C Cbl in Modulation of Cell Adhesion and Morphology

C Cbl 调节细胞粘附和形态

• 批准号: 6597789
• 负责人: ALEXANDER Y TSYGANKOV
• 金额: $ 3.09万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-07-01 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6597789
• 关键词: athymic mouse; biological signal transduction; cell adhesion; connective tissue neoplasm; enzyme activity; extracellular matrix proteins; fibroblasts; guanosinetriphosphatases; interleukin 3; oncoproteins; protein tyrosine kinase; ubiquitin; viral carcinogenesis; virus related neoplasm /cancer

DESCRIPTION: (Adapted from the investigator's abstract) The protooncogenic protein c-Cbl is tyrosine phosphorylated both in normal cells in response to various stimuli and in cells transformed by oncogenic protein tyrosine kinases (PTKs). Tyrosine phosphorylation of c-Cbl enhances its binding to crucial signaling proteins. c-Cbl also contains an SH2-like domain capable of binding to several PTKs and a large number of proline-rich motifs capable of binding to multiple SH3 domain-containing proteins. The ability of c-Cbl to interact with a wide variety o signaling proteins argues in favor of c-Cbl acting as a multivalent adaptor protein. Furthermore, c-Cbl is capable of inhibiting activities of some PTKs. This inhibition may be explained, at least partially, by the recently discovered ability of c-Cbl to facilitate ubiquitination of Cbl-associated PTKs, In spite of the abundance of biochemical data, biological functions of c-Cbl remain poorly understood. In particular, it remains unclear whether c-Cb1 can positively regulate cell functions. Furthermore, effects of protooncogenic c-Cb1 on cell transformation have not been characterized in detail. We have been studying tyrosine phosphorylation of c-Cb1 and its functional role in cell activation and transformation. Our analysis of biological functions of c-Cbl in v-Abl-transformed NIH 3T3 fibroblasts demonstrated that wild-type c-Cbl, but not its tyrosine phosphorylation-defective mutants, facilitates adhesion and spreading of transformed cells and reduces their anchorage independence, exhibiting an overall transformation-suppressing effect. We have recently shown that the c-Cb1-mediated reversion of morphological transformation of these cells is caused by an increase in extracellular matrix production, and that this increase is linked to the c-Cbl-dependent activation of small GTPases regulating cytoskeletal rearrangements. The current proposal is designed to further develop this research. Its overall objective is to understand the role of c-Cb1 in the regulation of cell adhesion and morphology and to determine the molecular basis of these effects. The hypothesis to be tested in the proposed study is that these biological effects of c-Cbl are mediated by triggering of P1-3' kinase, Vav2 and Crk-dependent signaling leading to activation of Rho-family GTPases and, possibly, Rap 1. We further hypothesize that ubiquitination-driven degradation of proteins interfering with the assembly offocal adhesions and stress fibers is involved in the effects of c-Cbl on v-Abl-transformed cells. Accordingly, the specific aims of our project are as follows: 1. To determine relative contributions of Rho-like and Rap GTPases to the observed c-Cbl-dependent facilitation of adhesion and spreading of v-Abl-transformed fibroblasts. 2. To elucidate the mechanisms of activation of small GTPases involved in c-Cbl-dependent facilitation of adhesion and spreading of v-Abl-transformed fibroblasts and to assess relative contributions of these mechanisms to the overall biological effect of c-Cbl. 3. To elucidate the mechanisms whereby c-Cbl-dependent ubiquitination is involved in the effects of c-Cbl on v-Abl-transformed fibroblasts. 4. To determine the effect of c-Cbl overexpression on adhesion and transformation potential of hematopoietic cells transformed with Bcr-Abl, a constitutively active PTK, which causes chronic myeologenous leukemia.

描述：（改编自研究者的摘要）原癌基因 蛋白质 c-Cbl 在正常细胞中被酪氨酸磷酸化，以响应 各种刺激以及致癌蛋白酪氨酸激酶转化的细胞中 （PTK）。 c-Cbl 的酪氨酸磷酸化增强了其与关键蛋白的结合 信号蛋白。 c-Cbl 还包含能够结合的 SH2 样结构域 多个 PTK 和大量富含脯氨酸的基序能够结合 多个含有 SH3 结构域的蛋白质。 c-Cbl 相互作用的能力 多种 o 信号蛋白都支持 c-Cbl 作为 多价接头蛋白。此外，c-Cbl 能够抑制 一些 PTK 的活动。这种抑制可以至少部分地解释， 最近发现 c-Cbl 能够促进泛素化 Cbl 相关的 PTK，尽管有丰富的生化数据，但生物学 c-Cbl 的功能仍然知之甚少。特别是目前还不清楚 c-Cb1是否能正向调节细胞功能。此外，影响 原癌基因 c-Cb1 对细胞转化的影响尚未在 细节。 我们一直在研究c-Cb1的酪氨酸磷酸化及其功能作用 在细胞活化和转化中。我们对生物功能的分析 v-Abl 转化的 NIH 3T3 成纤维细胞中的 c-Cbl 证明野生型 c-Cbl，但不是其酪氨酸磷酸化缺陷突变体，促进 转化细胞的粘附和扩散并减少其锚定 独立性，表现出整体的转化抑制作用。我们有 最近表明，c-Cb1 介导的形态逆转 这些细胞的转化是由细胞外基质的增加引起的 产生，并且这种增加与 c-Cbl 依赖性激活有关 调节细胞骨架重排的小 GTP 酶。 当前的提案旨在进一步发展这项研究。其总体 目的是了解 c-Cb1 在细胞粘附调节中的作用 和形态并确定这些效应的分子基础。这 在拟议的研究中要检验的假设是这些生物效应 c-Cbl 的作用是通过触发 P1-3' 激酶、Vav2 和 Crk 依赖性介导的 信号传导导致 Rho 家族 GTPases 以及可能的 Rap 1 的激活。我们 进一步假设泛素化驱动的蛋白质降解 干扰局灶粘连和应力纤维的组装 c-Cbl 对 v-Abl 转化细胞的影响。据此，具体 我们项目的目标如下： 1. 确定 Rho 样和 Rap GTP 酶对观察到的 c-Cbl 依赖性促进作用 v-Abl 转化的成纤维细胞的粘附和扩散。 2. 阐明小GTP酶参与的激活机制 c-Cbl依赖性促进v-Abl转化的粘附和扩散 成纤维细胞并评估这些机制对成纤维细胞的相对贡献 c-Cbl 的总体生物学效应。 3. 阐明c-Cbl依赖性泛素化的机制 参与 c-Cbl 对 v-Abl 转化的成纤维细胞的影响。 4. 确定 c-Cbl 过表达对粘附和粘附的影响 Bcr-Abl 转化造血细胞的转化潜力 组成型活性 PTK，导致慢性粒细胞白血病。