Mutational analysis of the regulatory function of the c-Abl Src homology 3 domain.
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 involve intramolecular binding to Pro242 in the linker region between the SH2 and catalytic domains as well as interactions with a trans-inhibitor. We analysed the effect of mutation or replacement of SH3 on c-Abl tyrosine kinase activity and transformation. Random mutagenesis of SH3 identified several novel point mutations that dysregulated c-Abl kinase activity in vivo, but the RT loop was insensitive to mutational activation. Activating SH3 mutations abolished binding of proline-rich SH3 ligands in vitro, while mutations at Ser140 in the connector between the SH3 and SH2 domains activated Abl kinase activity in vivo and in vitro but did not impair SH3 ligand-binding. Abl was regulated efficiently when its SH3 domain was replaced with a heterologous SH3 from c-Src that binds a different spectrum of proline-rich ligands, but not by substitution of a modular WW domain with similar ligand-binding specificity. These results suggest that the SH3 domain regulates Abl principally by binding to the atypical intramolecular ligand Pro242 rather than a canonical PxxP ligand. Coordination between the SH3 and SH2 domains mediated by the connector region may be required for regulation of Abl even in the absence of SH2 ligand binding.
Brasher
B B
BB
Enanta Pharmaceuticals, 500 Arsenal Street, Watertown, MA 02472, USA.
Roumiantsev
S
S
Van Etten
R A
RA
D000595:Amino Acid Sequence; D000818:Animals; D026481:Blotting, Far-Western; D017173:Caenorhabditis elegans; D002384:Catalysis; D002460:Cell Line; D002471:Cell Transformation, Neoplastic; D004789:Enzyme Activation; D007700:Kinetics; D008024:Ligands; D016296:Mutagenesis / Q000235:genetics*; D017354:Point Mutation / Q000235:genetics*; D011485:Protein Binding; D017434:Protein Structure, Tertiary / Q000235:genetics; D016315:Proto-Oncogene Proteins c-abl / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D018909:src Homology Domains / Q000235:genetics*
D008024:Ligands; D011993:Recombinant Fusion Proteins; D016315:Proto-Oncogene Proteins c-abl