IDENTIFICATION OF THE MAJOR SHPTP2-BINDING PROTEIN THAT IS TYROSINE-PHOSPHORYLATED IN RESPONSE TO INSULIN

Immunoprecipitation of the cytosolic Src homology 2 domain-containing protein-tyrosine phosphatase, SHPTP2, from insulin-stimulated 3T3L1 ad ipocytes or Chinese hamster ovary cells expressing the human insulin r eceptor resulted in the coimmunoprecipitation of a diffuse tyrosine-ph osphorylated band in the 115-kDa protein region on SDS-polyacrylamide gels. Although platelet-derived growth factor induced the tyrosine pho sphorylation of the platelet-derived growth factor receptor and SHPTP2 , there was no significant increase in the coimmunoprecipitation of ty rosine-phosphorylated pp115 with SHPTP2. SHPTP2 was also associated wi th tyrosine-phosphorylated insulin receptor substrate-1, but this only accounted for <2% of the total immunoreactive SHPTP2 protein. Similar ly, only a small fraction of the total amount of tyrosine-phosphorylat ed insulin receptor substrate-1 (<4%) was associated with SHPTP2. Expr ession and immunoprecipitation of a Myc epitope-tagged wild-type SHPTP 2 (Myc-WT-SHPTP2) and a catalytically inactive point mutant of SHPTP2 (Myc-C/S-SHPTP2) also demonstrated an insulin-dependent association of SHPTP2 with tyrosine-phosphorylated pp115. Furthermore, expression of the catalytically inactive SHPTP2 mutant resulted in a marked enhance ment in the amount of coimmunoprecipitated tyrosine-phosphorylated pp1 15 compared with the expression of wild-type SHPTP2. These data indica te that the insulin-stimulated tyrosine-phosphorylated 115-kDa protein is the predominant in vivo SHPTP2-binding protein and that pp115 may function as a physiological substrate for the SHPTP2 protein-tyrosine phosphatase.