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.
