The linker phosphorylation site Tyr292 mediates the negative regulatory effect of Cbl on ZAP-70 in T cells.
The protooncogene product Cbl has emerged as a negative regulator of tyrosine kinases. We have shown previously that Cbl binds to ZAP-70 through its N-terminal tyrosine kinase binding (TKB) domain. In this study, we demonstrate that overexpression of Cbl in Jurkat T cells decreases the TCR-induced phosphorylation of ZAP-70 and other cellular phosphoproteins. Coexpression of Cbl with ZAP-70 in COS cells reproduced the Cbl-induced reduction in the level of phosphorylated ZAP-70. The effect of Cbl was eliminated by the TKB-inactivating G306E mutation in Cbl as well as by a phenylalanine mutation of Tyr292 within the TKB domain binding site on ZAP-70. Notably, the oncogenic Cbl-70Z/3 mutant associated with ZAP-70, but did not reduce the levels of phosphorylated ZAP-70. Overexpression of Cbl, but not Cbl-G306E, in Jurkat T cells led to a decrease in the TCR-induced NF-AT luciferase reporter activity. Overexpression of the TKB domain itself, but not its G306E mutant, functioned in a dominant-negative manner and led to an increase in NF-AT reporter activity. Cbl-70Z/3-overexpressing cells exhibited an increase in both basal and TCR-induced NF-AT luciferase reporter activity, and this trend was reversed by the G306E mutation. Finally, by reconstituting a ZAP-70-deficient Jurkat T cell line, p116, we demonstrate that wild-type ZAP-70 is susceptible to the negative regulatory effect of Cbl, whereas the ZAP-70-Y292F mutant is resistant. Together, our results establish that the linker phosphorylation site Tyr292 mediates the negative regulatory effect of Cbl on ZAP-70 in T cells.
Rao
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Lymphocyte Biology Section, Division of Rheumatology, Immunology, and Allergy, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
Lupher
M L
ML
Ota
S
S
Reedquist
K A
KA
Druker
B J
BJ
Band
H
H
D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D017252:CD3 Complex / Q000276:immunology; D019556:COS Cells; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D015536:Down-Regulation / Q000276:immunology*; D004789:Enzyme Activation / Q000276:immunology; D017930:Genes, Reporter; D006801:Humans; D019169:Jurkat Cells; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D010766:Phosphorylation; D011485:Protein Binding / Q000276:immunology; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D050721:Proto-Oncogene Proteins c-cbl; D011948:Receptors, Antigen, T-Cell / Q000502:physiology; D013601:T-Lymphocytes / Q000201:enzymology* / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism* / Q000502:physiology; D044767:Ubiquitin-Protein Ligases; D051746:ZAP-70 Protein-Tyrosine Kinase
D000911:Antibodies, Monoclonal; D017252:CD3 Complex; D004268:DNA-Binding Proteins; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D011948:Receptors, Antigen, T-Cell; D014157:Transcription Factors; D014443:Tyrosine; D008156:Luciferases; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; D011505:Protein-Tyrosine Kinases; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human; D020928:Mitogen-Activated Protein Kinases; C067419:CBL protein, human