Mechanism of Normal and Ocogenic Kit Signaling in Vivo

描述（由申请人提供）：本提案的总体目标是继续我们对体外和体内Kit受体信号传导机制的研究，重点是造血。此外，我们将建立小鼠模型来研究Kit在肿瘤发生中的作用。在小鼠W位点编码的Kit受体酪氨酸激酶在配子发生、造血和黑色素形成中起作用。正常Kit受体介导的功能包括细胞增殖、细胞存活、细胞粘附、细胞迁移、分泌反应和分化。在人类肿瘤中，Kit的致癌激活被认为在肥大细胞增多症/肥大细胞白血病、急性骨髓性白血病、胃肠道间质肿瘤（GlST）和生殖细胞肿瘤中起作用。Kit受体的功能是通过激酶活化、受体自磷酸化和与各种信号分子的关联介导的，我们研究了PI 3-激酶和Src激酶在Kit介导的细胞增殖、抑制凋亡、细胞粘附和分泌反应中的作用。对表达突变Kit受体的Kit-/- BMMC的分析表明，这两种途径都有助于细胞的增殖和存活反应，消除这两种途径可以消除它们。这些研究还揭示了P1 3-激酶和Src激酶信号通路汇聚以激活Raci和JNK。此外，Fl 3-激酶的募集和激活在介导细胞粘附和分泌反应中起着关键作用。为了研究阻断Kit介导的Fl - 3激酶激活在体内的后果，我们在小鼠c-kit基因（KitY719F）中突变了酪氨酸719，这是Fl - 3激酶p85亚基的已知结合位点。对纯合突变体KitY719F/KitY719F小鼠的分析表明，Kit诱导的PIl 3-激酶活性在小鼠配子发生中起重要作用。在造血中，表型对腹膜肥大细胞数量的影响很小，没有其他表型。这些发现强调了细胞环境对体内Kit受体信号传导的重要性。这项应用的目的有两个：1)更精确地研究Kit介导的Fl - 3激酶和Kit介导的src信号在体内造血过程中的作用机制和后果，2)构建小鼠模型，研究Kit在肿瘤发生（造血恶性肿瘤和胃肠道间质肿瘤）中的作用，并阐明致癌激活的Kit受体的信号传导机制。

DESCRIPTION (provided by applicant): The overall objective of this proposal is to continue our investigations into the mechanisms of Kit receptor signaling in vitro and in vivo with emphasis on hematopoiesis. Furthermore, we will develop mouse models to investigate roles for Kit in oncogenesis. The Kit receptor tyrosine kinase encoded at the murine W locus functions in gametogenesis, hematopoiesis and melanogenesis Normal Kit receptor mediated functions include cell proliferation, cell survival, cell adhesion, cell migration, secretory response and differentiation. In human neoplasia oncogenic activation of Kit is thought to have roles in mastocytosis/mast cell leukemia, acute myelogenous leukemia, gastro intestinal stromal tumors (GlST) and germ cell tumors. Kit receptor functions are mediated by kinase activation, receptor autophosphorylation and association with various signaling molecules, We had investigated the role of PI 3-kinase and Src kinases in Kit mediated cell proliferation, suppression of apoptosis, cell adhesion and secretory responses. Analysis of Kit-/- BMMC expressing mutant Kit receptors indicated that both pathways contribute to the proliferative and the cell survival responses and that elimination of both pathways abolishes them. These studies also revealed that the P1 3-kinase and Src kinase signaling pathways converge to activate Raci and JNK. Moreover, recruitment and activation of Fl 3-kinase was shown to play a critical role in mediatingn cell adhesion and secretory responses. To investigate the consequences in vivo of blocking Kit mediated Fl 3-kinase activation we have mutated tyrosine 719 in the mouse c-kit gene (KitY719F), a known binding site for the p85 subunit of Fl 3-kinase. Analysis of homozygous mutant KitY719F/KitY719F mice indicated essential roles for Kit induced PIl 3-Kinase activity in mouse gametogenesis. In hematopoiesis phenotypes were minimal showing an effect on peritoneal mast cell numbers and no other phenotypes. These findings emphasize the importance of the cellular context for Kit receptor signaling in vivo. The purpose of this application is twofold: 1) to investigate more precisely the mechanism and the consequences of Kit mediated Fl 3-kinase and Kit mediated src signaling in vivo in hematopoiesis, and 2) to construct mouse models for investigating the role of Kit in oncogenesis (hematopoietic malignancies and gastrointestinal stromal tumors) and to elucidate the mechanisms of signaling by oncogenically activated Kit receptors.