EXPRESSION OF CATALYTICALLY INACTIVE SYP PHOSPHATASE IN 3T3 CELLS BLOCKS STIMULATION OF MITOGEN-ACTIVATED PROTEIN-KINASE BY INSULIN

To explore the role of the protein tyrosine phosphatase Syp in insulin signaling, a catalytically inert mutant Syp protein was expressed und er an inducible promoter in cells transfected with the human insulin r eceptor. Expression of the mutant phosphatase significantly reduced th e stimulation of mitogenesis by insulin; indicating that the mutation produced a dominant negative phenotype. Tyrosine phosphorylation of bo th the insulin receptor and its major substrates, She and insulin rece ptor substrate-1, were unaffected by the mutant phosphatase. However, both the insulin-dependent tyrosine phosphorylation and activation of mitogenactivated protein kinase were markedly attenuated. Expression o f the mutant phosphatase allowed the detection of a 120-kDa protein ph osphorylated in response to insulin that associated with the src homol ogy (SH) 2 domains of the phosphatase, suggesting a possible regulator y role for this protein. These results indicate that the activity of S yp plays a critical part in the mitogenic actions of insulin.