Yamamura Y, Noda M, Ikawa Y
Department of Biochemistry, Tokyo Medical and Dental University School of Medicine, Japan.
Proc Natl Acad Sci U S A. 1994 Sep 13;91(19):8866-70. doi: 10.1073/pnas.91.19.8866.
We have previously shown that expression of erythropoietin (EPO) receptor (EPOR) alone failed to confer EPO responsiveness on the interleukin 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, allowed the cells to proliferate in response to EPO. Here, we report that additional expression of v-Ki-Ras conferred EPO-dependent growth on CTLL-2 cells expressing the EPOR, with additional formation of a high-affinity EPOR. To investigate possible mechanisms of EPOR downstream signaling induced by v-Ki-Ras expression in these CTLL-2-derived cells, we carried out anti-phosphotyrosine immunoblot analysis of the EPOR complex immunoprecipitated with anti-EPOR antibody from lysates of cells with and without cytokine stimulation, revealing two 160-kDa and 130-kDa phosphotyrosyl proteins. An anti-JAK2 antibody did not react with these 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 cell lines, in which the Friend virus gp55/EPOR complex on the cell surface constitutively sends signals for cell growth.
我们之前已经表明，单独表达促红细胞生成素（EPO）受体（EPOR）无法使白细胞介素2依赖的T细胞系CTLL-2对EPO产生反应，而将EPOR导入白细胞介素3依赖的前B细胞系，如BAF-B03，则可使细胞对EPO产生增殖反应。在此，我们报告称，额外表达v-Ki-Ras可使表达EPOR的CTLL-2细胞产生依赖EPO的生长，并额外形成高亲和力的EPOR。为了研究在这些源自CTLL-2的细胞中v-Ki-Ras表达所诱导的EPOR下游信号传导的可能机制，我们对用抗EPOR抗体免疫沉淀的EPOR复合物进行了抗磷酸酪氨酸免疫印迹分析，该复合物来自有或无细胞因子刺激的细胞裂解物，结果显示有两种160 kDa和130 kDa的磷酸化酪氨酸蛋白。抗JAK2抗体与这些蛋白无反应，这表明它们可能代表参与v-Ki-Ras诱导的EPO-EPOR信号通路的细胞成分。在弗氏红白血病细胞系中也存在类似的磷酸化酪氨酸蛋白，其中细胞表面的弗氏病毒gp55/EPOR复合物可组成性地发出细胞生长信号。
