SIGNAL TRANSDUCTION THOUGH THE C-CBL PROTOONCOGENE

描述：p120 Cbl（Cbl）是一种高度酪氨酸磷酸化的蛋白质， bcrabl癌基因诱导的慢性粒细胞白血病， 在它自己的权利。 初步结果表明， 无论Cbl的粘附状态如何， 细胞，并使细胞贴壁不依赖性和致瘤性， 激活整合素依赖性途径。 相反，野生型形式 的Cbl以粘附依赖性方式被酪氨酸磷酸化，并且 通过SH 2介导的蛋白质-蛋白质连接到信号通路 交互. 这里提出的工作将详细探讨分子 Cbl介导粘附依赖性信号的基础和意义， 其如果被组成性激活，可介导恶性转化， 造血细胞 与酪氨酸磷酸化的Cbl相互作用的蛋白质将被直接 通过使用酪氨酸磷酸化的Cbl作为诱饵分子， 新的酵母双杂交系统。 在另一种方法中， 与Cbl以粘附依赖的方式，无论磷酸化 将确定CBL的状态。 一种新的哺乳动物双杂交表达系统 系统将用于这些研究。 具有介导蛋白质-蛋白质相互作用的Cbl的序列将是 鉴定;这些实验将包括定点诱变 在酵母双杂交系统中分析Cbl。 结果将提供 Cbl参与的蛋白质-蛋白质相互作用的性质的信息 在细胞粘附过程中，以及如何设计实验的信息， CBL功能的重要性 将采取几种方法来研究 整合素信号传导中的含cbl信号传导复合物。 组成型 Cbl的酪氨酸磷酸化形式（“功能获得性”突变体）将是 表达以激活Cbl介导的信号传导途径，而不管 细胞的粘附状态。 在另一种方法中， 将在细胞中研究野生型和突变型Cbl的表达 其中Cbl的内源性水平低或不存在。 此外，将产生“显性-阴性”形式的Cbl，以破坏 特异性含Cbl蛋白复合物在表达内源性 Cbl.在每种情况下，对各种生物化学和生物学的影响 将研究信号事件。

DESCRIPTION: p120Cbl (Cbl) is highly tyrosine phosphorylated protein in chronic myelogenous leukemia induced by bcrabl oncogene, and also oncogenic in its own right. Preliminary results demonstrate that the oncogenic form of Cbl is constitutively phosphorylated regardless of the adhesion status of the cells, and makes cells anchorage-independent and tumorigenic by activating an integrin-dependent pathway. In contrast, the wild-type form of Cbl is tyrosine phosphorylated in an adhesion-dependent manner, and connects to signaling pathways via SH2-mediated protein-protein interactions. The work proposed here will explore in detail the molecular basis and significance of Cbl in mediating adhesion-dependent signals, which, if constitutively activated, may mediate malignant transformation in hematopoietic cells. Proteins that interact with tyrosine-phosphorylated Cbl will be directly cloned by employing tyrosine-phosphorylated Cbl as a bait molecule in a novel yeast two-hybrid system. In another approach, proteins interacting with Cbl in an adhesion-dependent manner regardless of the phosphorylation status of Cbl will be identified. A novel mammalian two-hybrid expression system will be used in these studies. The sequences with Cbl that mediate the protein-protein interactions will be identified; these experiments will include site-directed mutagenesis analysis of Cbl in a yeast two-hybrid system. The results will provide information on the nature of the protein-protein interactions Cbl is engaged in during cell adhesion, and information on how to design experiments on functional importance of Cbl. Several approaches will be taken to study the functional significance of Cbl-containing signaling complexes in integrin signaling. Constitutively tyrosine-phosphorylated forms of Cbl ("gain-of function" mutants) will be expressed to activate Cbl-mediated signaling pathways regardless of the adhesion-status of the cells. In another approach, the effects of the expression of wild- type and mutant forms of Cbl will be studied in cells where endogenous levels of Cbl are low or absent. In addition, "dominant-negative" forms of Cbl will be generated to disrupt specific Cbl-containing protein complexes in cells expressing endogenous Cbl. In each case, the effects on various biochemical and biological signaling events will be studied.