Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1996 Aug;16(8):4147–4155. doi: 10.1128/mcb.16.8.4147

YMXM motifs and signaling by an insulin receptor substrate 1 molecule without tyrosine phosphorylation sites.

M G Myers Jr 1, Y Zhang 1, G A Aldaz 1, T Grammer 1, E M Glasheen 1, L Yenush 1, L M Wang 1, X J Sun 1, J Blenis 1, J H Pierce 1, M F White 1
PMCID: PMC231411  PMID: 8754813

Abstract

Tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1) by the activated receptors for insulin, IGF-1, and various cytokines creates binding sites for signaling proteins with Src homology 2 domains (SH2 proteins). Determining the role of specific SH2 proteins during insulin signaling has been difficult because IRS-1 possesses as many as 18 potential tyrosine phosphorylation sites, several of which contain redundant motifs. Using 32D cells, which contain no endogenous IRS proteins, we compared the signaling ability of an IRS-1 molecule in which 18 potential tyrosine phosphorylation sites were replaced by phenylalanine (IRS-1(F18)) with two derivative molecules which retained three YMXM motifs (IRS-1(3YMXM)) or the two COOH-terminal SHP2-Fyn binding sites (IRS-1(YCT)). During insulin stimulation, IRS-1(F18) failed to undergo tyrosine phosphorylation or mediate activation of the phosphotidylinositol (PI) 3'-kinase or p70(s6k); IRS-1(YCT) was tyrosine phosphorylated but also failed to mediate these signaling events. Neither IRS-1(3YMXM) nor IRS-1(YCT) mediated activation of mitogen-activated protein kinases. IRS-1(F18) and IRS-1(YCT) partially mediated similar levels of insulin-stimulated mitogenesis at high insulin concentrations, however, suggesting that IRS-1 contains phosphotyrosine-independent elements which effect mitogenic signals, and that the sites in IRS-l(YCT) do not augment this signal. IRS-1(3YMXM) mediated the maximal mitogenic response to insulin, although the response to insulin was more sensitive with wild-type IRS-1. By contrast, the association of IRS-1(3YMXM) with PI 3'-kinase was more sensitive to insulin than the association with IRS-1. Thus, the binding of SH2 proteins (such as PI 3'-kinase) by YMXM motifs in IRS-1 is an important element in the mitogenic response, but other elements are essential for full mitogenic sensitivity.

Full Text

The Full Text of this article is available as a PDF (603.8 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Araki E., Lipes M. A., Patti M. E., Brüning J. C., Haag B., 3rd, Johnson R. S., Kahn C. R. Alternative pathway of insulin signalling in mice with targeted disruption of the IRS-1 gene. Nature. 1994 Nov 10;372(6502):186–190. doi: 10.1038/372186a0. [DOI] [PubMed] [Google Scholar]
  2. Backer J. M., Myers M. G., Jr, Shoelson S. E., Chin D. J., Sun X. J., Miralpeix M., Hu P., Margolis B., Skolnik E. Y., Schlessinger J. Phosphatidylinositol 3'-kinase is activated by association with IRS-1 during insulin stimulation. EMBO J. 1992 Sep;11(9):3469–3479. doi: 10.1002/j.1460-2075.1992.tb05426.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Carpenter C. L., Auger K. R., Duckworth B. C., Hou W. M., Schaffhausen B., Cantley L. C. A tightly associated serine/threonine protein kinase regulates phosphoinositide 3-kinase activity. Mol Cell Biol. 1993 Mar;13(3):1657–1665. doi: 10.1128/mcb.13.3.1657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cheatham B., Vlahos C. J., Cheatham L., Wang L., Blenis J., Kahn C. R. Phosphatidylinositol 3-kinase activation is required for insulin stimulation of pp70 S6 kinase, DNA synthesis, and glucose transporter translocation. Mol Cell Biol. 1994 Jul;14(7):4902–4911. doi: 10.1128/mcb.14.7.4902. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chen R. H., Abate C., Blenis J. Phosphorylation of the c-Fos transrepression domain by mitogen-activated protein kinase and 90-kDa ribosomal S6 kinase. Proc Natl Acad Sci U S A. 1993 Dec 1;90(23):10952–10956. doi: 10.1073/pnas.90.23.10952. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chung J., Grammer T. C., Lemon K. P., Kazlauskas A., Blenis J. PDGF- and insulin-dependent pp70S6k activation mediated by phosphatidylinositol-3-OH kinase. Nature. 1994 Jul 7;370(6484):71–75. doi: 10.1038/370071a0. [DOI] [PubMed] [Google Scholar]
  7. Chung J., Kuo C. J., Crabtree G. R., Blenis J. Rapamycin-FKBP specifically blocks growth-dependent activation of and signaling by the 70 kd S6 protein kinases. Cell. 1992 Jun 26;69(7):1227–1236. doi: 10.1016/0092-8674(92)90643-q. [DOI] [PubMed] [Google Scholar]
  8. Dhand R., Hiles I., Panayotou G., Roche S., Fry M. J., Gout I., Totty N. F., Truong O., Vicendo P., Yonezawa K. PI 3-kinase is a dual specificity enzyme: autoregulation by an intrinsic protein-serine kinase activity. EMBO J. 1994 Feb 1;13(3):522–533. doi: 10.1002/j.1460-2075.1994.tb06290.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Escobedo J. A., Kaplan D. R., Kavanaugh W. M., Turck C. W., Williams L. T. A phosphatidylinositol-3 kinase binds to platelet-derived growth factor receptors through a specific receptor sequence containing phosphotyrosine. Mol Cell Biol. 1991 Feb;11(2):1125–1132. doi: 10.1128/mcb.11.2.1125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fantl W. J., Escobedo J. A., Martin G. A., Turck C. W., del Rosario M., McCormick F., Williams L. T. Distinct phosphotyrosines on a growth factor receptor bind to specific molecules that mediate different signaling pathways. Cell. 1992 May 1;69(3):413–423. doi: 10.1016/0092-8674(92)90444-h. [DOI] [PubMed] [Google Scholar]
  11. Fingar D. C., Hausdorff S. F., Blenis J., Birnbaum M. J. Dissociation of pp70 ribosomal protein S6 kinase from insulin-stimulated glucose transport in 3T3-L1 adipocytes. J Biol Chem. 1993 Feb 5;268(4):3005–3008. [PubMed] [Google Scholar]
  12. Hiles I. D., Otsu M., Volinia S., Fry M. J., Gout I., Dhand R., Panayotou G., Ruiz-Larrea F., Thompson A., Totty N. F. Phosphatidylinositol 3-kinase: structure and expression of the 110 kd catalytic subunit. Cell. 1992 Aug 7;70(3):419–429. doi: 10.1016/0092-8674(92)90166-a. [DOI] [PubMed] [Google Scholar]
  13. Joly M., Kazlauskas A., Fay F. S., Corvera S. Disruption of PDGF receptor trafficking by mutation of its PI-3 kinase binding sites. Science. 1994 Feb 4;263(5147):684–687. doi: 10.1126/science.8303278. [DOI] [PubMed] [Google Scholar]
  14. Kapeller R., Cantley L. C. Phosphatidylinositol 3-kinase. Bioessays. 1994 Aug;16(8):565–576. doi: 10.1002/bies.950160810. [DOI] [PubMed] [Google Scholar]
  15. Koch C. A., Anderson D., Moran M. F., Ellis C., Pawson T. SH2 and SH3 domains: elements that control interactions of cytoplasmic signaling proteins. Science. 1991 May 3;252(5006):668–674. doi: 10.1126/science.1708916. [DOI] [PubMed] [Google Scholar]
  16. Kundra V., Escobedo J. A., Kazlauskas A., Kim H. K., Rhee S. G., Williams L. T., Zetter B. R. Regulation of chemotaxis by the platelet-derived growth factor receptor-beta. Nature. 1994 Feb 3;367(6462):474–476. doi: 10.1038/367474a0. [DOI] [PubMed] [Google Scholar]
  17. Lam K., Carpenter C. L., Ruderman N. B., Friel J. C., Kelly K. L. The phosphatidylinositol 3-kinase serine kinase phosphorylates IRS-1. Stimulation by insulin and inhibition by Wortmannin. J Biol Chem. 1994 Aug 12;269(32):20648–20652. [PubMed] [Google Scholar]
  18. Miralpeix M., Sun X. J., Backer J. M., Myers M. G., Jr, Araki E., White M. F. Insulin stimulates tyrosine phosphorylation of multiple high molecular weight substrates in Fao hepatoma cells. Biochemistry. 1992 Sep 22;31(37):9031–9039. doi: 10.1021/bi00152a046. [DOI] [PubMed] [Google Scholar]
  19. Myers M. G., Jr, Backer J. M., Sun X. J., Shoelson S., Hu P., Schlessinger J., Yoakim M., Schaffhausen B., White M. F. IRS-1 activates phosphatidylinositol 3'-kinase by associating with src homology 2 domains of p85. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10350–10354. doi: 10.1073/pnas.89.21.10350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Myers M. G., Jr, Grammer T. C., Brooks J., Glasheen E. M., Wang L. M., Sun X. J., Blenis J., Pierce J. H., White M. F. The pleckstrin homology domain in insulin receptor substrate-1 sensitizes insulin signaling. J Biol Chem. 1995 May 19;270(20):11715–11718. doi: 10.1074/jbc.270.20.11715. [DOI] [PubMed] [Google Scholar]
  21. Myers M. G., Jr, Grammer T. C., Wang L. M., Sun X. J., Pierce J. H., Blenis J., White M. F. Insulin receptor substrate-1 mediates phosphatidylinositol 3'-kinase and p70S6k signaling during insulin, insulin-like growth factor-1, and interleukin-4 stimulation. J Biol Chem. 1994 Nov 18;269(46):28783–28789. [PubMed] [Google Scholar]
  22. 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]
  23. Myers M. G., Jr, Wang L. M., Sun X. J., Zhang Y., Yenush L., Schlessinger J., Pierce J. H., White M. F. Role of IRS-1-GRB-2 complexes in insulin signaling. Mol Cell Biol. 1994 Jun;14(6):3577–3587. doi: 10.1128/mcb.14.6.3577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Myers M. G., Jr, White M. F. The new elements of insulin signaling. Insulin receptor substrate-1 and proteins with SH2 domains. Diabetes. 1993 May;42(5):643–650. doi: 10.2337/diab.42.5.643. [DOI] [PubMed] [Google Scholar]
  25. Otsu M., Hiles I., Gout I., Fry M. J., Ruiz-Larrea F., Panayotou G., Thompson A., Dhand R., Hsuan J., Totty N. Characterization of two 85 kd proteins that associate with receptor tyrosine kinases, middle-T/pp60c-src complexes, and PI3-kinase. Cell. 1991 Apr 5;65(1):91–104. doi: 10.1016/0092-8674(91)90411-q. [DOI] [PubMed] [Google Scholar]
  26. Pluskey S., Wandless T. J., Walsh C. T., Shoelson S. E. Potent stimulation of SH-PTP2 phosphatase activity by simultaneous occupancy of both SH2 domains. J Biol Chem. 1995 Feb 17;270(7):2897–2900. doi: 10.1074/jbc.270.7.2897. [DOI] [PubMed] [Google Scholar]
  27. Pons S., Asano T., Glasheen E., Miralpeix M., Zhang Y., Fisher T. L., Myers M. G., Jr, Sun X. J., White M. F. The structure and function of p55PIK reveal a new regulatory subunit for phosphatidylinositol 3-kinase. Mol Cell Biol. 1995 Aug;15(8):4453–4465. doi: 10.1128/mcb.15.8.4453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Pronk G. J., McGlade J., Pelicci G., Pawson T., Bos J. L. Insulin-induced phosphorylation of the 46- and 52-kDa Shc proteins. J Biol Chem. 1993 Mar 15;268(8):5748–5753. [PubMed] [Google Scholar]
  29. Rameh L. E., Chen C. S., Cantley L. C. Phosphatidylinositol (3,4,5)P3 interacts with SH2 domains and modulates PI 3-kinase association with tyrosine-phosphorylated proteins. Cell. 1995 Dec 1;83(5):821–830. doi: 10.1016/0092-8674(95)90195-7. [DOI] [PubMed] [Google Scholar]
  30. Rordorf-Nikolic T., Van Horn D. J., Chen D., White M. F., Backer J. M. Regulation of phosphatidylinositol 3'-kinase by tyrosyl phosphoproteins. Full activation requires occupancy of both SH2 domains in the 85-kDa regulatory subunit. J Biol Chem. 1995 Feb 24;270(8):3662–3666. doi: 10.1074/jbc.270.8.3662. [DOI] [PubMed] [Google Scholar]
  31. Rosen O. M. After insulin binds. Science. 1987 Sep 18;237(4821):1452–1458. doi: 10.1126/science.2442814. [DOI] [PubMed] [Google Scholar]
  32. Ruderman N. B., Kapeller R., White M. F., Cantley L. C. Activation of phosphatidylinositol 3-kinase by insulin. Proc Natl Acad Sci U S A. 1990 Feb;87(4):1411–1415. doi: 10.1073/pnas.87.4.1411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Schu P. V., Takegawa K., Fry M. J., Stack J. H., Waterfield M. D., Emr S. D. Phosphatidylinositol 3-kinase encoded by yeast VPS34 gene essential for protein sorting. Science. 1993 Apr 2;260(5104):88–91. doi: 10.1126/science.8385367. [DOI] [PubMed] [Google Scholar]
  34. Skolnik E. Y., Lee C. H., Batzer A., Vicentini L. M., Zhou M., Daly R., Myers M. J., Jr, Backer J. M., Ullrich A., White M. F. The SH2/SH3 domain-containing protein GRB2 interacts with tyrosine-phosphorylated IRS1 and Shc: implications for insulin control of ras signalling. EMBO J. 1993 May;12(5):1929–1936. doi: 10.1002/j.1460-2075.1993.tb05842.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Songyang Z., Shoelson S. E., Chaudhuri M., Gish G., Pawson T., Haser W. G., King F., Roberts T., Ratnofsky S., Lechleider R. J. SH2 domains recognize specific phosphopeptide sequences. Cell. 1993 Mar 12;72(5):767–778. doi: 10.1016/0092-8674(93)90404-e. [DOI] [PubMed] [Google Scholar]
  36. Sugimoto S., Wandless T. J., Shoelson S. E., Neel B. G., Walsh C. T. Activation of the SH2-containing protein tyrosine phosphatase, SH-PTP2, by phosphotyrosine-containing peptides derived from insulin receptor substrate-1. J Biol Chem. 1994 May 6;269(18):13614–13622. [PubMed] [Google Scholar]
  37. Sun X. J., Crimmins D. L., Myers M. G., Jr, Miralpeix M., White M. F. Pleiotropic insulin signals are engaged by multisite phosphorylation of IRS-1. Mol Cell Biol. 1993 Dec;13(12):7418–7428. doi: 10.1128/mcb.13.12.7418. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sun X. J., Miralpeix M., Myers M. G., Jr, Glasheen E. M., Backer J. M., Kahn C. R., White M. F. Expression and function of IRS-1 in insulin signal transmission. J Biol Chem. 1992 Nov 5;267(31):22662–22672. [PubMed] [Google Scholar]
  39. Sun X. J., Pons S., Asano T., Myers M. G., Jr, Glasheen E., White M. F. The Fyn tyrosine kinase binds Irs-1 and forms a distinct signaling complex during insulin stimulation. J Biol Chem. 1996 May 3;271(18):10583–10587. doi: 10.1074/jbc.271.18.10583. [DOI] [PubMed] [Google Scholar]
  40. 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. Structure of the insulin receptor substrate IRS-1 defines a unique signal transduction protein. Nature. 1991 Jul 4;352(6330):73–77. doi: 10.1038/352073a0. [DOI] [PubMed] [Google Scholar]
  41. Sun X. J., Wang L. M., Zhang Y., Yenush L., Myers M. G., Jr, Glasheen E., Lane W. S., Pierce J. H., White M. F. Role of IRS-2 in insulin and cytokine signalling. Nature. 1995 Sep 14;377(6545):173–177. doi: 10.1038/377173a0. [DOI] [PubMed] [Google Scholar]
  42. Tamemoto H., Kadowaki T., Tobe K., Yagi T., Sakura H., Hayakawa T., Terauchi Y., Ueki K., Kaburagi Y., Satoh S. Insulin resistance and growth retardation in mice lacking insulin receptor substrate-1. Nature. 1994 Nov 10;372(6502):182–186. doi: 10.1038/372182a0. [DOI] [PubMed] [Google Scholar]
  43. Toker A., Meyer M., Reddy K. K., Falck J. R., Aneja R., Aneja S., Parra A., Burns D. J., Ballas L. M., Cantley L. C. Activation of protein kinase C family members by the novel polyphosphoinositides PtdIns-3,4-P2 and PtdIns-3,4,5-P3. J Biol Chem. 1994 Dec 23;269(51):32358–32367. [PubMed] [Google Scholar]
  44. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Uddin S., Katzav S., White M. F., Platanias L. C. Insulin-dependent tyrosine phosphorylation of the vav protooncogene product in cells of hematopoietic origin. J Biol Chem. 1995 Mar 31;270(13):7712–7716. doi: 10.1074/jbc.270.13.7712. [DOI] [PubMed] [Google Scholar]
  46. Uddin S., Yenush L., Sun X. J., Sweet M. E., White M. F., Platanias L. C. Interferon-alpha engages the insulin receptor substrate-1 to associate with the phosphatidylinositol 3'-kinase. J Biol Chem. 1995 Jul 7;270(27):15938–15941. doi: 10.1074/jbc.270.27.15938. [DOI] [PubMed] [Google Scholar]
  47. Wang L. M., Myers M. G., Jr, Sun X. J., Aaronson S. A., White M., Pierce J. H. IRS-1: essential for insulin- and IL-4-stimulated mitogenesis in hematopoietic cells. Science. 1993 Sep 17;261(5128):1591–1594. doi: 10.1126/science.8372354. [DOI] [PubMed] [Google Scholar]
  48. White M. F., Backer J. M. Preparation and use of anti-phosphotyrosine antibodies to study structure and function of insulin receptor. Methods Enzymol. 1991;201:65–79. doi: 10.1016/0076-6879(91)01009-q. [DOI] [PubMed] [Google Scholar]
  49. White M. F., Maron R., Kahn C. R. Insulin rapidly stimulates tyrosine phosphorylation of a Mr-185,000 protein in intact cells. Nature. 1985 Nov 14;318(6042):183–186. doi: 10.1038/318183a0. [DOI] [PubMed] [Google Scholar]
  50. Yenush L., Fernandez R., Myers M. G., Jr, Grammer T. C., Sun X. J., Blenis J., Pierce J. H., Schlessinger J., White M. F. The Drosophila insulin receptor activates multiple signaling pathways but requires insulin receptor substrate proteins for DNA synthesis. Mol Cell Biol. 1996 May;16(5):2509–2517. doi: 10.1128/mcb.16.5.2509. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES