Antisense-mediated suppression of the transmembrane protein-tyrosine phosphatase (PTPase) LAR has been shown previously to increase insulin-dependent phosphatidylinositol 3-kinase (PI 3-kinase) activation by greater than 300% in the rat hepatoma cell line McA-RH7777. Here, insulin-dependent insulin receptor tyrosine kinase activation was examined with recombinant insulin receptor substrate 1 (IRS-1) as the substrate and shown to be 3-fold greater in cells with suppressed LAR levels. Consistent with a receptor level effect, in vivo insulin-dependent tyrosine phosphorylation of both IRS-1 and Shc was increased by a similar 3-fold with LAR suppression. These increases in IRS-1 and Shc phosphorylation were paralleled by increases in insulin-dependent PI 3-kinase association with IRS-1 and activation of the MAP kinase pathway. Reduced LAR levels also resulted in increases of over 300% and 250% in epidermal growth factor (EGF)- and hepatocyte growth factor (HGF)-dependent receptor autophosphorylation, respectively, as well as a severalfold increase in substrate tyrosine phosphorylation. In a post-receptor response, EGF- and HGF-dependent MAP kinase activation was increased by 300% and 350%, respectively, with LAR suppression. Similarly, growth factor-dependent PI 3-kinase activation was increased in LAR antisense expressing cells when compared to null vector expressing cells. These results demonstrate that the transmembrane PTPase LAR modulates ligand-dependent activation of at least three receptor tyrosine kinases.