ACTIVATED KI-RAS COMPLEMENTS ERYTHROPOIETIN SIGNALING IN CTLL-2 CELLS, INDUCING TYROSINE PHOSPHORYLATION OF A 160-KDA PROTEIN

We have previously shown that expression of erythropoietin (EPO) recep tor (EPOR) alone failed to confer EPO responsiveness on the interleuki n 2-dependent T-cell line CTLL-2, whereas the introduction of the EPOR into interleukin 3 -dependent pro-B-cell lines, such as BAF-B03, allo wed the cells to proliferate in response to EPO. Here, we report that additional expression of v-Ki-Ras conferred EPO-dependent growth on CT LL-2 cells expressing the EPOR, with additional formation of a high-af finity EPOR. To investigate possible mechanisms of EPOR downstream sig naling induced by v-Ki-Ras expression in these CTLL-2-derived cells, w e carried out anti-phosphotyrosine inmunoblot analysis of the EPOR com plex immunoprecipitated with anti-EPOR antibody from lysates of cells with and without cytokine stimulation, revealing two 16O-kDa and 130-k Da phosphotyrosyl proteins. An anti-JAK2 antibody did not react with t hese proteins, suggesting that they may represent cellular components involved in an EPO-EPOR signaling pathway induced by v-Ki-Ras. Similar phosphotyrosyl proteins were present among Friend erythroleukemia cel l lines, in which the Friend virus gp55/EPOR complex on the cell surfa ce constitutively sends signals for cell growth.