Dominant-negative Zeta-associated protein 70 inhibits T cell antigen receptor signaling

Abstract

Zeta-associated protein (ZAP)-7 is a cytoplasmic protein tyrosine kinase required for T cell antigen receptor (TCR) signaling and development. Mutations in ZAP-70 result in severe combined immunodeficiency in humans. ZAP-70 interacts with the TCR by binding to tyrosine-phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs) present in the invariant subunits of the TCR complex. Here we report that two ZAP-70 mutants devoid of kinase activity, generated either by a point mutation in the kinase domain to create an inactive kinase, or by truncation of the kinase domain (SH2[N+C]), functioned as dominant-negative mutants to specifically suppress TCR-mediated activation NFAT, a nuclear factor essential for inducible interleukin-2 gene expression. Biochemical studies with the SH2(N+C) mutant showed that it also blocked early TCR signaling events, such as p95(vav) tyrosine phosphorylation, extracellular signal- regulated kinase 2 activation, and the association of a number of tyrosine- phosphorylated proteins with growth factor receptor binding protein 2 (GRB2). The inhibitor effects of the SH2(N+C) mutant revealed that is requires an intact phosphotyrosine-binding site in its COOH-terminal SH2 domain. Using a CD8-ζ chimeric receptor to analyze the interaction of the SH2(N+C) mutant with ITAMs of TCR-ζ, we found that this mutant was constitutively bound to the hyperphosphorylate CD8-ζ chimera. These results indicate that tyrosine-phosphorylated ITAM is the target for the action of this dominant-negative mutant, suggesting that the assembly of a functional receptor signaling complex on ITAMs is a critical proximal TCR signaling event leading to downstream activation.