Damen J E, Cutler R L, Jiao H, Yi T, Krystal G
Terry Fox Laboratory, British Columbia Cancer Research Centre, Vancouver, Canada.
J Biol Chem. 1995 Oct 6;270(40):23402-8. doi: 10.1074/jbc.270.40.23402.
We recently reported that phosphatidylinositol (PI) 3-kinase becomes associated with the activated erythropoietin receptor (EpR), most likely through the Src homology 2 (SH2) domains within the p85 subunit of PI-3 kinase and one or more phosphorylated tyrosines within the EpR. We have now investigated this interaction in more detail and have found, based on both blotting studies with glutathione S-transferase-p85-SH2 fusion proteins and binding of these fusion proteins to SDS-denatured EpRs, that this binding is direct. Moreover, both in vitro competition studies, involving phosphorylated peptides corresponding to the amino acid sequences flanking the eight tyrosines within the intracellular domain of the EpR, and in vivo studies with mutant EpRs bearing tyrosine to phenylalanine substitutions, indicate that phosphorylation of Tyr503 within the EpR is essential for the binding of PI 3-kinase. The presence of PI 3-kinase activity in EpR immunoprecipitates from DA-3 cells infected with wild-type but not Y503F EpRs confirms this finding. Our results demonstrate that the SH2 domains of p85 can bind, in addition to their well established Tyr-Met/Val-X-Met consensus binding sequence, a Tyr-Val-Ala-Cys motif that is present in the EpR. A comparison of erythropoietin-induced tyrosine phosphorylations and proliferation of wild-type and Y503F EpR-infected DA-3 cells revealed no differences. However, the PI-3 kinase inhibitor, wortmannin, markedly inhibited the erythropoietin-induced proliferation of both cell types, suggesting that PI 3-kinase is activated in Y503F EpR expressing cells. This was confirmed by carrying out PI 3-kinase assays with anti-phosphotyrosine immunoprecipitates from erythropoietin-stimulated Y503F EpR-infected DA-3 cells and suggested that PI 3-kinase has a role in regulating erythropoietin-induced proliferation, but at a site distinct from the EpR.
我们最近报道，磷脂酰肌醇（PI）3激酶与活化的促红细胞生成素受体（EpR）相关联，很可能是通过PI-3激酶p85亚基内的Src同源2（SH2）结构域以及EpR内一个或多个磷酸化酪氨酸实现的。我们现在对这种相互作用进行了更详细的研究，基于谷胱甘肽S-转移酶-p85-SH2融合蛋白的印迹研究以及这些融合蛋白与SDS变性EpR的结合，发现这种结合是直接的。此外，体外竞争研究（涉及与EpR胞内结构域中八个酪氨酸两侧氨基酸序列对应的磷酸化肽段）以及对携带酪氨酸到苯丙氨酸替代的突变EpR的体内研究均表明，EpR内Tyr503的磷酸化对于PI 3激酶的结合至关重要。从感染野生型而非Y503F EpR的DA-3细胞的EpR免疫沉淀物中存在PI 3激酶活性证实了这一发现。我们的结果表明，p85的SH2结构域除了能结合其已确定的Tyr-Met/Val-X-Met共有结合序列外，还能结合EpR中存在的Tyr-Val-Ala-Cys基序。对野生型和Y503F EpR感染的DA-3细胞中促红细胞生成素诱导的酪氨酸磷酸化和增殖的比较未发现差异。然而，PI-3激酶抑制剂渥曼青霉素显著抑制了两种细胞类型中促红细胞生成素诱导的增殖，这表明PI 3激酶在表达Y503F EpR的细胞中被激活。通过对促红细胞生成素刺激的Y503F EpR感染的DA-3细胞的抗磷酸酪氨酸免疫沉淀物进行PI 3激酶测定证实了这一点，这表明PI 3激酶在调节促红细胞生成素诱导的增殖中起作用，但作用位点与EpR不同。
