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The protein tyrosine kinase ZAP-70 is implicated in the early steps of the T-cell antigen receptor (TCR) signaling. Binding of ZAP-70 to the phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs) of the TCR zeta chain through its two src-homology 2 (SH2) domains results in its activation coupled to phosphorylation on multiple tyrosine residues, mediated by Src kinases including Lck as well as by autophosphorylation. The mechanism of ZAP-70 activation following receptor binding is still not completely understood. Here we investigated the effect of intramolecular interactions and autophosphorylation by following the kinetics of recombinant ZAP-70 activation in a spectrophotometric substrate phosphorylation assay. Under these conditions, we observed a lag phase of several minutes before full ZAP-70 activation, which was not observed using a truncated form lacking the first 254 residues, suggesting that it might be due to an intramolecular interaction involving the interdomain A and SH2 region. Accordingly, the lag phase could be reproduced by testing the truncated form in the presence of recombinant SH2 domains and was abolished by the addition of diphosphorylated ITAM peptide. Preincubation with ATP or phosphorylation by Lck also abolished the lag phase and resulted in a more active enzyme. The same results were obtained using a ZAP-70 mutant lacking the interdomain B tyrosines. These findings are consistent with a mechanism in which ZAP-70 phosphorylation/autophosphorylation on tyrosine(s) other than 292, 315, and 319, as well as engagement of the SH2 domains by the phosphorylated TCR, can induce a conformational change leading to accelerated enzyme kinetics and higher catalytic efficiency.

Type

Journal article

Journal

Biochemistry

Publication Date

14/03/2000

Volume

39

Pages

2784 - 2791

Keywords

Adenosine Triphosphate, Amino Acid Sequence, Animals, COS Cells, Circular Dichroism, Enzyme Activation, Humans, Jurkat Cells, Kinetics, Lymphocyte Specific Protein Tyrosine Kinase p56(lck), Membrane Proteins, Molecular Sequence Data, Phosphorylation, Protein Binding, Protein Structure, Secondary, Protein-Tyrosine Kinases, Receptors, Antigen, T-Cell, Recombinant Proteins, Sequence Deletion, Tyrosine, ZAP-70 Protein-Tyrosine Kinase, src Homology Domains