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

J Mol Biol. 2001 Nov 16;314(1):129-38. doi: 10.1006/jmbi.2001.5126.

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 (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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Binding Sites
  • CSK Tyrosine-Protein Kinase
  • Catalytic Domain*
  • Enzyme Activation
  • Humans
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed*
  • Nuclear Magnetic Resonance, Biomolecular*
  • Point Mutation / genetics
  • Proline / metabolism
  • Protein Structure, Quaternary
  • Protein Tyrosine Phosphatases / chemistry
  • Protein Tyrosine Phosphatases / metabolism
  • Protein-Tyrosine Kinases
  • Sequence Alignment
  • Structure-Activity Relationship
  • src Homology Domains*
  • src-Family Kinases / chemistry*
  • src-Family Kinases / genetics
  • src-Family Kinases / metabolism*

Substances

  • Proline
  • Protein-Tyrosine Kinases
  • CSK Tyrosine-Protein Kinase
  • src-Family Kinases
  • CSK protein, human
  • Protein Tyrosine Phosphatases