Screening of signal transducing molecules by using transient expression

为了寻找对细胞增殖或分化起重要作用的酪氨酸激酶的新底物，我们建立了以活性c-kit为诱饵的双杂交筛选方法。我们克隆了含有SH2结构域和PH结构域的新的接头分子APS和STAP-1。RT-POR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。但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，提示STAP-1与未分化的细胞类型有关。在293细胞中，STAP-1被激活的c-kit蛋白酪氨酸磷酸化。体外结合实验表明，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。这些结果表明STAP-1具有…的功能在造血干细胞中c-kit下游有更多的daptor分子。此外，我们最近克隆了一种新的c-kit或其他酪氨酸激酶底物。这个基因，我们称之为WARS，不包含任何SH2或PH结构域，但有一个新的结构域与果蝇中Ras-MAP激酶的负调节基因相关。我们将试图发现该基因在癌症发生发展中的作用。我们克隆了一个新的SH2蛋白Jab，它与JAK2的激活域相结合。Jab与JAK酪氨酸激酶相互作用并抑制其活性。通过SH2结构域，Jab与JAK激酶激活环中的磷酸酪氨酸结合并抑制其催化活性。我们已经证明，干扰素-γ强烈地诱导了JAB。我们证明，JAB缺陷小鼠在围产期死亡，淋巴发育改变，包括体内激活的T细胞的产生。这种致命性在RAG2或干扰素-γ上被消除。背景不足。根据这一结果，我们认为Jab在干扰素-γ的负反馈调节中起着重要作用。较少

To identify the novel substrate of tyrosine kinases which is important for proliferation or differentiation, we developed a two-hybrid screening using active c-kit as bait. We cloned new adaptor molecules, APS and STAP-1 which contain SH2 domain and PH domain. RT-POR 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- hematopoietic 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 associated 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 protein including c-kit and STAT5. These suggest that STAP-1 function as a a … More daptor molecule downstream of c-kit in hematopoietic stem cells.Furthermore, we recently cloned a novel substrate of c-kit or other tyrosine kinases. This gene, we call WARS, does not contain any SH2 or PH domain but has a novel domain related to a gene that is a negative regulator of Ras-MAP kinase in Drosophila. We will try to find the function of this gene in development of cancer.We cloned a novel SH2 protein JAB, that binds to the kinase domain of JAK2. JAB interacts with and inhibits JAK tyrosine kinases. Through the SH2 domain, JAB binds to a phosphotyrosine in the activation loop of the JAK kinases and suppresses their catalytic activity. We have shown that JAB is strongly induced by interferon-γ. We demonstrate that JAB deficient mice die perinatally with altered lymphoid development including the generation of activated T cells in vivo. The lethality is eliminated on a Rag2 or interferon-γ. Deficient background . Based on the results, we propose that JAB plays an essential role in negative feedback regulation of interferon-γ. Less