Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1989 Oct;86(19):7306–7310. doi: 10.1073/pnas.86.19.7306

In vitro tyrosine phosphorylation studies on RAS proteins and calmodulin suggest that polylysine-like basic peptides or domains may be involved in interactions between insulin receptor kinase and its substrate.

Y Fujita-Yamaguchi 1, S Kathuria 1, Q Y Xu 1, J M McDonald 1, H Nakano 1, T Kamata 1
PMCID: PMC298050  PMID: 2552437

Abstract

We have investigated the in vitro tyrosine phosphorylation of the HRAS and KRAS proteins by human placental insulin receptor kinase. Purified HRAS proteins are not phosphorylated by purified insulin receptor kinase. Since the tyrosine phosphorylation of calmodulin by the insulin receptor kinase in vitro requires cofactors such as protamine and poly(L-lysine), we examined the possibility that poly(L-lysine) may also potentiate the interaction between RAS proteins and the insulin receptor. We found that purified HRAS proteins are indeed phosphorylated by purified insulin receptor kinase in the presence of poly(L-lysine). In contrast, the KRAS protein, which carries an extremely basic domain (residues 172-182, Lys-Asp-Glu-Lys6-Ser-Arg), is phosphorylated by the receptor kinase without the addition of basic proteins. We then determined whether the KRAS basic domain peptide plays a role similar to that of poly(L-lysine) and found that both the HRAS protein and calmodulin are phosphorylated by the receptor kinase in the presence of the KRAS basic domain peptide. Further examination of the role of poly(L-lysine) in potentiating tyrosine phosphorylation of the HRAS protein and calmodulin by purified insulin receptor kinase indicates that poly(L-lysine) affects the conformation of these protein substrates as well as that of the receptor kinase domain. These studies suggest that polylysine-like basic proteins or domains are required to establish the interaction between insulin receptor kinase and its substrate.

Full text

PDF
7306

Images in this article

7307Image
on p.7307
7307Image
on p.7307
7308Image
on p.7308
7308Image
on p.7308

Selected References

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

  1. Barbacid M. ras genes. Annu Rev Biochem. 1987;56:779–827. doi: 10.1146/annurev.bi.56.070187.004023. [DOI] [PubMed] [Google Scholar]
  2. Blackshear P. J., Haupt D. M. Evidence against insulin-stimulated phosphorylation of calmodulin in 3T3-L1 adipocytes. J Biol Chem. 1989 Mar 5;264(7):3854–3858. [PubMed] [Google Scholar]
  3. Casnellie J. E., Harrison M. L., Pike L. J., Hellström K. E., Krebs E. G. Phosphorylation of synthetic peptides by a tyrosine protein kinase from the particulate fraction of a lymphoma cell line. Proc Natl Acad Sci U S A. 1982 Jan;79(2):282–286. doi: 10.1073/pnas.79.2.282. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Colca J. R., DeWald D. B., Pearson J. D., Palazuk B. J., Laurino J. P., McDonald J. M. Insulin stimulates the phosphorylation of calmodulin in intact adipocytes. J Biol Chem. 1987 Aug 25;262(24):11399–11402. [PubMed] [Google Scholar]
  5. Deshpande A. K., Kung H. F. Insulin induction of Xenopus laevis oocyte maturation is inhibited by monoclonal antibody against p21 ras proteins. Mol Cell Biol. 1987 Mar;7(3):1285–1288. doi: 10.1128/mcb.7.3.1285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Fujita-Yamaguchi Y. Characterization of purified insulin receptor subunits. J Biol Chem. 1984 Jan 25;259(2):1206–1211. [PubMed] [Google Scholar]
  8. Fujita-Yamaguchi Y., Choi S., Sakamoto Y., Itakura K. Purification of insulin receptor with full binding activity. J Biol Chem. 1983 Apr 25;258(8):5045–5049. [PubMed] [Google Scholar]
  9. Fujita-Yamaguchi Y., Kathuria S. The monomeric alpha beta form of the insulin receptor exhibits much higher insulin-dependent tyrosine-specific protein kinase activity than the intact alpha 2 beta 2 form of the receptor. Proc Natl Acad Sci U S A. 1985 Sep;82(18):6095–6099. doi: 10.1073/pnas.82.18.6095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gatica M., Allende C. C., Antonelli M., Allende J. E. Polylysine-containing peptides, including the carboxyl-terminal segment of the human c-Ki-ras 2 protein, affect the activity of some key membrane enzymes. Proc Natl Acad Sci U S A. 1987 Jan;84(2):324–328. doi: 10.1073/pnas.84.2.324. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gross M., Sweet R. W., Sathe G., Yokoyama S., Fasano O., Goldfarb M., Wigler M., Rosenberg M. Purification and characterization of human H-ras proteins expressed in Escherichia coli. Mol Cell Biol. 1985 May;5(5):1015–1024. doi: 10.1128/mcb.5.5.1015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Grunfeld C., Shigenaga J. K., Huang B. J., Fujita-Yamaguchi Y., McFarland K. C., Burnier J., Ramachandran J. Identification of the intact insulin receptor using a sequence-specific antibody directed against the C-terminus of the beta-subunit. Endocrinology. 1987 Sep;121(3):948–957. doi: 10.1210/endo-121-3-948. [DOI] [PubMed] [Google Scholar]
  13. Hara M., Tamaoki T., Nakano H. Guanine nucleotide binding properties of purified v-Ki-ras p21 protein produced in Escherichia coli. Oncogene Res. 1988 May;2(4):325–333. [PubMed] [Google Scholar]
  14. Hurley J. B., Simon M. I., Teplow D. B., Robishaw J. D., Gilman A. G. Homologies between signal transducing G proteins and ras gene products. Science. 1984 Nov 16;226(4676):860–862. doi: 10.1126/science.6436980. [DOI] [PubMed] [Google Scholar]
  15. Kamata T., Kathuria S., Fujita-Yamaguchi Y. Insulin stimulates the phosphorylation level of v-Ha-ras protein in membrane fraction. Biochem Biophys Res Commun. 1987 Apr 14;144(1):19–25. doi: 10.1016/s0006-291x(87)80469-2. [DOI] [PubMed] [Google Scholar]
  16. Kasuga M., Fujita-Yamaguchi Y., Blithe D. L., White M. F., Kahn C. R. Characterization of the insulin receptor kinase purified from human placental membranes. J Biol Chem. 1983 Sep 25;258(18):10973–10980. [PubMed] [Google Scholar]
  17. Kathuria S., Hartman S., Grunfeld C., Ramachandran J., Fujita-Yamaguchi Y. Differential sensitivity of two functions of the insulin receptor to the associated proteolysis: kinase action and hormone binding. Proc Natl Acad Sci U S A. 1986 Nov;83(22):8570–8574. doi: 10.1073/pnas.83.22.8570. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Korn L. J., Siebel C. W., McCormick F., Roth R. A. Ras p21 as a potential mediator of insulin action in Xenopus oocytes. Science. 1987 May 15;236(4803):840–843. doi: 10.1126/science.3554510. [DOI] [PubMed] [Google Scholar]
  19. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  20. O'Brien R. M., Siddle K., Houslay M. D., Hall A. Interaction of the human insulin receptor with the ras oncogene product p21. FEBS Lett. 1987 Jun 15;217(2):253–259. doi: 10.1016/0014-5793(87)80673-7. [DOI] [PubMed] [Google Scholar]
  21. Sacks D. B., McDonald J. M. Insulin-stimulated phosphorylation of calmodulin by rat liver insulin receptor preparations. J Biol Chem. 1988 Feb 15;263(5):2377–2383. [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES