CONSTITUTIVE PHOSPHORYLATION OF EPS8 IN TUMOR-CELL LINES - RELEVANCE TO MALIGNANT TRANSFORMATION

eps8, a recently identified tyrosine kinase substrate, has been shown to augment epidermal growth factor (EGF) responsiveness, implicating i t in EGF receptor (EGFR)-mediated mitogenic signaling. We investigated the status of eps8 phosphorylation in normal and transformed cells an d the role of eps8 in transformation. In NIH 3T3 cells overexpressing EGFR (NIH-EGFR), eps8 becomes rapidly phosphorylated upon EGF stimulat ion. At receptor-saturating doses of EGF, similar to 30% of the eps8 p ool is tyrosine phosphorylated. Under physiological conditions of acti vation (i.e., at low receptor occupancy), corresponding to the 50% eff ective dose of EGF for mitogenesis, similar to 3 to 4% of the eps8 con tains phosphotyrosine. In human tumor cell lines, we detected constitu tive tyrosine phosphorylation of eps8, with a stoichiometry (similar t o 5%) similar to that associated with potent mitogenic response in NIH -EGFR cells. Overexpression of eps8 was able to transform NIH 3T3 cell s under limiting conditions of activation of the EGFR pathway. Concomi tant tyrosine phosphorylation of eps8 and she, but not of rasGAP, phos pholipase C-gamma, and eps15, was frequently detected in tumor cells. This suggested that eps8 and she might be part of a pathway which is p referentially selected in some tumors. Cooperation between these two t ransducers was further indicated by the finding of their in vivo assoc iation. This association was, at least in part, dependent on recogniti on of shc by the SH3 domain of eps8. Our results indicate that eps8 is physiologically part of the EGFR-activated signaling and that its alt erations can contribute to the malignant phenotype.