Screening of signal transducing molecules by using transient expression

为了鉴定对增殖或分化至关重要的新型酪氨酸激酶底物，我们开发了一种以活性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结构域的新接头分子APS和STAP-1。RT-POR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、STAT5等多种酪氨酸磷酸化蛋白相互作用。这些结果表明，在造血干细胞中，STAP-1作为c-kit下游的一个更多的受体分子发挥作用。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，但具有与果蝇Ras-MAP激酶负调控基因相关的新结构域。我们将试图找出这个基因在癌症发展中的作用。我们克隆了一个新的SH2蛋白JAB，它与JAK2的激酶结构域结合。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强烈诱导JAB。我们证明，JAB缺陷小鼠在围产期死亡时淋巴细胞发育发生改变，包括体内活化T细胞的产生。在Rag2或干扰素-γ上消除致死率。背景不足。基于这些结果，我们认为JAB在干扰素-γ的负反馈调控中发挥了重要作用。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