C Cbl in Modulation of Cell Adhesion and Morphology

描述：（改编自研究者摘要） 蛋白质c-Cbl在正常细胞中被酪氨酸磷酸化， 在各种刺激和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其与关键蛋白的结合。 信号蛋白c-Cbl还含有能够结合 与几种PTK和大量富含脯氨酸的基序结合， 多种含SH3结构域的蛋白质。c-Cbl与 各种各样的信号蛋白支持c-Cbl作为一种 多价衔接蛋白。此外，c-Cbl能够抑制 一些PTK的活动。这种抑制作用至少可以部分地解释为， 通过最近发现的c-Cbl促进泛素化的能力， 尽管有丰富的生物化学数据，但生物学上， c-Cbl的功能仍然知之甚少。尤其是， c-Cb1是否能积极调节细胞功能。此外， 原癌基因c-Cb 1对细胞转化的作用尚未在 详细 我们一直在研究c-Cb 1的酪氨酸磷酸化及其功能作用 在细胞活化和转化中。我们分析了 在v-Abl转化的NIH 3T3成纤维细胞中的c-Cbl证实野生型c-Cbl表达增加。 c-Cbl，而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效应。我们有 最近表明，c-Cb 1介导的形态学逆转 这些细胞的转化是由细胞外基质的增加引起的。 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。 目前的建议旨在进一步发展这项研究。其整体 目的了解c-Cb 1在细胞粘附调节中的作用 和形态，并确定这些影响的分子基础。的 在拟议的研究中要检验的假设是，这些生物效应 c-Cbl的表达是通过触发P1 - 3 '激酶、Vav 2和Crk依赖性激酶介导的。 信号传导导致Rho家族GTP酶和可能的Rap 1的活化。我们 进一步假设泛素化驱动的蛋白质降解 干扰组装的焦点粘连和应力纤维是参与 c-Cbl对v-Abl转化细胞的影响。因此，具体 本课题的研究目的如下：1.确定相对贡献 Rho样和Rap GTP酶对所观察到的c-Cbl依赖性的 v-Abl转化的成纤维细胞的粘附和铺展。 2.为了阐明小GTP酶的激活机制， c-Cbl依赖性促进v-Abl转化的细胞粘附和铺展 成纤维细胞，并评估这些机制的相对贡献， c-Cbl的总体生物学效应。 3.为了阐明c-Cbl依赖的泛素化的机制， 参与c-Cbl对v-Abl转化的成纤维细胞的作用。 4.为了确定c-Cbl过表达对粘附的影响， 用Bcr-Abl，a 组成型活性PTK，其导致慢性髓性白血病。

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.