In vitro phosphorylation of the erythropoietin receptor and an associated protein, pp130-猫眼课题宝

促红细胞生成素受体和相关蛋白 pp130 的体外磷酸化

DOI：

发表时间：

1992

期刊：

Molecular and Cellular Biology

影响因子：

5.3

通讯作者：

中科院分区：

生物学2区

文献类型：

作者：

Akihiko Yoshimura;H. F. Lodish

克隆的促红细胞生成素 (EPO) 受体 (EPOR) 的胞质结构域不含蛋白激酶基序，但向 EPO 反应细胞添加 EPO 会导致蛋白酪氨酸磷酸化增加。在这里，我们表明，向表达野生型 EPOR 的 IL-3 依赖性细胞系添加 EPO 或白细胞介素 3 (IL-3) 会导致总细胞 EPOR 的一小部分（小于 5%）凝胶迁移率从 66 kDa 转变为 72 kDa，这至少部分是由于磷酸化。使用生物素化 EPO 作为亲和试剂，我们发现 72 kDa 的物质在细胞表面大量富集。为了证明蛋白激酶活性与细胞表面 EPOR 相关，将细胞与生物素化的 EPO 一起孵育，然后与硫醇可裂解的化学交联剂交联。将亲和素-琼脂糖选择的复合物与[γ-32P]ATP一起孵育。在没有还原剂的体外磷酸化和变性后，抗磷酸酪氨酸和抗 EPOR 抗体都免疫沉淀标记的 72-kDa EPOR 和未鉴定的 130-kDa 磷蛋白 (pp130)，表明蛋白激酶与细胞表面 EPOR 相关，并且一部分 EPOR 在细胞内或在无细胞磷酸化反应期间在酪氨酸残基上被磷酸化。在还原条件下，用抗 EPOR 抗体对 72 kDa 磷酸化 EPOR 而不是 pp130 进行免疫沉淀，表明 pp130 通过硫醇可裂解的化学交联剂与 EPOR 结合。此前，我们发现删除 EPOR 的 42 个羧基端氨基酸可使细胞在正常 EPO 浓度的 1/10 下生长，而不影响受体数量或亲和力。对 EPO 高反应性的两个羧基端截短的 EPO 受体在体外反应过程中磷酸化不良，表明 EPOR 的羧基端区域包含磷酸化位点或与蛋白激酶相互作用的位点。我们的数据表明，羧基末端区域的磷酸化或与蛋白激酶的相互作用可能会下调 EPOR 的增殖作用。

The cytoplasmic domain of the cloned erythropoietin (EPO) receptor (EPOR) contains no protein kinase motif, yet addition of EPO to EPO-responsive cells causes an increase in protein-tyrosine phosphorylation. Here we show that addition of EPO or interleukin-3 (IL-3) to an IL-3-dependent cell line expressing the wild-type EPOR causes a small fraction (less than 5%) of total cellular EPOR to shift in gel mobility from 66 to 72 kDa, due at least in part to phosphorylation. Using biotinylated EPO as an affinity reagent, we show that the 72-kDa species is greatly enriched on the cell surface. To demonstrate that a protein kinase activity associates with cell surface EPOR, cells were incubated with biotinylated EPO and then cross-linked with a thiol-cleavable chemical cross-linker. The avidin-agarose-selected complexes were incubated with [gamma-32P]ATP. After in vitro phosphorylation and denaturation without reducing agent, both antiphosphotyrosine and anti-EPOR antibodies immunoprecipitated labeled 72-kDa EPOR and an unidentified 130-kDa phosphoprotein (pp130), indicating that a protein kinase is associated with cell surface EPOR and that a fraction of the EPOR was phosphorylated on tyrosine residues either in the cells or during the cell-free phosphorylation reaction. Under reducing conditions, the 72-kDa phosphorylated EPOR but not pp130 was immunoprecipitated with an anti-EPOR antibody, suggesting that the pp130 is bound to the EPOR by the thiol-cleavable chemical cross-linker. Previously, we showed that deletion of the 42 carboxy-terminal amino acids of the EPOR allows cells to grow in 1/10 the normal EPO concentration, without affecting receptor number or affinity. Two carboxy-terminal truncated EPO receptors that are hyperresponsive to EPO were poorly phosphorylated during the in vitro reaction, suggesting that the carboxy-terminal region of the EPOR contains a site for phosphorylation or a site for interaction with a protein kinase. Our data suggests that phosphorylation or interaction with a protein kinase in the carboxy-terminal region may down-modulate the proliferative action of the EPOR.