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具有重要意义的c-kit的新型底物，采用荧光激活细胞分选仪对小鼠cd34阴性、Sca-1阳性、c-kit阳性和谱系标记阴性（CD34-Sca-1+ c-kit - lin -）细胞进行了分选，并构建了两个杂交cDNA文库。以c-kit为诱饵，通过筛选，我们克隆了一段新的cDNA，并设计了STAP-1，编码一个具有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衔接蛋白。RT-PCR分析显示，STAP-1在表达c-kit的骨髓细胞组分中表达受限，在CD34-Sca-1+ c-kit +Lin-造血干细胞富集组分中表达最高。小鼠髓系M1高表达STAP-1。然而，在白血病抑制因子诱导M1细胞单核细胞分化时，STAP-1的表达被强烈抑制，这表明STAP-1与未分化的细胞类型有关。在293细胞中，STAP-1被活化的c-kit酪氨酸磷酸化。体外结合实验表明，STAP-1 SH2结构域与c-kit和STAT5等多种酪氨酸磷酸化蛋白相互作用。研究表明，在造血干细胞中，STAP-1作为c-kit下游的一个接头分子起作用。CIS3/SOC53是含有SH2的小蛋白，与JAK酪氨酸激酶相互作用并抑制其活性。在胚胎发育过程中，CIS53/SOCS-3在胎儿肝脏的部分红系细胞中高度表达，并且这种表达不依赖于促红细胞生成素（EPO）信号传导。转基因介导的组成表达阻断胎儿红细胞生成，导致胚胎死亡。CIS3/SOCS-3的缺失导致12-16天的胚胎死亡，并伴有明显的红细胞增多。此外，胎儿肝祖细胞的个体体外增殖能力大大提高。结果表明，CIS3/SOCS-3在负性调节胎儿肝脏造血中起关键作用。我们还发现，在脾脏和表达EPOR的Ba/F3细胞（BF-ER）的红系祖细胞中，CIS3-SOCS3与EPO受体和JAK2结合，表明CIS3/SOCS3对EPO信号传导具有特异性的负调控作用。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