p120(cbl) is a cytosolic adapter protein that associates with phosphoinositide 3-kinase in response to epidermal growth factor in PC12 and other cells

Although epidermal growth factor (EGF) activates phosphoinositide (PI) 3-kinase activity in a number of types of cells or cell lines, in most cases that we have investigated the p85 regulatory subunit of PI 3-kinase does not appear to bind directly to the EGF receptor. Previously we demonstrated that EGF dependent activation of PI 3-kinase activity in A431 cells is accompanied by the binding of p85 to ErbB3, an EGF receptor homologue. However, this mechanism did not explain the large activation of PI 3-kinase activity that was found in PC12 and A549 cells, which possess little or no ErbB3. Here we provide evidence that the p120(cbl) protooncoprotein is an intracellular adapter protein that associates with PI 3-kinase and thus is involved in the EGF-dependent activation of this enzyme in these two cell lines. Using an anti-p120(cbl) antibody, we immunoprecipitated the EGF receptor from PC12 cells and PI 3-kinase activity from PC12 and A549 cells in an EGF-dependent fashion. Treatment of PC12 cells with nerve growth factor or insulin stimulated large increases in PI 3-kinase activity that was immunoprecipitated using anti-Tyr(P) antibody but not using anti p120(cbl) antibody. In EGF-treated PC12 cells, the tyrosine phosphorylation of p120(cbl) displayed similar kinetics to the activation of PI 3-kinase as measured by both in vivo lipid production and lipid kinase assays conducted using anti-p120(cbl) and anti-Tyr(P) immunoprecipitates. The use of glutathione S-transferase fusion proteins of various domains of p85 demonstrated that p120(cbl) associated with both the SH2 and SH3 domains of p85, p120(cbl) was also present in A431 cells and offers an additional pathway by which EGF can activate PI 3-kinase in these cells.