Characterization of the Abl -interactor (Abi) proteins

Abl-interactor (Abi) proteins are targets of Abl-family non-receptor tyrosine kinases. Abl tyrosine kinases are ubiquitously expressed proto-oncoproteins with pleiotropic biological effects, including regulation of cell growth, transformation, and cytoskeletal dynamics. Abi proteins are adaptors and substrates for the Abl kinases that have been implicated in the regulation of Abl transformation. Abi proteins are also involved in signaling events that regulate cell motility. This work describes attempts to further characterize the Abi proteins and their interactions with c-Abl and Abl-derived oncoproteins in normal development and tumorigenesis. We show that Abi proteins are degraded in an ubiquitin-dependent fashion in the presence of oncogenic forms of the Abl and Src tyrosine kinases. Significantly, Abi protein expression is lost in cell lines and bone marrow from patients with aggressive Bcr-Abl-positive leukemias. Loss of Abi proteins may therefore contribute to the progression of certain cancers. To investigate possible developmental contributions of Abi proteins, this work examines the temporal-spatial pattern of expression, phosphorylation, and sub-cellular localization of Abi-1 and Abi-2 throughout mouse nervous system development. Our findings suggest that Abi-1 and Abi-2 make unique and shared contributions to nervous system development, and that Abi adaptors exhibit proper expression patterns and sub-cellular localization to participate in Abl kinase signaling in the nervous system. To study the connection between Abi proteins and cytoskeletal reorganization and cell motility, we examined their localization in live, moving cells. Abi-1 and Abi-2b fused to enhanced yellow fluorescent protein (EYFP) are specifically recruited to the tips of protrusive elements termed lamellipodia and filopodia, but not to sites of cell retraction. Targeting to these sites of protrusion is attributed to the homologous amino-terminus of Abi-1 and Abi-2b, a novel function for this region of Abi proteins. These data implicate Abi proteins in processes that regulate actin polymerization dynamics in lamellipodia and filopodia and provide further support for the Abi proteins as regulators of cell motility. Lastly, a yeast two-hybrid screen was undertaken to identify novel Abi-interacting proteins. Several cDNA clones that encode proteins capable of binding a region of Abi-2b were isolated using this method. One clone encodes a partial protein with significant identity to Zygin II/FEZ 2, a human homologue of the Caenorhabditis elegans UNC-76 protein. Abi proteins interact with this clone in in vitro binding assays, as well as in the two-hybrid screen. UNC-76 family proteins contribute to axon outgrowth and fasciculation and therefore provide a novel connection between the Abi proteins and events that regulate cell motility and adhesion. Together these data provide novel insight into the potential contributions of Abi proteins to neuronal development, transformation, and cell motility.