Mutation of tyrosines 492/493 in the kinase domain of ZAP-70 affects multiple T-cell receptor signaling pathways.
The protein-tyrosine kinase ZAP-70 is implicated, together with the Src kinase p56(lck), in controlling the early steps of the T-cell antigen receptor (TCR) signaling cascade. To help elucidate further the mechanism by which ZAP-70 regulates these initial events, we used a dominant-negative mutant approach. We overexpressed in the Jurkat T-cell line ZAP-70 mutated on Tyr-492 and Tyr-493 in the putative regulatory loop of its kinase domain. This mutant inhibited TCR-induced activation of nuclear factor of activated T cells by interfering with both intracellular calcium increase and Ras-regulated activation of extracellular signal-regulated kinases. Moreover, TCR-induced phosphorylation of pp36-38, thought to play a role upstream of these pathways, was found to be reduced. In contrast, overexpression of wild-type ZAP-70 induced constitutive activation of nuclear factor of activated T cells. The ZAP-70 mutant studied here could be phosphorylated on tyrosine when associated to the TCR zeta chain and was able to bind p56(lck). This result demonstrates that Tyr-492 and Tyr-493 are not responsible for the Src homology domain 2-mediated association of p56(lck) with ZAP-70. Our data are most consistent with a model in which recruitment to the TCR allows ZAP-70 autophosphorylation and binding to p56(lck), which in turn phosphorylates Tyr-492 and/or Tyr-493 with consequent up-regulation of the ZAP-70 kinase activity. ZAP-70 will then be able to effectively control phosphorylation of its substrates and lead to gene activation.
Mège
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Molecular Immunology Unit, Department of Immunology, Pasteur Institute 25, rue du Dr. Roux, 75724 Paris, Cedex 15, France. oacuto@pasteur.fr
Di Bartolo
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Germain
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Tuosto
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Michel
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Acuto
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D048868:Adaptor Proteins, Signal Transducing; D002118:Calcium / Q000502:physiology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D051380:GRB2 Adaptor Protein; D006801:Humans; D008213:Lymphocyte Activation; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D046911:Macromolecular Substances; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D019000:Phosphotyrosine / Q000502:physiology; D011505:Protein-Tyrosine Kinases / Q000737:chemistry* / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D011948:Receptors, Antigen, T-Cell / Q000502:physiology*; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D013601:T-Lymphocytes / Q000502:physiology*; D014157:Transcription Factors / Q000378:metabolism; D014407:Tumor Cells, Cultured; D051746:ZAP-70 Protein-Tyrosine Kinase; D019061:src-Family Kinases / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D004268:DNA-Binding Proteins; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D046911:Macromolecular Substances; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D011506:Proteins; D011948:Receptors, Antigen, T-Cell; D014157:Transcription Factors; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human; D019061:src-Family Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium