ABSTRACT
The protein tyrosine kinase C-terminal Src kinase (Csk) is activated by the engagement of its Src homology (SH) 2 domain. However, the molecular mechanism required for this is not completely understood. The crystal structure of the active Csk indicates that Csk could be activated by contact between the SH2 domain and the beta3-alphaC loop in the N-terminal lobe of the kinase domain. To study the importance of this interaction for the SH2-domain-mediated activation of Csk, we mutated the amino acid residues forming the contacts between the SH2 domain and the beta3-alphaC loop. The mutation of the beta3-alphaC loop Ala228 to glycine and of the SH2 domain Tyr116, Tyr133, Leu138, and Leu149 to alanine resulted in the inability of the SH2 domain ligand to activate Csk. Furthermore, the overexpressed Csk mutants A228G, Y133A/Y116A, L138A, and L149A were unable to efficiently inactivate endogenous Src in human embryonic kidney 293 cells. The results suggest that the SH2-domain-mediated activation of Csk is dependent on the binding of the beta3-alphaC loop Ala228 to the hydrophobic pocket formed by the side chains of Tyr116, Tyr133, Leu138, and Leu149 on the surface of the SH2 domain.
Subject(s)
Protein-Tyrosine Kinases/chemistry , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins/chemistry , Proto-Oncogene Proteins/metabolism , Amino Acid Sequence , Amino Acid Substitution , Binding Sites/genetics , CSK Tyrosine-Protein Kinase , Cell Line , Enzyme Activation , Humans , Hydrophobic and Hydrophilic Interactions , Kinetics , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutant Proteins/chemistry , Mutant Proteins/genetics , Mutant Proteins/metabolism , Protein Folding , Protein Structure, Tertiary , Protein-Tyrosine Kinases/genetics , Proto-Oncogene Proteins/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Spectrometry, Fluorescence , Tryptophan/chemistry , src Homology Domains , src-Family KinasesABSTRACT
The crystal structure of full-length Csk (C-terminal Src kinase) molecules shows a hydrophobic interaction between the SH2-kinase linker residue Phe183 and the alphaC-helix of the catalytic domain. To study the possible involvement of this contact in the regulation of the activity of Csk and CHK (Csk homologous kinase), a Csk SH2-kinase linker deletion mutant, Csk Phe183 and CHK Leu223 point mutants were analyzed. It was observed that a residue with a long hydrophobic side chain in position 183 (Csk) and 223 (CHK) is required to sustain the catalytic activity of Csk and CHK. These results suggest that Csk Phe183 and CHK Leu223 stabilize the movement of the alphaC-helix of these protein tyrosine kinases.
