TY - JOUR
T1 - Novel mechanism of regulation of the non-receptor protein tyrosine kinase Csk
T2 - Insights from NMR mapping studies and site-directed mutagenesis
AU - Shekhtman, Alexander
AU - Ghose, Ranajeet
AU - Wang, Dongxia
AU - Cole, Philip A.
AU - Cowburn, David
N1 - Funding Information:
This work was supported by NIH grants GM-47021, CA-37244, NCI Fellowship F037244, and the Burroughs Wellcome Fund. We acknowledge helpful discussions with Tom Muir, Ronnie Xu, and Hong Ji.
PY - 2001/11/16
Y1 - 2001/11/16
N2 - 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 15N) 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.
AB - 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 15N) 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.
KW - Heteronuclear NMR
KW - Protein tyrosine kinase
KW - SH3 domain
KW - Src-family kinases
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U2 - 10.1006/jmbi.2001.5126
DO - 10.1006/jmbi.2001.5126
M3 - Article
C2 - 11724538
AN - SCOPUS:0035900556
SN - 0022-2836
VL - 314
SP - 129
EP - 138
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 1
ER -