Growth and differentiation signals of the erythropoietin receptor

促红细胞生成素受体的生长和分化信号

• 批准号: 09470036
• 负责人: YOSHIMURA Akihiko
• 金额: $ 8.58万
• 依托单位: Kurume university
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09470036/
• 关键词: tyrosine kinase; JAK; STAT; CIS; cytokine; knockout mice; erythropoietin; fetal liver hematopoiesis; エリスポエチン; エリスロポエチン受容体; シグナル伝達; 細胞分化; SH2ドメイン; りん酸化; 情報伝達; CISファミリー

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

为了筛选对造血干细胞自我更新或分化具有重要作用的c-kit新底物，用荧光激活细胞分选仪分选小鼠CD 34阴性、Sca-1阳性、c-KIT阳性和Lineage标记阴性（CD 34-Sca-1+c-Kit-Lin-）细胞，构建双杂交cDNA文库。以c-kit为诱饵，通过筛选，我们克隆了一个新的cDNA，命名为STAP-1，它编码一个含有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的骨髓细胞组分，并且在CD 34-Sca-1+c-Kit+Lin-造血干细胞富集组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，当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，这表明STAP-1与M1细胞的分化有关。 ...更多信息 未分化细胞类型的艾德。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的SH 2结构域与c-kit和STAT 5等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下游的衔接分子; CIS 3/SOC 53是含SH 2的小分子蛋白，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，CIS 53/SOCS-3在胎肝的一些但不是所有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。转基因介导的组成型表达阻断胎儿红细胞生成，导致胚胎死亡。CIS 3/SOCS-3的缺失导致在12-16天时的胚胎致死，其与显著的红细胞增多相关。此外，胎肝祖细胞的个体体外增殖能力大大增加。结果表明，CIS 3/SOCS-3在负调控胎肝造血中起关键作用。我们还证明了CIS 3-SOCS 3与来自脾的红系祖细胞和表达EPOR的Ba/F3细胞（BF-ER）中的EPO受体以及JAK 2结合，表明CIS 3/SOCS 3对EPO信号传导具有特异性负调节作用。少

项目成果

Misawa,H.,et al: "Cloning and Characterization of a novel class II Phosphoinositide 3-kinase containing C2 domein." Biochem.Biophys.Res.Comm.(in press). (1998)

Misawa,H.,et al：“含有 C2 结构域的新型 II 类磷酸肌醇 3-激酶的克隆和表征。”

Yasukawa H, et al.: "The JAK-Binding Protein JAB Inhibits Janus Tyrosine Kinase Activity Through Binding in the Activation Loop."EMBO J.. 18, 5. 1309-1320 (1999)

Yasukawa H 等人：“JAK 结合蛋白 JAB 通过与激活环中的结合抑制 Janus 酪氨酸激酶活性。”EMBO J.. 18, 5. 1309-1320 (1999)

Matsumoto A, et al.: "Suppression of STAT5 Functions in Liver, Mammary Glands, and T Cells in Cytokine-Inducible SH2-Containing Protein 1 Transgenic Mice"Mol. Cell. Biol. 19・9. 6396-6407 (1999)

Matsumoto A 等人：“含有细胞因子诱导的 SH2 蛋白的转基因小鼠中 STAT5 功能的抑制”，Mol. 19·9。

Wakioka T,et al.: "APS,an Adaptor Protein Containing PH and SH2 Domains Inhibits the JAK-STAT Pathway in Collaboration with c-Chl" Leukemia. in press. (1999)

Wakioka T 等人：“APS，一种含有 PH 和 SH2 结构域的接头蛋白，与 c-Chl 协同抑制 JAK-STAT 通路”白血病。

Masuhara,M.,et al: "Cloning and Characterization of Novel CIS Family Genes." Biochem.Biophys.Res.Comm.239・2. 439-446 (1997)

Masuhara, M., 等人：“新型 CIS 家族基因的克隆和表征。”Biochem.Biophys.Res.Comm.239·2（1997）。