Growth and differentiation signals of the erythropoietin receptor

为了鉴定对造血干细胞自我更新或分化很重要的c-kit新型底物，通过荧光激活细胞分选仪对小鼠的CD34阴性、Sca-1阳性、c-KIT阳性和谱系标记阴性(CD34-Sca-1 c-Kit-Lin-)细胞进行分选，并构建了两个杂交cDNA文库。通过以c-kit为诱饵筛选，我们克隆了一个新的cDNA，设计了STAP-1，编码具有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表达仅限于表达c-kit的骨髓细胞级分中，并且在CD34-Sca-1 c-Kit Lin-造血干细胞富集级分中观察到最高表达。小鼠骨髓细胞系 M1 表达高水平的 STAP-1。然而，该表达受到白血病抑制因子的强烈抑制，该因子诱导 M1 细胞的单核细胞分化，这表明 STAP-1 与未分化的细胞类型相关。在 293 细胞中，STAP-1 被激活的 c-kit 酪氨酸磷酸化。体外结合实验表明，STAP-1 SH2 结构域与包括 c-kit 和 STAT5 在内的多种酪氨酸磷酸化蛋白相互作用。表明 STAP-1 在造血干细胞中充当 c-kit 下游的衔接分子。CIS3/SOC53 是含有 SH2 的小蛋白，可与 JAK 酪氨酸激酶相互作用并抑制 JAK 酪氨酸激酶。在胚胎发育过程中，CIS53/SOCS-3 在胎儿肝脏的一些（但不是全部）红系细胞中高度表达，并且这种表达独立于促红细胞生成素 (EPO) 信号传导。转基因介导的组成型表达阻断胎儿红细胞生成，导致胚胎致死。 CIS3/SOCS-3 的缺失会导致 12-16 天的胚胎死亡，并伴有明显的红细胞增多症。而且，胎肝祖细胞个体体外增殖能力大大增强。结果表明CIS3/SOCS-3在负调节胎儿肝脏造血中发挥着关键作用。我们还证明，CIS3-SOCS3 与表达 EPOR (BF-ER) 的脾脏红系祖细胞和表达 EPOR (BF-ER) 的 Ba/F3 细胞中的 EPO 受体以及 JAK2 结合，表明 CIS3/SOCS3 对 EPO 信号传导具有特定的负调节作用。较少的

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