title: Activation of the Src family kinase Hck without SH3-linker release.
abstract: Src family protein-tyrosine kinases are regulated by intramolecular binding of the SH2 domain to the C-terminal tail and association of the SH3 domain with the SH2 kinase-linker. The presence of two regulatory interactions raises the question of whether disruption of both is required for kinase activation. To address this question, we engineered a high affinity linker (HAL) mutant of the Src family member Hck in which an optimal SH3 ligand was substituted for the natural linker. Surface plasmon resonance analysis demonstrated tight intramolecular binding of the modified HAL sequence to SH3. Hck-HAL was then combined with a tail tyrosine mutation (Y501F) and expressed in Rat-2 fibroblasts. Surprisingly, Hck-HAL-Y501F showed strong transforming and kinase activities, demonstrating that intramolecular SH3-linker release is not required for SH2-based kinase activation. In Saccharomyces cerevisiae, which lacks the negative regulatory tail kinase Csk, wild-type Hck was more strongly activated in the presence of an SH3-binding protein (human immunodeficiency virus-1 Nef), indicating persistence of native SH3-linker interaction in an active Hck conformation. Taken together, these data support the existence of multiple active conformations of Src family kinases that may generate unique downstream signals.
authors.affiliation: Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA.
authors.forename: Edwina C
authors.initials: EC
authors.lastname: Lerner
authors.forename: Ronald P
authors.initials: RP
authors.lastname: Trible
authors.forename: Anthony P
authors.initials: AP
authors.lastname: Schiavone
authors.forename: James M
authors.initials: JM
authors.lastname: Hochrein
authors.forename: John R
authors.initials: JR
authors.lastname: Engen
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Smithgall
mesh_terms: D000818:Animals; D001665:Binding Sites; D019921:Biotinylation; D002460:Cell Line; D018360:Crystallography, X-Ray; D004789:Enzyme Activation; D015870:Gene Expression; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D010446:Peptide Fragments / Q000235:genetics; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D051572:Proto-Oncogene Proteins c-hck / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017422:Sequence Analysis, DNA; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D020349:Surface Plasmon Resonance; D014162:Transfection; D018909:src Homology Domains / Q000502:physiology*
chemical_list: D010446:Peptide Fragments; D011994:Recombinant Proteins; C496631:HCK protein, human; D051572:Proto-Oncogene Proteins c-hck