Novel mechanism of regulation of the non-receptor protein tyrosine kinase Csk: Insights from NMR mapping studies and site-directed mutagenesis

Alexander Shekhtman, Ranajeet Ghose, Dongxia Wang, Philip A. Cole, David Cowburn

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)129-138
Number of pages10
JournalJournal of Molecular Biology
Volume314
Issue number1
DOIs
StatePublished - Nov 16 2001

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Site-Directed Mutagenesis
Magnetic Resonance Spectroscopy
src-Family Kinases
src Homology Domains
Catalytic Domain
Phosphotransferases
CSK tyrosine-protein kinase
Mutation
Down-Regulation
Binding Sites

Keywords

  • Heteronuclear NMR
  • Protein tyrosine kinase
  • SH3 domain
  • Src-family kinases

ASJC Scopus subject areas

  • Virology

Cite this

Novel mechanism of regulation of the non-receptor protein tyrosine kinase Csk : Insights from NMR mapping studies and site-directed mutagenesis. / Shekhtman, Alexander; Ghose, Ranajeet; Wang, Dongxia; Cole, Philip A.; Cowburn, David.

In: Journal of Molecular Biology, Vol. 314, No. 1, 16.11.2001, p. 129-138.

Research output: Contribution to journalArticle

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