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 repression of catalytic activity. The crystal structure of the Src family kinase Hck, with its regulatory domains intact, has been solved. It predicts that a conserved residue, Trp260, at the end of the linker between the SH2 and the catalytic domains plays an important role in regulation by the SH3 and SH2 domains. We have mutated this residue and compared the activities of C-terminally phosphorylated wild type Hck and W260A Hck. The W260A mutant has a higher specific activity than wild type Hck. The W260A mutant requires autophosphorylation at Tyr416 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 Trp260 plays a critical role in the coupling of the regulatory domains to the catalytic domain, as well as in positioning the ligand binding surfaces.
LaFevre-Bernt
M
M
Department of Physiology and Biophysics, School of Medicine, State University of New York at Stony Brook, Stony Brook, New York 11794, USA.
Sicheri
F
F
Pico
A
A
Porter
M
M
Kuriyan
J
J
Miller
W T
WT
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D020134:Catalytic Domain; D002460:Cell Line; D017124:Conserved Sequence; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D017354:Point Mutation; D017433:Protein Structure, Secondary; D011505:Protein-Tyrosine Kinases / Q000737:chemistry* / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000378:metabolism*; D051572:Proto-Oncogene Proteins c-hck; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D018411:Spodoptera; D013379:Substrate Specificity; D014162:Transfection; D014364:Tryptophan; D018909:src Homology Domains
D009842:Oligopeptides; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D014364:Tryptophan; D011505:Protein-Tyrosine Kinases; D051572:Proto-Oncogene Proteins c-hck