| To investigate the sufficiency of dimerization for Abl kinase activation, I utilized a conditional system of oligomerization. This system consists a chemical dimerizer, a compound with two binding surfaces that can bind simultaneously to two protein targets, and a target domain that binds the dimerizing ligand (FKBP). A chimeric Abl-FKBP protein was constructed and characterized in a variety of cellular transformation assays. Using this system I demonstrated that oligomerization was sufficient to activate the Abl kinase in vivo and induce transformation of fibroblasts and hematopoeitic cells. This system could be a useful tool for investigating the primary consequences of activating Abl kinase in vivo as well as understanding the regulation of c-Abl.; While the characterization of Abl-FKBP chimeric proteins suggests that oligomerization is sufficient for kinase activation, it did not exclude a role for other Bcr domains for transformation and leukemogenesis by Bcr-Abl. To investigate the effects of oligomerization on Bcr-Abl, I replaced the coiled-coil domain of Bcr with FKBP to create a Bcr-Abl-FKBP chimera that could be inducibly dimerized. Like Abl-FKBP, the chimeric proteins were able to induce kinase activation and transformation of fibroblasts, hematopoetic cells, and primary B-lymphoid cells in a ligand-dependent manner. These results suggest the Bcr-Abl-FKBP conditional mutant will be a valuable reagent for studying the mechanism of cellular transformation and resistance to apoptosis induced by Bcr-Abl, and may be useful for generation of conditional animal models of Ph1 positive leukemia, particularly by transgenic approaches.; I generated a Bcr mutant with alanines substituted for the hydrophobic residues of the coiled-coil domain, a second mutant with substitution of a single proline in the middle of the domain, and a third mutant lacking the coiled-coil domain. Results suggest that oligomerization of Bcr-Abl stimulates Abl kinase activity by promoting intermolecular autophosphorylation, but abrogation of the intramolecular inhibitory function of the SH3 domain can substitute for oligomerization.; Paradoxically, the proline substitution mutant transformed fibroblasts and primary B cells in vitro and efficiently induced B-lymphoid leukemia instead of CML-like disease in mice. Myeloid leukemogenesis was partially restored by SH3 mutation. This mutant may retain some oligomerization activity in vivo although it was not detected in a coprecipitation assay. I also identified a revertant of a Bcr-Abl coiled-coil mutant that efficiently transforms fibroblasts, and localized the reversion mutation to a novel region in the Abl COOH-terminus. (Abstract shortened by UMI.)... |
