pallas, M. White, L. Cantley and T.M. Roberts, Common elements in growth factor stimulation and oncogenic transformation: 85 kd phosphoprotein and phosphatidylinositol kinase activity, Cell 50:1021 (1987).PubMedCrossRefGoogle Scholar
  14. 14.
    C.J. Molloy, D.P. Bottaro, T.P. Fleming, M.S. Marshall, J.B. Gibbs and S.A. Aaronson, PDGF induction of tyrosine phosphorylation of GTPase activating protein, Nature 342:711 (1989).PubMedCrossRefGoogle Scholar
  15. 15.
    C. Ellis, M. Moran, F. McCormick and T. Pawson, Phosphorylation of GAP and GAP-associated proteins by transforming and mitogenic tyrosine kinases, Nature 343:377 (1990).PubMedCrossRefGoogle Scholar
  16. 16.
    A.J. Rossomando, D.M. Payne, M.J. Weber and T.W. Sturgill, Evidence that pp42, a major tyrosine kinase target protein, is a mitogen-activated serine/threonine protein kinase, Proc. Natl. Acad. Sci. USA. 86:6940 (1989).PubMedCrossRefGoogle Scholar
  17. 17.
    D.K. Morrison, D.R. Kaplan, U. Rapp and T.M. Roberts, Signal transduction from membrane to cytoplasm: Growth factors and membrane-bound oncogene products increase Raf-1 phosphorylation and associated protein kinase activity, Proc. Natl. Acad. Sci. USA. 85:8855 (1988).PubMedCrossRefGoogle Scholar
  18. 18.
    A.O. Morla, G. Draetta, D. Beach and J.Y.J. Wang, Reversible tyrosine phosphorylation of cdc2: dephosphorylation accompanies activation during entry into mitosis, Cell 58: 193 (1989).PubMedCrossRefGoogle Scholar
  19. 19.
    K.L. Gould and P. Nurse, Tyrosine phosphorylation of the fission yeast cdc2+ protein kinase regulates entry into mitosis, Nature, 342:39 (1989).PubMedCrossRefGoogle Scholar
  20. 20.
    B. Margolis, A. Zilberstein, C. Franks, S. Felder, S. Kremer, A. Ullrich, S.G. Rhee, K. Skorecki, J. Schlessinger, Effect of phospholipase C-γ overexpression on PDGF-induced second messengers and mitogenesis, Science 248:607 (1990).PubMedCrossRefGoogle Scholar
  21. 21.
    D.K. Morrison, D.R. Kaplan, J.A. Escobedo, U.R. Rapp, T.M. Roberts and L.T. Williams, Direct activation of the serine/threonine kinase activity of Raf-1 through tyrosine phosphorylation by the PDGF β-receptor, Cell 58:649 (1989).PubMedCrossRefGoogle Scholar
  22. 22.
    N.G. Anderson, J.L. Mailer, N.K. Tonks and T.W. Sturgill, Requirement for integration of signals from two distinct phosphorylation pathways for activation of MAP kinase, Nature 343:651 (1990).PubMedCrossRefGoogle Scholar
  23. 23.
    K. Yonezawa, G. Endemann, K.S. Kovacina, J.E. Chin, C. Stover and R.A. Roth, Substrates of the insulin receptor kinase, in: “The Biology and Medicine of Signal Transduction,” Y. Nishizuka et al., Publisher, New York (1990).Google Scholar
  24. 24.
    P.J. Blackshear, D. McNeill Haupt, H. App and U.R. Rapp, Insulin activates the Raf-1 protein kinase, J. Biol. Chem. 265:12131 (1990).PubMedGoogle Scholar
  25. 25.
    K.S. Kovacina, K. Yonezawa, D.L. Brautigan, N.K. Tonks, U.R. Rapp and R.A. Roth, Insulin activates the kinase activity of the Raf-1 proto-oncogene by increasing its serine phosphorylation, J. Biol. Chem. 265:12115 (1990).PubMedGoogle Scholar
  26. 26.
    G. Endemann, K. Yonezawa and R.A. Roth, Phosphatidylinositol kinase or an associated protein is a substrate for the insulin receptor tyrosine kinase, J. Biol. Chem., 265:396 (1990).PubMedGoogle Scholar
  27. 27.
    N.B. Ruderman, R. Kapeller, M.F. White and L.C. Cantley, Activation of phosphatidylinositol 3-kinase by insulin, Proc. Natl. Acad. Sci. USA. 87:1411 (1990).PubMedCrossRefGoogle Scholar
  28. 28.
    G. Steele-Perkins and R.A. Roth, Monoclonal antibody aIR-3 inhibits the ability of insulin-like growth factor II to stimulate a signal from the type I receptor without inhibiting its binding, Biochem. Biophvs. Res. Comm., in press.Google Scholar
  29. 29.
    O.P. Pignataro and M. Ascoli, Mol. Endocrinol. 4:758 (1990).PubMedCrossRefGoogle Scholar
  30. 30.
    R.A. Roth, G. Steele-Perkins, J. Hari, C. Stover, S. Pierce, J. Turner, J.C. Edman and W.J. Rutter, Insulin and insulin-like growth factor receptors and responses, Cold Spring Harbor Svmp., LIII:537 (1988).CrossRefGoogle Scholar
  31. 31.
    L. Ellis, E. Clauser, D.O. Morgan, M. Edery, R.A. Roth and W.J. Rutter, Replacement of insulin receptor tyrosine residues 1162 and 1163 compromises insulin-stimulated kinase activity and uptake of 2-deoxyglucose, Cell. 45:721 (1986).PubMedCrossRefGoogle Scholar
  32. 32.
    D.M. Hawley, B.A. Maddux, R.G. Patel, K-Y. Wong, P.W. Mamula, G.L. Firestone, A.Brunetti, E. Verspohl and I.D. Goldfine, Insulin receptor monoclonal antibodies that mimic insulin action without activating tyrosine kinase, J. Biol. Chem., 264:2438 (1989).PubMedGoogle Scholar
  33. 33.
    J. Whittaker, A.K. Okamoto, R. Thys, G.I. Bell, D.F. Steiner and C.A. Hofmann, High-level expression of human insulin receptor cDNA in mouse NIH 3T3 cells, Proc. Natl. Acad. Sci. USA 84:5237 (1987).PubMedCrossRefGoogle Scholar
  34. 34.
    G. Swarup, S. Cohen, D.L. Garbers, Inhibition of membrane phosphotyrosyl protein phosphatase activity by vanadate, Biochem. Biophys. Res. Commun.. 107:1104 (1982).PubMedCrossRefGoogle Scholar
  35. 35.
    J. Avruch, H.E. Tornqvist, J.R. Gunsalus, E.J. Yurkow, Insulin regulation of protein phosphorylation, im “Insulin,” P. Cuatrecasas and S. Jacobs, ed., Springer-Verlag Berlin Heidelberg (1990).Google Scholar
  36. 36.
    R.E. Lewis, L. Cao, D. Perregaux and M.P. Czech, Threonine 1336 of the human insulin receptor is a major target for phosphorylation by protein kinase C, Biochemistry. 29: 1807 (1990).PubMedCrossRefGoogle Scholar
  37. 37.
    R.S. Thies, A. Ullrich and D.A. McClain, Augmented mitogenesis and impaired metabolic signaling mediated by a truncated insulin receptor, J. Biol. Chem. 264:12820 (1989).PubMedGoogle Scholar
  38. 38.
    K. Yonezawa and R.A. Roth, Assessment of the in situ tyrosine kinase activity of mutant insulin receptors lacking tyrosine autophosphorylation sites 1162 and 1163, Mol. Endocrinol., Submitted (1990).Google Scholar
  39. 39.
    C. Reynet, M. Caron, J. Magre, G. Cherqui, E. Clauser, J. Picard and J. Capeau, Mutation of tyrosine residues 1162 and 1163 of the insulin receptor affects hormone and receptor internalization, Mol. Endocrinol., 304 (1990).Google Scholar
  40. 40.
    A. Debant, E. Clauser, G. Ponzio, C. Filloux, C. Auzan, J.O. Contreres, B. Rossi, Replacement of insulin receptor tyrosine residues 1162 and 1163 does not alter the mitogenic effect of the hormone, Proc. Natl. Acad. Sci. USA. 85:8032 (1988).PubMedCrossRefGoogle Scholar

Copyright information

© Plenum Press, New York 1991

Authors and Affiliations

  • Kazuyoshi Yonezawa
    • 1
  • Sarah Pierce
    • 1
  • Cynthia Stover
    • 1
  • Martine Aggerbeck
    • 2
  • William J. Rutter
    • 2
  • Richard A. Roth
    • 1
  1. 1.Department of PharmacologyStanford University School of MedicineStanfordUSA
  2. 2.Hormone Research Institute and Department of Biochemistry and BiophysicsUniversity of California, San FranciscoSan FranciscoUSA

Personalised recommendations