ACTIVATION OF THE RAS MITOGEN-ACTIVATED PROTEIN-KINASE PATHWAY BY KINASE-DEFECTIVE EPIDERMAL GROWTH-FACTOR RECEPTORS RESULTS IN CELL-SURVIVAL BUT NOT PROLIFERATION/

Signalling by the epidermal growth factor (EGF) receptor (EGFR) has be en studied intensively, but for most cell types the analysis is compli cated by the fact that EGFR not only homodimerizes but can also form h eterodimers with other EGFR family members. Heterodimerization is a pa rticular problem in the study of EGFR mutants, where the true phenotyp e of the mutants is confounded by the contribution of the heterodimer partner to signal transduction. We have made use of the murine hemopoi etic cell line BaF/3, which does not express EGFR family members, to e xpress wild-type (WT) EGFR, three kinase-defective EGFR mutants (V741G , Y740F, and K721R), or a C-terminally truncated EGFR (CT957) and have measured their responses to EGF, We found that under the appropriate conditions EGF ran stimulate cell proliferation of BaF/3 cells express ing WT or CT957 EGFRs but not that of cells expressing the kinase-defe ctive mutants. However, EGF promotes the survival of BaF/3 cells expre ssing either of the kinase-defective receptors (V741G and Y740F), indi cating that these receptors can still transmit a survival signal, Anal ysis of the early signalling events by the WT, V741G, and Y740F mutant EGF receptors indicated that EGF stimulates comparable levels of Shc phosphorylation, Shc-GRB-2 association, and activation of Pas, B-Raf, and Erk-1. Blocking the mitogen-activated protein kinase (MAPK) signal ling pathway with the specific inhibitor PD98059 abrogates completely the EGF-dependent survival of cells expressing the kinase-defective EG FR mutants but has no effect on the EGF dependent proliferation mediat ed by WT and CT957 EGFRs. Similarly, the Src family kinase inhibitor P P1 abrogates EGF-dependent survival without affecting proliferation. H owever blocking phosphatidylinositol-3-kinase or JAK-2 kinase with spe cific inhibitors does arrest growth factor-dependent cell proliferatio n. Thus, EGFR-mediated mitogenic signalling in BaF/3 cells requires an intact EGFR tyrosine kinase activity and appears to depend on the act ivation of both the JAK-2 and PI-3 kinase pathways. Activation of the Src family of kinases or of the Ras/MAPK pathway can, however, be init iated by a kinase-impaired EGFR and is linked to survival.