Mutational analysis of the regulatory function of the c-Abl Src homology 3domain

The catalytic activity of the c-Abl tyrosine kinase is tightly regulated by its Src homology 3 (SH3) domain through a complex mechanism that may invol ve intramolecular binding to Pro242 in the linker region between the SH2 an d catalytic domains as well as interactions with a trans-inhibitor. We anal ysed the effect of mutation or replacement of SH3 on c-Abl tyrosine kinase activity and transformation. Random mutagenesis of SH3 identified several n ovel point mutations that dysregulated c-Abl kinase activity in vivo, but t he RT loop was insensitive to mutational activation. Activating SH3 mutatio ns abolished binding of proline-rich SH3 ligands in vitro, while mutations at Ser140 in the connector between the SH3 and SH2 domains activated Abl ki nase activity in vivo and in vitro but did not impair SH3 ligand-binding. A bl was regulated efficiently when its SH3 domain was replaced with a hetero logous SH3 from c-Src that binds a different spectrum of proline-rich ligan ds, but not by substitution of a modular WW domain with similar ligand-bind ing specificity. These results suggest that the SH3 domain regulates Abl pr incipally by binding to the atypical intramolecular ligand Pro242 rather th an a canonical PxxP ligand. Coordination between the SH3 and SH2 domains me diated by the connector region may be required for regulation of Abl even i n the absence of SH2 ligand binding.