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. 1996 Feb 1;97(3):613–620. doi: 10.1172/JCI118457

Glucose-induced phosphorylation of the insulin receptor. Functional effects and characterization of phosphorylation sites.

T S Pillay 1, S Xiao 1, J M Olefsky 1
PMCID: PMC507096  PMID: 8609215

Abstract

Elevated glucose concentrations have been reported to inhibit insulin receptor kinase activity. We studied the effects of high glucose on insulin action in Rat1 fibroblasts transfected with wild-type human insulin receptor (HIRcB) and a truncated receptor lacking the COOH-terminal 43 amino acids (delta CT). In both cell lines, 25 mM glucose impaired receptor and insulin receptor substrate-1 phosphorylation by 34%, but IGF-1 receptor phosphorylation was unaffected. Phosphatidylinositol 3-kinase activity and bromodeoxyuridine uptake were decreased by 85 and 35%, respectively. This was reversed by coincubation with a protein kinase C (PKC) inhibitor or microinjection of a PKC inhibitor peptide. Phosphopeptide mapping revealed that high glucose or PMA led to serine/threonine phosphorylation of similar peptides. Inhibition of the microtubule-associated protein (MAP) kinase cascade by the MAP kinase kinase inhibitor PD98059 did not reverse the impaired phosphorylation. We conclude that high glucose inhibits insulin action by inducing serine phosphorylation through a PKC-mediated mechanism at the level of the receptor at sites proximal to the COOH-terminal 43 amino acids. This effect is independent of activation of the MAP kinase cascade. Proportionately, the impairment of insulin receptor substrate-1 tyrosine phosphorylation is greater than that of the insulin receptor resulting in attenuated phosphatidylinositol 3-kinase activation and mitogenic signaling.

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Selected References

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  1. Ahn J., Donner D. B., Rosen O. M. Interaction of the human insulin receptor tyrosine kinase from the baculovirus expression system with protein kinase C in a cell-free system. J Biol Chem. 1993 Apr 5;268(10):7571–7576. [PubMed] [Google Scholar]
  2. Berti L., Mosthaf L., Kroder G., Kellerer M., Tippmer S., Mushack J., Seffer E., Seedorf K., Häring H. Glucose-induced translocation of protein kinase C isoforms in rat-1 fibroblasts is paralleled by inhibition of the insulin receptor tyrosine kinase. J Biol Chem. 1994 Feb 4;269(5):3381–3386. [PubMed] [Google Scholar]
  3. Bollag G. E., Roth R. A., Beaudoin J., Mochly-Rosen D., Koshland D. E., Jr Protein kinase C directly phosphorylates the insulin receptor in vitro and reduces its protein-tyrosine kinase activity. Proc Natl Acad Sci U S A. 1986 Aug;83(16):5822–5824. doi: 10.1073/pnas.83.16.5822. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Campbell J. S., Seger R., Graves J. D., Graves L. M., Jensen A. M., Krebs E. G. The MAP kinase cascade. Recent Prog Horm Res. 1995;50:131–159. doi: 10.1016/b978-0-12-571150-0.50011-1. [DOI] [PubMed] [Google Scholar]
  5. Chin J. E., Dickens M., Tavare J. M., Roth R. A. Overexpression of protein kinase C isoenzymes alpha, beta I, gamma, and epsilon in cells overexpressing the insulin receptor. Effects on receptor phosphorylation and signaling. J Biol Chem. 1993 Mar 25;268(9):6338–6347. [PubMed] [Google Scholar]
  6. Chin J. E., Liu F., Roth R. A. Activation of protein kinase C alpha inhibits insulin-stimulated tyrosine phosphorylation of insulin receptor substrate-1. Mol Endocrinol. 1994 Jan;8(1):51–58. doi: 10.1210/mend.8.1.7512195. [DOI] [PubMed] [Google Scholar]
  7. Coghlan M. P., Pillay T. S., Tavaré J. M., Siddle K. Site-specific anti-phosphopeptide antibodies: use in assessing insulin receptor serine/threonine phosphorylation state and identification of serine-1327 as a novel site of phorbol ester-induced phosphorylation. Biochem J. 1994 Nov 1;303(Pt 3):893–899. doi: 10.1042/bj3030893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cosio F. G. Effects of high glucose concentrations on human mesangial cell proliferation. J Am Soc Nephrol. 1995 Feb;5(8):1600–1609. doi: 10.1681/ASN.V581600. [DOI] [PubMed] [Google Scholar]
  9. Ebina Y., Araki E., Taira M., Shimada F., Mori M., Craik C. S., Siddle K., Pierce S. B., Roth R. A., Rutter W. J. Replacement of lysine residue 1030 in the putative ATP-binding region of the insulin receptor abolishes insulin- and antibody-stimulated glucose uptake and receptor kinase activity. Proc Natl Acad Sci U S A. 1987 Feb;84(3):704–708. doi: 10.1073/pnas.84.3.704. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ebina Y., Ellis L., Jarnagin K., Edery M., Graf L., Clauser E., Ou J. H., Masiarz F., Kan Y. W., Goldfine I. D. The human insulin receptor cDNA: the structural basis for hormone-activated transmembrane signalling. Cell. 1985 Apr;40(4):747–758. doi: 10.1016/0092-8674(85)90334-4. [DOI] [PubMed] [Google Scholar]
  11. Feener E. P., Shiba T., Hu K. Q., Wilden P. A., White M. F., King G. L. Characterization of phorbol ester-stimulated serine phosphorylation of the human insulin receptor. Biochem J. 1994 Oct 1;303(Pt 1):43–50. doi: 10.1042/bj3030043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gustafson T. A., He W., Craparo A., Schaub C. D., O'Neill T. J. Phosphotyrosine-dependent interaction of SHC and insulin receptor substrate 1 with the NPEY motif of the insulin receptor via a novel non-SH2 domain. Mol Cell Biol. 1995 May;15(5):2500–2508. doi: 10.1128/mcb.15.5.2500. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Häring H., Kirsch D., Obermaier B., Ermel B., Machicao F. Tumor-promoting phorbol esters increase the Km of the ATP-binding site of the insulin receptor kinase from rat adipocytes. J Biol Chem. 1986 Mar 15;261(8):3869–3875. [PubMed] [Google Scholar]
  14. Ide R., Maegawa H., Kikkawa R., Shigeta Y., Kashiwagi A. High glucose condition activates protein tyrosine phosphatases and deactivates insulin receptor function in insulin-sensitive rat 1 fibroblasts. Biochem Biophys Res Commun. 1994 May 30;201(1):71–77. doi: 10.1006/bbrc.1994.1670. [DOI] [PubMed] [Google Scholar]
  15. Inoguchi T., Xia P., Kunisaki M., Higashi S., Feener E. P., King G. L. Insulin's effect on protein kinase C and diacylglycerol induced by diabetes and glucose in vascular tissues. Am J Physiol. 1994 Sep;267(3 Pt 1):E369–E379. doi: 10.1152/ajpendo.1994.267.3.E369. [DOI] [PubMed] [Google Scholar]
  16. Kellerer M., Coghlan M., Capp E., Mühlhöfer A., Kroder G., Mosthaf L., Galante P., Siddle K., Häring H. U. Mechanism of insulin receptor kinase inhibition in non-insulin-dependent diabetes mellitus patients. Phosphorylation of serine 1327 or threonine 1348 is unaltered. J Clin Invest. 1995 Jul;96(1):6–11. doi: 10.1172/JCI118073. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kikkawa R., Haneda M., Uzu T., Koya D., Sugimoto T., Shigeta Y. Translocation of protein kinase C alpha and zeta in rat glomerular mesangial cells cultured under high glucose conditions. Diabetologia. 1994 Aug;37(8):838–841. doi: 10.1007/BF00404342. [DOI] [PubMed] [Google Scholar]
  18. L'Allemain G. Deciphering the MAP kinase pathway. Prog Growth Factor Res. 1994;5(3):291–334. doi: 10.1016/0955-2235(94)90011-6. [DOI] [PubMed] [Google Scholar]
  19. Lewis R. E., Cao L., Perregaux D., Czech M. P. Threonine 1336 of the human insulin receptor is a major target for phosphorylation by protein kinase C. Biochemistry. 1990 Feb 20;29(7):1807–1813. doi: 10.1021/bi00459a020. [DOI] [PubMed] [Google Scholar]
  20. Lewis R. E., Volle D. J., Sanderson S. D. Phorbol ester stimulates phosphorylation on serine 1327 of the human insulin receptor. J Biol Chem. 1994 Oct 21;269(42):26259–26266. [PubMed] [Google Scholar]
  21. Lewis R. E., Wu G. P., MacDonald R. G., Czech M. P. Insulin-sensitive phosphorylation of serine 1293/1294 on the human insulin receptor by a tightly associated serine kinase. J Biol Chem. 1990 Jan 15;265(2):947–954. [PubMed] [Google Scholar]
  22. Liu F., Roth R. A. Identification of serines-1035/1037 in the kinase domain of the insulin receptor as protein kinase C alpha mediated phosphorylation sites. FEBS Lett. 1994 Oct 3;352(3):389–392. doi: 10.1016/0014-5793(94)00996-1. [DOI] [PubMed] [Google Scholar]
  23. Liu F., Roth R. A. Identification of serines-967/968 in the juxtamembrane region of the insulin receptor as insulin-stimulated phosphorylation sites. Biochem J. 1994 Mar 1;298(Pt 2):471–477. doi: 10.1042/bj2980471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Maegawa H., McClain D. A., Freidenberg G., Olefsky J. M., Napier M., Lipari T., Dull T. J., Lee J., Ullrich A. Properties of a human insulin receptor with a COOH-terminal truncation. II. Truncated receptors have normal kinase activity but are defective in signaling metabolic effects. J Biol Chem. 1988 Jun 25;263(18):8912–8917. [PubMed] [Google Scholar]
  25. Maegawa H., Olefsky J. M., Thies S., Boyd D., Ullrich A., McClain D. A. Insulin receptors with defective tyrosine kinase inhibit normal receptor function at the level of substrate phosphorylation. J Biol Chem. 1988 Sep 5;263(25):12629–12637. [PubMed] [Google Scholar]
  26. McClain D. A., Maegawa H., Levy J., Huecksteadt T., Dull T. J., Lee J., Ullrich A., Olefsky J. M. Properties of a human insulin receptor with a COOH-terminal truncation. I. Insulin binding, autophosphorylation, and endocytosis. J Biol Chem. 1988 Jun 25;263(18):8904–8911. [PubMed] [Google Scholar]
  27. Morgan S. J., Smith A. D., Parker P. J. Purification and characterization of bovine brain type I phosphatidylinositol kinase. Eur J Biochem. 1990 Aug 17;191(3):761–767. doi: 10.1111/j.1432-1033.1990.tb19185.x. [DOI] [PubMed] [Google Scholar]
  28. Mosthaf L., Berti L., Kellerer M., Mushack J., Seffer E., Bossenmaier B., Coghlan M., Siddle K., Ullrich A., Häring H. U. C-terminus or juxtamembrane deletions in the insulin receptor do not affect the glucose-dependent inhibition of the tyrosine kinase activity. Eur J Biochem. 1995 Feb 1;227(3):787–791. doi: 10.1111/j.1432-1033.1995.tb20202.x. [DOI] [PubMed] [Google Scholar]
  29. Myers M. G., Jr, Sun X. J., White M. F. The IRS-1 signaling system. Trends Biochem Sci. 1994 Jul;19(7):289–293. doi: 10.1016/0968-0004(94)90007-8. [DOI] [PubMed] [Google Scholar]
  30. Müller H. K., Kellerer M., Ermel B., Mühlhöfer A., Obermaier-Kusser B., Vogt B., Häring H. U. Prevention by protein kinase C inhibitors of glucose-induced insulin-receptor tyrosine kinase resistance in rat fat cells. Diabetes. 1991 Nov;40(11):1440–1448. doi: 10.2337/diab.40.11.1440. [DOI] [PubMed] [Google Scholar]
  31. Nishizuka Y. Protein kinase C and lipid signaling for sustained cellular responses. FASEB J. 1995 Apr;9(7):484–496. [PubMed] [Google Scholar]
  32. O'Neill T. J., Craparo A., Gustafson T. A. Characterization of an interaction between insulin receptor substrate 1 and the insulin receptor by using the two-hybrid system. Mol Cell Biol. 1994 Oct;14(10):6433–6442. doi: 10.1128/mcb.14.10.6433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Olefsky J. M., Nolan J. J. Insulin resistance and non-insulin-dependent diabetes mellitus: cellular and molecular mechanisms. Am J Clin Nutr. 1995 Apr;61(4 Suppl):980S–986S. doi: 10.1093/ajcn/61.4.980S. [DOI] [PubMed] [Google Scholar]
  34. Pang L., Sawada T., Decker S. J., Saltiel A. R. Inhibition of MAP kinase kinase blocks the differentiation of PC-12 cells induced by nerve growth factor. J Biol Chem. 1995 Jun 9;270(23):13585–13588. doi: 10.1074/jbc.270.23.13585. [DOI] [PubMed] [Google Scholar]
  35. Pillay T. S., Makgoba M. W. Enhancement of epidermal growth factor (EGF) and insulin-stimulated tyrosine phosphorylation of endogenous substrates by sodium selenate. FEBS Lett. 1992 Aug 10;308(1):38–42. doi: 10.1016/0014-5793(92)81045-n. [DOI] [PubMed] [Google Scholar]
  36. Pillay T. S., Sasaoka T., Olefsky J. M. Insulin stimulates the tyrosine dephosphorylation of pp125 focal adhesion kinase. J Biol Chem. 1995 Jan 20;270(3):991–994. doi: 10.1074/jbc.270.3.991. [DOI] [PubMed] [Google Scholar]
  37. Pillay T. S., Siddle K. Insulin-stimulated serine/threonine phosphorylation of the insulin receptor: paucity of threonine 1348 phosphorylation in vitro indicates the involvement of more than one serine/threonine kinase in vivo. Biochem Biophys Res Commun. 1991 Sep 16;179(2):962–971. doi: 10.1016/0006-291x(91)91912-v. [DOI] [PubMed] [Google Scholar]
  38. Pillay T. S., Whittaker J., Lammers R., Ullrich A., Siddle K. Multisite serine phosphorylation of the insulin and IGF-I receptors in transfected cells. FEBS Lett. 1991 Aug 19;288(1-2):206–211. doi: 10.1016/0014-5793(91)81035-7. [DOI] [PubMed] [Google Scholar]
  39. Roth R. A., Liu F., Chin J. E. Biochemical mechanisms of insulin resistance. Horm Res. 1994;41 (Suppl 2):51–55. doi: 10.1159/000183961. [DOI] [PubMed] [Google Scholar]
  40. Songyang Z., Margolis B., Chaudhuri M., Shoelson S. E., Cantley L. C. The phosphotyrosine interaction domain of SHC recognizes tyrosine-phosphorylated NPXY motif. J Biol Chem. 1995 Jun 23;270(25):14863–14866. doi: 10.1074/jbc.270.25.14863. [DOI] [PubMed] [Google Scholar]
  41. Stabnel S. Protein kinase C--an enzyme and its relatives. Semin Cancer Biol. 1994 Aug;5(4):277–284. [PubMed] [Google Scholar]
  42. Takayama S., White M. F., Kahn C. R. Phorbol ester-induced serine phosphorylation of the insulin receptor decreases its tyrosine kinase activity. J Biol Chem. 1988 Mar 5;263(7):3440–3447. [PubMed] [Google Scholar]
  43. Tavaré J. M., Zhang B., Ellis L., Roth R. A. Insulin-stimulated serine and threonine phosphorylation of the human insulin receptor. An assessment of the role of serines 1305/1306 and threonine 1348 by their replacement with neutral or negatively charged amino acids. J Biol Chem. 1991 Nov 15;266(32):21804–21809. [PubMed] [Google Scholar]
  44. Thies R. S., Ullrich A., McClain D. A. Augmented mitogenesis and impaired metabolic signaling mediated by a truncated insulin receptor. J Biol Chem. 1989 Aug 5;264(22):12820–12825. [PubMed] [Google Scholar]
  45. Toullec D., Pianetti P., Coste H., Bellevergue P., Grand-Perret T., Ajakane M., Baudet V., Boissin P., Boursier E., Loriolle F. The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C. J Biol Chem. 1991 Aug 25;266(24):15771–15781. [PubMed] [Google Scholar]
  46. Ullrich A., Bell J. R., Chen E. Y., Herrera R., Petruzzelli L. M., Dull T. J., Gray A., Coussens L., Liao Y. C., Tsubokawa M. Human insulin receptor and its relationship to the tyrosine kinase family of oncogenes. 1985 Feb 28-Mar 6Nature. 313(6005):756–761. doi: 10.1038/313756a0. [DOI] [PubMed] [Google Scholar]
  47. Ullrich A., Gray A., Tam A. W., Yang-Feng T., Tsubokawa M., Collins C., Henzel W., Le Bon T., Kathuria S., Chen E. Insulin-like growth factor I receptor primary structure: comparison with insulin receptor suggests structural determinants that define functional specificity. EMBO J. 1986 Oct;5(10):2503–2512. doi: 10.1002/j.1460-2075.1986.tb04528.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Wilkinson S. E., Parker P. J., Nixon J. S. Isoenzyme specificity of bisindolylmaleimides, selective inhibitors of protein kinase C. Biochem J. 1993 Sep 1;294(Pt 2):335–337. doi: 10.1042/bj2940335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Yamaguchi K., Ogita K., Nakamura S., Nishizuka Y. The protein kinase C isoforms leading to MAP-kinase activation in CHO cells. Biochem Biophys Res Commun. 1995 May 25;210(3):639–647. doi: 10.1006/bbrc.1995.1708. [DOI] [PubMed] [Google Scholar]

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