Insulin-induced tyrosine phosphorylation of a M(r) 70,000 protein revealed by association with the Src homology 2 (SH2) and SH3 domains of p120GAP and Grb2.
We have used two approaches to identify possible substrates of the insulin receptor (IR) tyrosine kinase. First, we used a potent tyrosine phosphatase inhibitor, phenylarsine oxide (PAO), which is reported to be specific for the insulin-induced signal transduction route, to augment tyrosine phosphorylation. Second, we used src homology 2 (SH2) domains fused to glutathione S-transferase as high affinity binding agents for tyrosine-phosphorylated proteins. Using the SH2 domain-containing region of p120 GTPase-activating protein and growth factor-bound protein 2, we observed a tyrosine-phosphorylated M(r) 70,000 protein in insulin- plus PAO-treated NIH3T3 cells overexpressing the IR. This M(r) 70,000 protein, which migrated as a doublet on SDS-polyacrylamide gels, efficiently bound to polyuridylic acid-Sepharose but is distinct from similar-size RNA-binding proteins such as p62 (sam68) and heterogeneous nuclear ribonucleoproteins I, K, L, and M. In addition, it differs from other M(r) 70,000 tyrosine-phosphorylated proteins, such as SH2-containing tyrosine phosphatase, raf1, and paxillin. Tyrosine phosphorylation of this protein was hardly observed after epidermal growth factor treatment. This suggests that the M(r) 70,000 protein is a novel and specific substrate for the IR kinase or an insulin-induced tyrosine kinase. The requirement for PAO to identify this tyrosine phosphorylation indicates a high turnover rate of the tyrosine phosphate.
Medema
J P
JP
Laboratory for Physiological Chemistry, Utrecht University, the Netherlands.
Pronk
G J
GJ
de Vries-Smits
A M
AM
Clark
R
R
McCormick
F
F
Bos
J L
JL
D016475:3T3 Cells; D000818:Animals; D001152:Arsenicals / Q000494:pharmacology; D004791:Enzyme Inhibitors; D051383:GRB10 Adaptor Protein; D020690:GTPase-Activating Proteins; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D007328:Insulin / Q000494:pharmacology*; D051379:Mice; D010766:Phosphorylation; D011072:Poly U / Q000378:metabolism; D011506:Proteins / Q000235:genetics*; D020794:Receptor Protein-Tyrosine Kinases / Q000187:drug effects / Q000378:metabolism*; D012261:Ribonucleoproteins; D018909:src Homology Domains / Q000502:physiology*
D001152:Arsenicals; D004791:Enzyme Inhibitors; D020690:GTPase-Activating Proteins; C495959:Grb10 protein, mouse; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D007328:Insulin; D011506:Proteins; D012261:Ribonucleoproteins; C029341:oxophenylarsine; D051383:GRB10 Adaptor Protein; D011072:Poly U; D020794:Receptor Protein-Tyrosine Kinases