Dominant-negative zeta-associated protein 70 inhibits T cell antigen receptor signaling.
Zeta-associated protein (ZAP)-70 is a cytoplasmic protein tyrosine 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 entire kinase domain (SH2[N+C]), functioned as dominant-negative mutants to specifically suppress TCR-mediated activation of 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 p95vav 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 inhibitory effects of the SH2(N+C) mutant revealed that it requires an intact phosphotyrosine-binding site in its COOH-terminal SH2 domain. Using a CD8-zeta chimeric receptor to analyze the interaction of the SH2(N+C) mutant with ITAMs of TCR-zeta, we found that this mutant was constitutively bound to the hyperphosphorylated CD8-zeta 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.
Qian
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Department of Microbiology and Immunology, Howard Hughes Medical Institute, University of California, San Francisco 94143, USA.
Mollenauer
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MN
Weiss
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A
D048868:Adaptor Proteins, Signal Transducing; D016827:CD8 Antigens / Q000235:genetics / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D018797:Cell Cycle Proteins; D051380:GRB2 Adaptor Protein; D017930:Genes, Reporter; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1; D009154:Mutation; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051567:Proto-Oncogene Proteins c-vav; D011948:Receptors, Antigen, T-Cell / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics; D013601:T-Lymphocytes / Q000276:immunology*; D014162:Transfection; D014407:Tumor Cells, Cultured; D051746:ZAP-70 Protein-Tyrosine Kinase; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D016827:CD8 Antigens; D018797:Cell Cycle Proteins; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D008565:Membrane Proteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D051567:Proto-Oncogene Proteins c-vav; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins; C496618:VAV1 protein, human; C054979:antigen T cell receptor, zeta chain; D011505:Protein-Tyrosine Kinases; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1