Identification and characterization of proteins which interact with the Arg protein-tyrosine kinase

Arg and c-Abl represent the mammalian members of Abl family of nonreceptor protein-tyrosine kinases (PTKs). To gain insight into Arg function, the Arg subcellular localization, protein binding domains and interacting proteins were analyzed. We show here that unlike c-Abl, Arg is localized in cytoplasm and like c-Abl, the IB form of Arg also undergoes myristoylation on glycine-2. The exclusive cytoplasmic localization of Arg suggests that Arg and Abl may play different roles in cell growth and differentiation. To define Arg interacting domains, the interaction of Arg with Crk was studied. In vitro protein binding assay revealed that there are three SH3-binding sites for SH3 domains of Crk, Grb2 and Nck on an Arg C-terminal region (ArgSH3BR). Arg coimmunoprecipitates with Crk in sf9 cells and phosphorylates Tyr-221 of Crk in cotransfected COS cells. These findings suggest that Arg may play a role in the regulation of Crk activity. Arg function was further explored by using the yeast two-hybrid system to identify its interacting proteins. Using the ArgSH3BR bait, we identified two novel interacting proteins, ArgBP1 and ArgBP2 (Arg Binding Protein). ArgBP1 contains a C-terminal SH3 domain, several PEST sequences, a serine-rich region and an SH3-binding site. ArgBP1 is highly expressed in brain, heart and testis. ArgBP1 coimmunoprecipitates with Arg. The ArgBP1 SH3 domain binds to a C-terminal Arg SH3-binding site, and an N-terminal ArgBP1 proline-rich sequence binds to the Arg SH3 domain. ArgBP1 is localized in the cytoplasm. The similarity of the ArgBP1 expression pattern and subcellular localization to those of Arg and potentially strong association suggest that ArgBP1 is likely to be a regulator and/or effector of Arg function. ArgBP2, the second novel protein, contains three C-terminal SH3 domains, four potential phosphorylation sites for Arg and Abl. The first and third SH3 domains of ArgBP2 bind Arg in vitro. ArgBP2 is expressed at an extremely high level in heart. ArgBP2 is specifically localized to Z-band. These observations strongly suggest that ArgBP2 plays an important role in heart function and Arg and/or c-Abl regulate ArgBP2 function. Studies in this dissertation provide evidence for the possible function of Arg