Growth and differentiation signals of the erythropoietin receptor

为了筛选对造血干细胞自我更新或分化起重要作用的c-kit新底物，用荧光激活细胞分选仪对CD34阴性、SCA-1阳性、c-kit阳性和谱系标志物阴性(CD34-SCA-1+c-Kit-Lin-)的小鼠细胞进行分选，构建了CD34-SCA-1+c-Kit-Lin-细胞。通过以c-kit为诱饵的筛选，我们克隆了一个新的cDNA，设计了STAP-1，编码一个带有Pleckstrin同源结构域、Src同源2结构域和多个酪氨酸磷酸化位点的接头蛋白。RT-PCR分析显示STAP-1仅在表达c-kit的骨髓细胞组分表达，CD34-SCA-1+c-Kit+LIN-造血干细胞富集组表达最强。小鼠髓系细胞株M1高表达STAP-1。然而，在白血病抑制因子诱导M1细胞向单核细胞分化时，STAP-1的表达被强烈抑制，表明STAP-1与…有关更多的是未分化细胞类型。在293细胞中，STAP-1被激活的c-kit蛋白酪氨酸磷酸化。体外结合分析表明，STAP-1SH2结构域与c-kit和STAT5等多种酪氨酸磷酸化蛋白相互作用，提示STAP-1在造血干细胞中是c-kit下游的适配分子。在胚胎发育过程中，胎肝的部分红系细胞(而不是全部红系细胞)高表达Cis53/SOCS-3，这种表达不依赖于促红细胞生成素(EPO)信号。转基因介导的结构性表达阻断了胎儿的红细胞生成，导致胚胎死亡。Cis3/SOCS-3基因缺失会导致12-16天的胚胎死亡，这与明显的红细胞增多症有关。此外，胎肝祖细胞的个体体外增殖能力也大大提高。结果表明，Cis3/SOCS-3在胎肝造血的负性调控中起重要作用。我们还发现，在脾红系祖细胞和表达EPOR(BF-ER)的BA/F3细胞中，CIS3-SOCS3与EPO受体和JAK2结合，提示CIS3/SOCS3对EPO信号具有特异的负性调节作用。较少

To identify the novel substrate of c-kit which is important for hematopoietic stem cell self-renewal or differentiation, CD34-negative, Sca-1 positive, c-KIT-positive, and Lineage marker-negative (CD34-Sca-1+c-Kit-Lin- )cells were sorted by a fluorescence-activated cell sorter from mouse and two hybrid cDNA library was constructed. By the screening using c-kit as bait, we cloned a novel cDNA, designed STAP-1, encoding an adaptor protein with a Pleckstrin homology domain, Src homology 2 domain, and a number of tyrosine phosphorylation sites. RT-PCR analysis revealed that STAP-1 expression is restricted in bone marrow cell fraction expressing c-kit, and the highest expression was observed in CD34-Sca-1+c-Kit+Lin- hematopietic stem cell enriched fraction. Murine myeloid cell line, M1 expressed high level of STAP-1. However, the expression was strongly repressed in response to leukemia inhibitory factor which induced monocytic differentiation of M1 cells, suggesting that STAP-1 is associat … More ed with undifferentiated cell type. In 293 cells, STAP-1 was tyrosine phosphorylated by activated c-kit. In vitro binding assay suggested that STAP-1 SH2 domain interacted with several tyrosine phosphorylated proteins including c-kit and STAT5.There suggest that STAP-1 functions as an adaptor molecule downstream of c-kit in hematopoietic stem cells.CIS3/SOC53 are small SH2 containing proteins that interact with and inhibit JAK tyrosine kinases. During embryonic development, CIS53/SOCS-3 is highly expressed in some but no all erythroid lineage cells of the fetal liver and this expression is independent of erythropoietin (EPO) signaling. Transgene mediated constitutive expression blocks fetal erythropoiesis resulting in an embryonic lethality. Deletion of CIS3/SOCS-3 results in an embryonic lethality at 12-16 days that is associated with a marked erythrocytosis. Moreover, the individual in vitro proliferative capacity of fetal liver progenitors is greatly increased. The results demonstrate that CIS3/SOCS-3 play a critical role in negatively regulating fetal liver hematopoiesis. We also demonstrated that CIS3-SOCS3 bound to the EPO receptor as well as JAK2 in erythroid progenitors from spleen and Ba/F3 cells expressing the EPOR (BF-ER), suggesting a specific negative regulatory effect of CIS3/SOCS3 on EPO signaling. Less