Novel mechanism of regulation of the non-receptor protein tyrosine kinase Csk: insights from NMR mapping studies and site-directed mutagenesis.
Csk (C-terminal Src kinase), a protein tyrosine kinase, consisting of the Src homology 2 and 3 (SH2 and SH3) domains and a catalytic domain, phosphorylates the C-terminal tail of Src-family members, resulting in downregulation of the Src family kinase activity. The Src family kinases share 37 % homology with Csk but, unlike Src-family kinases, the catalytic domain of Csk alone is weakly active and can be stimulated in trans by interacting with the Csk-SH3 domain, suggesting a mode of intradomain regulation different from that of Src family kinases. The structural determinants of this intermolecular interaction were studied by nuclear magnetic resonance (NMR) and site-directed mutagenesis techniques. Chemical shift perturbation of backbone nuclei (H' and (15)N) has been used to map the Csk catalytic domain binding site on the Csk-SH3. The experimentally determined interaction surface includes three structural elements: the N-terminal tail, a small part of the RT-loop, and the C-terminal SH3-SH2 linker. Site-directed mutagenesis revealed that mutations in the SH3-SH2 linker of the wild-type Csk decrease Csk kinase activity up to fivefold, whereas mutations in the RT-loop left Csk kinase activity largely unaffected. We conclude that the SH3-SH2 linker plays a major role in the activation of the Csk catalytic domain.
Shekhtman
A
A
The Rockefeller University, 1230 York Ave., New York, NY 10021-6399, USA.
Ghose
R
R
Wang
D
D
Cole
P A
PA
Cowburn
D
D
D000595:Amino Acid Sequence; D001665:Binding Sites; D000081247:CSK Tyrosine-Protein Kinase; D020134:Catalytic Domain; D004789:Enzyme Activation; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D017354:Point Mutation / Q000235:genetics; D011392:Proline / Q000378:metabolism; D020836:Protein Structure, Quaternary; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry / Q000378:metabolism; D011505:Protein-Tyrosine Kinases; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D018909:src Homology Domains; D019061:src-Family Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D011392:Proline; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C000631346:CSK protein, human; D017027:Protein Tyrosine Phosphatases