INTRAMOLECULAR REGULATORY INTERACTIONS IN THE SRC FAMILY KINASE HCK PROBED BY MUTAGENESIS OF A CONSERVED TRYPTOPHAN RESIDUE

Intramolecular interactions between the Src homology domains (SH2 and SH3) and the catalytic domains of Src family kinases result in repress ion of catalytic activity. The crystal structure of the Src family kin ase Hck, with its regulatory domains intact, has been solved. It predi cts that a conserved residue, Trp(260), at the end of the linker betwe en the SH2 and the catalytic domains plays an important role in regula tion by the SH3 and SH2 domains. We have mutated this residue and comp ared the activities of C-terminally phosphorylated wild type Hck and W 260A Hck, The W260A mutant has a higher specific activity than wild ty pe Hck. The W260A mutant requires autophosphorylation at Tyr(416) for full activity, but it is not activated by ligand binding to the SH3 or SH2 domains. This mutation also changes the accessibility of the SH2 and SH3 domains to their cognate peptide ligands, Our results indicate that Trp(260) plays a critical role in the coupling of the regulatory domains to the catalytic domain, as well. as in positioning the ligan d binding surfaces.