Interdomain B in ZAP-70 regulates but is not required for ZAP-70 signaling function in lymphocytes.
The protein tyrosine kinase ZAP-70 plays an important role in T-cell activation and development. After T-cell receptor stimulation, ZAP-70 associates with the receptor and is phosphorylated on many tyrosines, including Y292, Y315, and Y319 within interdomain B. Previously, we demonstrated that Y292 negatively regulates ZAP-70 function and that Y315 positively regulates ZAP-70 function by interacting with Vav. Recent studies have suggested that Y319 also positively regulate ZAP-70 function. Paradoxically, removal of interdomain B (to create the construct designated Delta), containing the Y292, Y315, and Y319 sites, did not eliminate the ability of ZAP-70 to induce multiple gene reporters in Syk-deficient DT-40 B cells and ZAP-70/Syk-deficient Jurkat cells. Here we show that Delta still utilizes the same pathways as wild-type ZAP-70 to mediate NF-AT induction. This is manifested by the ability of Delta to restore induction of calcium fluxes and mitogen-activated protein kinase activation and by the ability of dominant negative Ras and FK506 to block the induction of NF-AT activity mediated by Delta. Biochemically we show that the stimulated tyrosine phosphorylation of Vav, Shc, and ZAP-70 itself is diminished, whereas that of Slp-76 is increased in cells reconstituted with Delta. Deletion of interdomain B did not affect the ability of ZAP-70 to bind to the receptor. The in vitro kinase activity of ZAP-70 lacking interdomain B was markedly reduced, but the kinase activity was still required for the protein's in vivo activity. Based on these data, we concluded that interdomain B regulates but is not required for ZAP-70 signaling function leading to cellular responses.
Zhao
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Department of Medicine, Howard Hughes Medical Institute, University of California at San Francisco, San Francisco, California 94143-0795, USA.
Williams
B L
BL
Abraham
R T
RT
Weiss
A
A
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D001402:B-Lymphocytes / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D018797:Cell Cycle Proteins; D002460:Cell Line; D004268:DNA-Binding Proteins; D006801:Humans; D019169:Jurkat Cells; D008214:Lymphocytes; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051567:Proto-Oncogene Proteins c-vav; D020794:Receptor Protein-Tyrosine Kinases / Q000502:physiology; D011947:Receptors, Antigen, B-Cell / Q000378:metabolism; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*; D055765:Shc Signaling Adaptor Proteins; D015398:Signal Transduction; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D014157:Transcription Factors; D014443:Tyrosine / Q000378:metabolism; D051746:ZAP-70 Protein-Tyrosine Kinase; D018631:ras Proteins / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D010750:Phosphoproteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D051567:Proto-Oncogene Proteins c-vav; D011947:Receptors, Antigen, B-Cell; D011948:Receptors, Antigen, T-Cell; C528024:SHC1 protein, human; C092947:SLP-76 signal Transducing adaptor proteins; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D014157:Transcription Factors; C496618:VAV1 protein, human; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D020794:Receptor Protein-Tyrosine Kinases; C084944:TYRO3 protein, human; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human; D018631:ras Proteins; D002118:Calcium