Miura O, Nakamura N, Quelle F W, Witthuhn B A, Ihle J N, Aoki N
First Department of Internal Medicine, Tokyo Medical and Dental University, Japan.
Blood. 1994 Sep 1;84(5):1501-7.
Protein tyrosine phosphorylation has been hypothesized to play a key role in the growth signaling induced by erythropoietin (Epo), although the Epo receptor (EpoR), a member of the cytokine receptor superfamily, lacks a tyrosine kinase domain. Recently, the JAK2 tyrosine kinase was shown to be activated on Epo stimulation and to bind to the cytoplasmic domain of EpoR in vitro. To further explore the mechanisms of activation of JAK2 in EpoR-mediated signal transduction, we assessed the conditions for association of JAK2 with EpoR in vivo. Epo stimulation rapidly induced association of JAK2 with the EpoR in an interleukin 3 (IL-3)-dependent cell line transfected with the wild-type EpoR. On Epo stimulation JAK2 also associated with a truncated mutant EpoR (H-mutant), which is mitogenetically active but not tyrosine phosphorylated, indicating that association does not require receptor phosphorylation and occurs in the membrane proximal region. However, association was not detected with mutant receptors inactivated by an internal deletion or a point mutation, Trp282 to Arg, in a membrane-proximal cytoplasmic region (PB or PM4 mutant, respectively). Immune complex kinase assays of anti-EpoR immunoprecipitates also revealed that activated JAK2 associates with the EpoR in Epo-stimulated cells. By this approach, association also occurred with the mitogenically active H mutant but not with the mitogenically inactive PB or PM4 mutants. In the immune complex kinases assays, EpoR, JAK2, and a 150-kD protein were phosphorylated on tyrosine. Taken together, the results further support the hypothesis that, on Epo stimulation, JAK2 associates with the membrane-proximal cytoplasmic region of the EpoR to be activated and induces tyrosine phosphorylation of cellular substrates, including the EpoR, to transduce a growth signal.
蛋白酪氨酸磷酸化被认为在促红细胞生成素（Epo）诱导的生长信号传导中起关键作用，尽管细胞因子受体超家族成员之一的Epo受体（EpoR）缺乏酪氨酸激酶结构域。最近，JAK2酪氨酸激酶在Epo刺激下被证明可被激活，并在体外与EpoR的胞质结构域结合。为了进一步探索JAK2在EpoR介导的信号转导中的激活机制，我们评估了JAK2与EpoR在体内结合的条件。在转染了野生型EpoR的白细胞介素3（IL-3）依赖性细胞系中，Epo刺激迅速诱导JAK2与EpoR结合。在Epo刺激下，JAK2也与截短的突变型EpoR（H-突变体）结合，该突变体具有促有丝分裂活性但未发生酪氨酸磷酸化，这表明结合不需要受体磷酸化，且发生在膜近端区域。然而，在膜近端胞质区域内通过内部缺失或点突变（分别为PB或PM4突变体，色氨酸282突变为精氨酸）而失活的突变型受体中未检测到结合。抗EpoR免疫沉淀的免疫复合物激酶分析还显示，在Epo刺激的细胞中，活化的JAK2与EpoR结合。通过这种方法，结合也发生在具有促有丝分裂活性的H突变体上，但未发生在无促有丝分裂活性的PB或PM4突变体上。在免疫复合物激酶分析中，EpoR、JAK2和一种150-kD的蛋白在酪氨酸上被磷酸化。综上所述，这些结果进一步支持了以下假说：在Epo刺激下，JAK2与EpoR的膜近端胞质区域结合并被激活，进而诱导包括EpoR在内的细胞底物的酪氨酸磷酸化，以转导生长信号。
