Li Ke, Miller Chris, Hegde Shailajia, Wojchowski Don
Department of Veterinary Science, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
J Biol Chem. 2003 Oct 17;278(42):40702-9. doi: 10.1074/jbc.M307182200. Epub 2003 Aug 9.
Erythroid progenitor cell expansion depends upon co-signaling by Epo receptor (EpoR) and Kit, but underlying mechanisms are incompletely understood. To quantitatively analyze EpoR contributions to co-signaling, phosphotyrosine (Tyr(P)) mutants were expressed as human epidermal growth factor (hEGF) receptor-mEpoR EE chimeras at matched and physiological levels in FDCW2 hematopoietic progenitor cells and were assayed for proliferative activities in the absence or presence of endogenous Kit stimulation. Two Tyr(P)-null (but Jak2-coupled) EpoR forms each retained <or=25% of the wild-type activity, whereas the add-back of single Tyr(P) sites in the EpoR forms EE-T-Y343 (Stat5 binding site), EE-Y479 (p85/phosphatidylinositol 3-kinase binding site), or EE-Y464 (Src kinase binding site) significantly enhanced activities (to 100, 95, and 50% of EE-WT (wild type) levels, respectively). EE-Y343&Y401 and EEF343&F401 double add-back and deletion constructs were also prepared and were shown to possess 90 and <or=50% of wild-type activity. In contrast, efficient Kit co-signaling activity was retained only by EE-T-Y343 and EE-Y343&Y401 EpoR forms. EE-T-Y343 together with EE-T-Y343F and EE-WT EpoR forms were also analyzed in embryonic stem cell-derived erythroid G1E-2 cells with highly comparable outcomes, including the ability of EE-T-Y343 (but not EE-T-Y-343F) to synergize with Kit. Despite specific connection of EE-T-Y343 to Stat5, the contributions of Kit to EpoR-dependent proliferation did not involve Kit effects on Stat5 activation (but was limited by the mutation of Kit Tyr(P)-567 and Tyr(P)-569 Src kinase recruitment sites). Instead, co-signaling appears to depend upon the downstream integration of Kit signals with the targets of an EpoR/Jak2/Y343/Stat 5 response axis.
红系祖细胞的扩增依赖于促红细胞生成素受体（EpoR）和Kit的共信号传导，但其潜在机制尚未完全阐明。为了定量分析EpoR对共信号传导的贡献，在FDCW2造血祖细胞中，将磷酸酪氨酸（Tyr(P)）突变体作为人表皮生长因子（hEGF）受体-mEpoR EE嵌合体以匹配的生理水平表达，并在有无内源性Kit刺激的情况下检测其增殖活性。两种Tyr(P)缺失（但与Jak2偶联）的EpoR形式各自保留的活性均≤野生型活性的25%，而在EpoR形式EE-T-Y343（Stat5结合位点）、EE-Y479（p85/磷脂酰肌醇3激酶结合位点）或EE-Y464（Src激酶结合位点）中单个Tyr(P)位点的回补显著增强了活性（分别达到EE-WT（野生型）水平的100%、95%和50%）。还制备了EE-Y343&Y401和EEF343&F401双回补和缺失构建体，结果显示它们分别具有野生型活性的90%和≤50%。相反，只有EE-T-Y343和EE-Y343&Y401 EpoR形式保留了有效的Kit共信号传导活性。在胚胎干细胞来源的红系G1E-2细胞中也分析了EE-T-Y343以及EE-T-Y343F和EE-WT EpoR形式，结果高度可比，包括EE-T-Y343（而非EE-T-Y-343F）与Kit协同作用的能力。尽管EE-T-Y343与Stat5有特定联系，但Kit对EpoR依赖性增殖的贡献并不涉及Kit对Stat5激活的影响（而是受到Kit Tyr(P)-567和Tyr(P)-569 Src激酶募集位点突变的限制）。相反，共信号传导似乎依赖于Kit信号与EpoR/Jak2/Y343/Stat 5反应轴靶点的下游整合。
