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.
Showing the most recent 10 out of 18 publications
