Analysis of the interaction of ZAP-70 and syk protein-tyrosine kinases with the T-cell antigen receptor by plasmon resonance.

Tyrosine phosphorylation of a 17-amino acid immunoreceptor tyrosine-based activation motif (ITAM), conserved in each of the signaling subunits of the T-cell antigen receptor (TCR), mediates the recruitment of ZAP-70 and syk protein-tyrosine kinases (PTKs) to the activated receptor. The interaction between the two tandemly arranged Src-homology 2 (SH2) domains of ...
this family of PTKs and each of the phosphotyrosine-containing ITAMs was examined by real-time measurements of kinetic parameters. The association rate and equilibrium binding constants for the ZAP-70 and syk SH2 domains were determined for the CD3 epsilon ITAM. Both PTKs bound with ka and Kd values of 5 x 10(6) M-1.sec-1 and approximately 25 nM, respectively. Bindings to the other TCR ITAMs (zeta 1, zeta 2, gamma, and delta ITAMs) were comparable, although the zeta 3 ITAM bound approximately 2.5-fold less well. Studies of the affinity of a single functional SH2 domain of ZAP-70 provided evidence for the cooperative nature of binding of the dual SH2 domains. Mutation of either single SH2 domain decreased the Kd by > 100-fold. Finally, the critical features of the ITAM for syk binding were found to be similar to those required for ZAP-70 binding. These data provide insight into the mechanism by which the multisubunit TCR interacts with downstream effector molecules.
Mesh Terms:
Amino Acid Sequence, Binding Sites, Electrophoresis, Polyacrylamide Gel, Enzyme Precursors, Glutathione Transferase, Humans, Intracellular Signaling Peptides and Proteins, Kinetics, Molecular Sequence Data, Protein-Tyrosine Kinases, Receptor-CD3 Complex, Antigen, T-Cell, Receptors, Antigen, T-Cell, Recombinant Fusion Proteins, Sequence Homology, Amino Acid, ZAP-70 Protein-Tyrosine Kinase
Proc. Natl. Acad. Sci. U.S.A.
Date: May. 23, 1995
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