Role of the Src homology 2 domains and interdomain regions in ZAP-70 phosphorylation and enzymatic activity

Eur J Biochem. 1999 Dec;266(3):1166-73. doi: 10.1046/j.1432-1327.1999.00973.x.

Abstract

The protein tyrosine kinase ZAP-70, which mediates T-cell antigen receptor (TCR) signalling, contains three distinct functional modules, two tandemly arranged SH2 domains, a kinase domain and a linker region (interdomain B) that connects them. ZAP-70 enzymatic activation is strictly dependent on the binding, via its SH2 domains, to the triggered TCR and on tyrosine phosphorylation. Here we utilized recombinant ZAP-70 and carried out a mutational analysis to understand the structural requirements for its activation. We show that deletion of both SH2 domains corresponding to the first 254 residues moderately increases ZAP-70 enzymatic activity on an exogenous substrate in vitro, results in increased tyrosine phosphorylation and produces subtle conformational changes, as judged by altered SDS/PAGE migration. Mutation of Tyr292, 315 and 319 to Phe in the interdomain B region, which constitute the major phosphorylation sites both in vitro and in vivo, did not affect ZAP-70 enzymatic activity. Moreover, deletion analysis of the interdomain B region established residues 320-619 as a minimal region endowed with full kinase activity. We propose that binding of ZAP-70 to the TCR promotes, through conformational changes, its extensive phosphorylation on tyrosine. However, Tyr292, 315 and 319 do not affect ZAP-70 enzymatic activity and may influence ZAP-70 signalling only indirectly by mediating its association with intracellular transducers.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Binding Sites / genetics
  • COS Cells
  • Catalytic Domain / genetics
  • Humans
  • In Vitro Techniques
  • Jurkat Cells
  • Mutation
  • Phosphorylation
  • Protein-Tyrosine Kinases / chemistry*
  • Protein-Tyrosine Kinases / genetics
  • Protein-Tyrosine Kinases / metabolism*
  • Receptors, Antigen, T-Cell / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Signal Transduction
  • Transfection
  • Tyrosine / metabolism
  • ZAP-70 Protein-Tyrosine Kinase
  • src Homology Domains / genetics

Substances

  • Receptors, Antigen, T-Cell
  • Recombinant Proteins
  • Tyrosine
  • Protein-Tyrosine Kinases
  • ZAP-70 Protein-Tyrosine Kinase
  • ZAP70 protein, human