Mechanism of Normal and Ocogenic Kit Signaling in Vivo

描述(申请人提供)：这项建议的总体目标是继续我们在体外和体内对Kit受体信号转导机制的研究，重点是造血。此外，我们将开发小鼠模型来研究Kit在肿瘤发生中的作用。小鼠W基因座编码的Kit受体酪氨酸激酶在配子发生、造血和黑素生成中的功能正常的Kit受体介导的功能包括细胞增殖、细胞存活、细胞黏附、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、急性髓系白血病、胃肠间质瘤(GlST)和生殖细胞肿瘤中起作用。Kit受体的功能是通过激活Kit、受体自身磷酸化以及与多种信号分子结合来实现的，我们研究了PI3-Kis和Src-Kines在Kit介导的细胞增殖、抑制细胞凋亡、细胞黏附和分泌反应中的作用。对Kit-/-BMMC表达突变Kit受体的分析表明，这两条途径都参与了增殖反应和细胞存活反应，而这两条途径的取消就取消了它们。这些研究还表明，P13K和Src信号通路汇聚在一起，激活了RACI和JNK。此外，F13-激酶的募集和激活在调节细胞黏附和分泌反应中起着关键作用。为了研究阻断Kit介导的F13-激酶激活在体内的后果，我们在小鼠c-Kit基因(KitY719F)上突变了酪氨酸719，该基因是F13-Kit的P85亚单位的已知结合部位。对纯合子突变的KitY719F/KitY719F小鼠的分析表明，Kit诱导的PIL-3-Kinase活性在小鼠配子发生中起重要作用。在造血方面，表型很少，表明对腹膜肥大细胞数量有影响，但不影响其他表型。这些发现强调了细胞环境对体内Kit受体信号的重要性。这一应用的目的有两个：1)更准确地研究Kit介导的F13-K和Kit介导的体内src信号在造血系统中的机制和后果；2)建立Kit在肿瘤发生(血液系统恶性肿瘤和胃肠道间质瘤)中的作用模型，并阐明肿瘤激活的Kit受体的信号转导机制。

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.