SIGNAL TRANSDUCTION THOUGH THE C-CBL PROTOONCOGENE

描述：p120Cbl(Cbl)是一种高度酪氨酸磷酸化的蛋白。 闸蟹癌基因诱发慢性髓系白血病及其致癌作用 就凭它自己的力量。初步结果表明，致癌形式 无论Cb1的黏附状态如何，Cb1的结构上都是磷酸化的 细胞，并通过以下方式使细胞独立于锚定和致瘤 激活整合素依赖的途径。相比之下，野生型 Cb1的酪氨酸以黏附依赖的方式被磷酸化，并且 通过SH2介导的蛋白质-蛋白质连接到信号通路 互动。这里提出的工作将详细地探索分子 Cbl在介导黏附依赖信号中的基础和意义， 如果被结构性激活，可能会介导恶性转化。 造血细胞。 与酪氨酸磷酸化的Cbl相互作用的蛋白质将直接 利用酪氨酸磷酸化的Cbl作为诱饵分子在 新型酵母双杂交系统。在另一种方法中，相互作用的蛋白质 与Cb1以黏附依赖的方式结合，而不考虑磷酸化 Cbl的状态将被确定。一种新的哺乳动物双杂交表达系统 系统将在这些研究中使用。 带有Cb1的序列将介导蛋白质-蛋白质相互作用 鉴定；这些实验将包括定点突变 酵母双杂交系统中Cbl的分析。结果将提供 关于Cbl参与的蛋白质-蛋白质相互作用的性质的信息 在细胞黏附过程中，以及如何设计实验的信息 Cbl的功能重要性。 我们将采取几种方法来研究其功能意义 整合素信号中含有CBL的信号复合体。构成要素 酪氨酸磷酸化形式的Cbl(功能增益突变体)将是 表达以激活Cb1介导的信号通路，而不管 粘附性-细胞的状态。在另一种方法中， 野生型和突变型Cbl的表达将在细胞中进行研究 内源性Cbl水平低或不存在。 此外，还会产生“显性-负面”形式的Cbl，以扰乱 表达内源性Cbl的细胞内特异性Cbl蛋白复合体 CBL.在每种情况下，对各种生化和生物的影响 将对信号事件进行研究。

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.