Miura Y, Miura O, Ihle J N, Aoki N
First Department of Internal Medicine, Tokyo Medical and Dental University, Japan.
J Biol Chem. 1994 Nov 25;269(47):29962-9.
The erythropoietin receptor (EpoR) belongs to the cytokine receptor family, members of which lack a tyrosine kinase domain. Recent studies, however, have shown that a cytoplasmic tyrosine kinase, JAK2, interacts with the cytoplasmic domain of the EpoR and becomes activated upon binding of Epo to the receptor. Epo has also been shown to stimulate activation of Ras and Raf-1. The present studies were undertaken to examine the possible involvement of Epo-induced tyrosine phosphorylation in activation of the Ras/mitogen-activated protein kinase (MAP kinase) pathway and to determine its significance on the growth signaling from the EpoR. In an interleukin (IL)-3-dependent cell line expressing the transfected wild-type EpoR, Epo, or IL-3 induced tyrosine phosphorylation of Shc and its association with Grb2. These cytokines also induced tyrosine phosphorylation and activation of MAP kinase isoforms ERK1 and ERK2. A mutant EpoR with a carboxyl-terminal deletion of 108 amino acids (H mutant), which is mitogenically functional but lacks tyrosine phosphorylation sites in the carboxyl-terminal region, showed markedly diminished abilities to induce tyrosine phosphorylation of Shc and to phosphorylate and activate MAP kinases. A mutant receptor (PM4 mutant) inactivated by a point mutation, Trp282 to Arg, which abrogates the interaction with JAK2, failed to induce any effect on Shc or MAP kinases. In cells expressing a mutant EpoR that is constitutively activated by a point mutation, Arg129 to Cys, in the extracellular portion of the receptor, neither tyrosine phosphorylation of Shc nor activation of MAP kinases by phosphorylation was detectable without stimulation with Epo or IL-3. These results suggest that the carboxyl-terminal region of EpoR may play a crucial role in activation of MAP kinases through the Ras signaling pathway which may be activated by tyrosine phosphorylation of Shc and its association with Grb2. The activation of MAP kinases, however, failed to correlate with the mitogenic activity of mutant EpoRs and thus may not be required for growth signaling from the EpoR.
促红细胞生成素受体（EpoR）属于细胞因子受体家族，该家族成员缺乏酪氨酸激酶结构域。然而，最近的研究表明，一种细胞质酪氨酸激酶JAK2与EpoR的细胞质结构域相互作用，并在促红细胞生成素（Epo）与受体结合后被激活。Epo还被证明能刺激Ras和Raf-1的激活。本研究旨在探讨Epo诱导的酪氨酸磷酸化在Ras/丝裂原活化蛋白激酶（MAP激酶）途径激活中的可能作用，并确定其对EpoR生长信号传导的意义。在表达转染野生型EpoR的白细胞介素（IL）-3依赖性细胞系中，Epo或IL-3诱导Shc的酪氨酸磷酸化及其与Grb2的结合。这些细胞因子还诱导MAP激酶亚型ERK1和ERK2的酪氨酸磷酸化和激活。一种羧基末端缺失108个氨基酸的突变型EpoR（H突变体），其具有促有丝分裂功能，但在羧基末端区域缺乏酪氨酸磷酸化位点，显示出诱导Shc酪氨酸磷酸化以及磷酸化和激活MAP激酶的能力明显减弱。一种因点突变（Trp282突变为Arg）而失活的突变受体（PM4突变体），该突变消除了与JAK2的相互作用，未能对Shc或MAP激酶产生任何影响。在表达一种因受体细胞外部分的点突变（Arg129突变为Cys）而组成性激活的突变型EpoR的细胞中，在没有Epo或IL-3刺激的情况下，既检测不到Shc的酪氨酸磷酸化，也检测不到通过磷酸化激活的MAP激酶。这些结果表明，EpoR的羧基末端区域可能在通过Ras信号通路激活MAP激酶中起关键作用，该信号通路可能通过Shc的酪氨酸磷酸化及其与Grb2的结合而被激活。然而，MAP激酶的激活与突变型EpoR的促有丝分裂活性不相关，因此可能不是EpoR生长信号传导所必需的。
