Evidence for two distinct 60-kilodalton substrates of the SRC tyrosine kinase.
A monoclonal antibody to a 60-kDa substrate of the insulin receptor tyrosine kinase is utilized in the present studies to examine this molecule in 3T3 cells expressing either the transforming chicken c-Src (mutant Phe-527), the wild type molecule, or the parental cells. The tyrosine phosphorylation of this 60-kDa protein was greatly increased in cells expressing transforming Src and partially increased in cells expressing wild type enzyme. This tyrosine phosphorylation correlated with an increased association with the GTPase-activating protein of p21ras (GAP). However, this 60-kDa protein did not react with antibodies to another 62-kDa tyrosine-phosphorylated protein previously isolated from 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 anti-phosphotyrosine precipitates from cells expressing transforming c-Src but not the parental cells. These two proteins could also be distinguished by their subcellular location, the ability of the latter but not the former protein to bind RNA, and their migration in SDS gels. Moreover, the 62-kDa RNA-binding phosphoprotein could be almost completely 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. When the two proteins were phosphorylated in vitro with purified c-Src, they were both found to bind directly to the amino-terminal SH2 domain of GAP, although the RNA-binding protein was found to have a weaker affinity. These results indicate that two distinct 60-kDa proteins are substrates for the Src tyrosine kinase, one which binds RNA and the other which constitutes the major GAP-associated 60-kDa phosphoprotein.
Ogawa
W
W
Department of Molecular Pharmacology, Stanford University School of Medicine, California 94305.
Hosomi
Y
Y
Shii
K
K
Roth
R A
RA
D016475:3T3 Cells; D000818:Animals; D002645:Chickens; D020690:GTPase-Activating Proteins; D051379:Mice; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism*; D013379:Substrate Specificity; D014443:Tyrosine / Q000378:metabolism
D020690:GTPase-Activating Proteins; D010750:Phosphoproteins; D011506:Proteins; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D016392:Proto-Oncogene Proteins pp60(c-src)