title: The oncogenic 70Z Cbl mutation blocks the phosphotyrosine binding domain-dependent negative regulation of ZAP-70 by c-Cbl in Jurkat T cells.
abstract: T-cell receptor (TCR) engagement results in the activation of Src family (Lck and Fyn) and ZAP-70 protein tyrosine kinases, leading to tyrosine phosphorylation of multiple cellular substrates including the complex adapter protein c-Cbl. Moreover, Cbl is tyrosine phosphorylated upon engagement of growth factor receptors, cytokine receptors, and immunoreceptors and functions as a negative regulator of tyrosine kinase signalling pathways. Cbl associates via its phosphotyrosine binding (PTB) domain to the ZAP-70 pY292 negative regulatory phosphotyrosine. We recently demonstrated that the oncogenic Cbl mutant, 70Z Cbl, requires its PTB domain to upregulate NFAT in unstimulated Jurkat T cells. Here, we demonstrate that kinase-dead but not wild-type forms of Fyn, Lck, and ZAP-70 block 70Z Cbl-mediated NFAT activation. Moreover, 70Z Cbl does not upregulate NFAT in the ZAP-70-deficient P116 Jurkat T-cell line. The requirement for Fyn, Lck, and ZAP-70 is not due to tyrosine phosphorylation of 70Z Cbl, as mutation of all tyrosines in, or deletion of, the C-terminal region of 70Z Cbl (amino acids 655 to 906) blocks 70Z Cbl tyrosine phosphorylation but enhances 70Z Cbl-mediated NFAT activation. Further, 70Z Cbl does not cooperate with ZAP-70 Y292F to upregulate NFAT, indicating that 70Z Cbl and ZAP-70 do not activate parallel signalling pathways. Finally, the upregulation of NFAT observed upon ZAP-70 overexpression is blocked by Cbl in a PTB domain-dependent manner. We conclude that oncogenic 70Z Cbl acts as a dominant negative to block the PTB domain-dependent negative regulatory role of endogenous Cbl on ZAP-70, leading to constitutive ZAP-70 signalling and activation of transcription factors.
authors.affiliation: Laboratory of Cellular and Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA. vanleeuj@box-v.nih.gov
authors.forename: J E
authors.initials: JE
authors.lastname: van Leeuwen
authors.forename: P K
authors.initials: PK
authors.lastname: Paik
authors.forename: L E
authors.initials: LE
authors.lastname: Samelson
mesh_terms: D001665:Binding Sites; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D008954:Models, Biological; D009154:Mutation; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D010766:Phosphorylation; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D050721:Proto-Oncogene Proteins c-cbl; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases; D051746:ZAP-70 Protein-Tyrosine Kinase; D019061:src-Family Kinases / Q000378:metabolism
chemical_list: 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; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; D011505:Protein-Tyrosine Kinases; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human; D019061:src-Family Kinases; C067419:CBL protein, human