title: Usp12 stabilizes the T-cell receptor complex at the cell surface during signaling.
abstract: Posttranslational modifications are central to the spatial and temporal regulation of protein function. Among others, phosphorylation and ubiquitylation are known to regulate proximal T-cell receptor (TCR) signaling. Here we used a systematic and unbiased approach to uncover deubiquitylating enzymes (DUBs) that participate during TCR signaling in primary mouse T lymphocytes. Using a C-terminally modified vinyl methyl ester variant of ubiquitin (HA-Ub-VME), we captured DUBs that are differentially recruited to the cytosol on TCR activation. We identified ubiquitin-specific peptidase (Usp) 12 and Usp46, which had not been previously described in this pathway. Stimulation with anti-CD3 resulted in phosphorylation and time-dependent translocation of Usp12 from the nucleus to the cytosol. Usp12(-/-) Jurkat cells displayed defective NFκB, NFAT, and MAPK activities owing to attenuated surface expression of TCR, which were rescued on reconstitution of wild type Usp12. Proximity-based labeling with BirA-Usp12 revealed several TCR adaptor proteins acting as interactors in stimulated cells, of which LAT and Trat1 displayed reduced expression in Usp12(-/-) cells. We demonstrate that Usp12 deubiquitylates and prevents lysosomal degradation of LAT and Trat1 to maintain the proximal TCR complex for the duration of signaling. Our approach benefits from the use of activity-based probes in primary cells without any previous genome modification, and underscores the importance of ubiquitin-mediated regulation to refine signaling cascades.
authors.affiliation: HKU-Pasteur Research Pole and Center for Influenza Research, School of Public Health, LKS Faculty of Medicine, University of Hong Kong, Hong Kong;
authors.forename: Akhee S
authors.initials: AS
authors.lastname: Jahan
authors.affiliation: HKU-Pasteur Research Pole and Center for Influenza Research, School of Public Health, LKS Faculty of Medicine, University of Hong Kong, Hong Kong;
authors.forename: Maxime
authors.initials: M
authors.lastname: Lestra
authors.affiliation: Whitehead Institute for Biomedical Research, Cambridge, MA 02142;
authors.forename: Lee Kim
authors.initials: LK
authors.lastname: Swee
authors.affiliation: HKU-Pasteur Research Pole and Center for Influenza Research, School of Public Health, LKS Faculty of Medicine, University of Hong Kong, Hong Kong;
authors.forename: Ying
authors.initials: Y
authors.lastname: Fan
authors.affiliation: HKU-Pasteur Research Pole and Center for Influenza Research, School of Public Health, LKS Faculty of Medicine, University of Hong Kong, Hong Kong;
authors.forename: Mart M
authors.initials: MM
authors.lastname: Lamers
authors.affiliation: Whitehead Institute for Biomedical Research, Cambridge, MA 02142;
authors.forename: Fikadu G
authors.initials: FG
authors.lastname: Tafesse
authors.affiliation: Whitehead Institute for Biomedical Research, Cambridge, MA 02142;
authors.forename: Christopher S
authors.initials: CS
authors.lastname: Theile
authors.affiliation: Whitehead Institute for Biomedical Research, Cambridge, MA 02142;
authors.forename: Eric
authors.initials: E
authors.lastname: Spooner
authors.affiliation: HKU-Pasteur Research Pole and Center for Influenza Research, School of Public Health, LKS Faculty of Medicine, University of Hong Kong, Hong Kong; Department of Cell Biology and Infection, Institut Pasteur, 75015 Paris, France;
authors.forename: Roberto
authors.initials: R
authors.lastname: Bruzzone
authors.affiliation: Whitehead Institute for Biomedical Research, Cambridge, MA 02142; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142.
authors.forename: Hidde L
authors.initials: HL
authors.lastname: Ploegh
authors.affiliation: HKU-Pasteur Research Pole and Center for Influenza Research, School of Public Health, LKS Faculty of Medicine, University of Hong Kong, Hong Kong; Department of Cell Biology and Infection, Institut Pasteur, 75015 Paris, France; sanyal@hku.hk.
authors.forename: Sumana
authors.initials: S
authors.lastname: Sanyal
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems / Q000235:genetics; D002462:Cell Membrane / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D002469:Cell Separation; D003600:Cytosol / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D006801:Humans; D019169:Jurkat Cells; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008954:Models, Biological; D016328:NF-kappa B / Q000378:metabolism; D050778:NFATC Transcription Factors / Q000378:metabolism; D021381:Protein Transport; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*; D015203:Reproducibility of Results; D015398:Signal Transduction; D013379:Substrate Specificity / Q000187:drug effects; D013601:T-Lymphocytes / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism; D043222:Ubiquitin Thiolesterase / Q000172:deficiency / Q000378:metabolism*; D064570:Ubiquitin-Specific Proteases / Q000378:metabolism
chemical_list: D005231:Fatty Acids, Unsaturated; D016328:NF-kappa B; D050778:NFATC Transcription Factors; D011948:Receptors, Antigen, T-Cell; C486402:USP12 protein, human; D025801:Ubiquitin; D020928:Mitogen-Activated Protein Kinases; D010450:Endopeptidases; C562185:ubiquitin-specific peptidase 46, human; D043222:Ubiquitin Thiolesterase; D064570:Ubiquitin-Specific Proteases; C038753:leptomycin B
keywords: TCR signaling; Usp12; deubiquitylases
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