EVIDENCE FOR 2 DISTINCT 60-KILODALTON SUBSTRATES OF THE SRC TYROSINE KINASE

A monoclonal antibody to a 60-kDa substrate of the insulin receptor ty rosine kinase is utilized in the present studies to examine this molec ule in 3T3 cells expressing either the transforming chicken c-Src (mut ant Phe-527), the wild type molecule, or the parental cells. The tyros ine phosphorylation of this 60-kDa protein was greatly increased in ce lls expressing transforming Src and partially increased in cells expre ssing wild type enzyme. This tyrosine phosphorylation correlated with an increased association with the GTPase-activating protein of p21(ras ) (GAP). However, this 60-kDa protein did not react with antibodies to another 62-kDa tyrosine-phosphorylated protein previously isolated fr om Src-transformed cells (Wong, G., Muller, O., Clark, R., Conroy, L., Moran, M. F., Polakis, P., and McCormick, F. (1992) Cell 69, 551-558) , although this latter antibody did react with a 62-kDa protein in ant i-phosphotyrosine precipitates from cells expressing transforming c-Sr c but not the parental cells. These two proteins could also be disting uished by their subcellular location, the ability of the latter but no t the former protein to bind RNA, and their migration in SDS gels. Mor eover, the 62-kDa RNA-binding phosphoprotein could be almost completel y depleted from cell lysates with poly(U)-Sepharose without affecting the amount of either the GAP-associated 60-kDa tyrosine-phosphorylated protein or the protein precipitated with the monoclonal antibody. whe n the two proteins were phosphorylated in vitro with purified c-Src, t hey were both found to bind directly to the aminoterminal SH2 domain o f GAP, although the RNA-binding protein was found to have a weaker aff inity. These results indicate that two distinct 60-kDa proteins are su bstrates for the Src tyrosine kinase, one which binds RNA and the othe r which constitutes the major GAP-associated 60-kDa phosphoprotein.