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

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 86; no. 19; pp. 7306 - 7310
• Main Authors: Fujita-Yamaguchi, Yoko, Kathuria, Satish, Xu, Qin-Yu, McDonald, Jay M., Nakano, Hirofumi, Kamata, Tohru
• Format: Journal Article
• Language: English
• Published: Washington, DC National Academy of Sciences of the United States of America 01.10.1989; National Acad Sciences
• Subjects: Adipocytes; Amino acids; Analytical, structural and metabolic biochemistry; Biochemistry; Biological and medical sciences; Calmodulin - metabolism; Cell Membrane - metabolism; Enzymes and enzyme inhibitors; Female; Fundamental and applied biological sciences. Psychology; Humans; Insulin; Oncogene Protein p21(ras) - metabolism; Phosphorylation; Placenta - metabolism; Polylysine - metabolism; Polylysine - pharmacology; Pregnancy; Protein-Tyrosine Kinases - isolation & purification; Protein-Tyrosine Kinases - metabolism; Proteins; Receptor, Insulin - metabolism; Receptors; Transferases; Tyrosine; Human; Phosphorylation; Membrane receptor; Insulin; Protein-tyrosine kinase; Calmodulin
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.86.19.7306

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.