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 ins ulin receptor (IR) tyrosine kinase. First, we used a potent tyrosine p hosphatase inhibitor, phenylarsine oxide (PAO), which is reported to b e specific for the insulin-induced signal transduction route, to augme nt tyrosine phosphorylation. Second, we used src homology 2 (SH2) doma ins 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 prot ein 2, we observed a tyrosine-phosphorylated M(r) 70,000 protein in in sulin- 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 fro m similar-size RNA-binding proteins such as p62 (sam68) and heterogene ous 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 phosphor ylation of this protein was hardly observed after epidermal growth fac tor treatment, This suggests that the M(r) 70,000 protein is a novel a nd specific substrate for the IR kinase or an insulin-induced tyrosine kinase, The requirement for PAO to identify this tyrosine phosphoryla tion indicates a high turnover rate of the tyrosine phosphate.