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首页> 外文期刊>Molecular and Cellular Biology >In vitro phosphorylation of the erythropoietin receptor and an associated protein, pp130.
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In vitro phosphorylation of the erythropoietin receptor and an associated protein, pp130.

机译：促红细胞生成素受体和相关蛋白pp130的体外磷酸化。

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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.
机译：克隆的促红细胞生成素（EPO）受体（EPOR）的胞质域不包含蛋白激酶基序，但是EPO反应性细胞中添加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依赖性细胞系中添加EPO或白介素3（IL-3）会导致总细胞EPOR的一小部分（少于5％）发生凝胶迁移从66到72kDa，至少部分由于磷酸化。使用生物素化的EPO作为亲和试剂，我们表明72 kDa物种在细胞表面上大量富集。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孵育，然后与硫醇可裂解的化学交联剂交联。将抗生物素蛋白-琼脂糖选择的复合物与[γ-32P] ATP一起孵育。在没有还原剂的情况下进行体外磷酸化和变性后，抗磷酸酪氨酸和抗EPOR抗体都免疫沉淀了标记的72 kDa EPOR和未鉴定的130 kDa磷蛋白（pp130），这表明蛋白激酶与细胞表面EPOR相关，并且一部分EPOR在细胞中或无细胞磷酸化反应过程中的酪氨酸残基上被磷酸化。在还原条件下，用抗EPOR抗体免疫沉淀72-kDa磷酸化的EPOR，但未将pp130免疫沉淀，这表明pp130通过硫醇可裂解的化学交联剂与EPOR结合。以前，我们表明删除EPOR的42个羧基末端氨基酸可使细胞以正常EPO浓度的1/10生长，而不会影响受体数目或亲和力。对EPO高度敏感的两个羧基末端截短的EPO受体在体外反应过程中磷酸化较弱，这表明EPOR的羧基末端区域包含一个磷酸化位点或一个与蛋白激酶相互作用的位点。我们的数据表明，磷酸化或与羧基末端区域中的蛋白激酶相互作用可能会下调EPOR的增殖作用。

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