Regulation of CYLD activity and specificity by phosphorylation and ubiquitin-binding CAP-Gly domains
Paul R. Elliott,
Derek Leske,
Jane Wagstaff,
Lisa Schlicher,
Georgina Berridge,
Sarah Maslen,
Frederik Timmermann,
Biao Ma,
Roman Fischer,
Stefan M.V. Freund,
David Komander,
Mads Gyrd-Hansen
Affiliations
Paul R. Elliott
Division of Protein and Nucleic Acid Chemistry, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK; Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK; Corresponding author
Derek Leske
Ludwig Institute for Cancer Research, University of Oxford, Old Road Campus Research Building, Off-Roosevelt Drive, Oxford OX3 7DQ, UK
Jane Wagstaff
Division of Protein and Nucleic Acid Chemistry, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK
Lisa Schlicher
Ludwig Institute for Cancer Research, University of Oxford, Old Road Campus Research Building, Off-Roosevelt Drive, Oxford OX3 7DQ, UK
Georgina Berridge
TDI Mass Spectrometry Laboratory, Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7FZ, UK
Sarah Maslen
Division of Protein and Nucleic Acid Chemistry, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK
Frederik Timmermann
Ludwig Institute for Cancer Research, University of Oxford, Old Road Campus Research Building, Off-Roosevelt Drive, Oxford OX3 7DQ, UK
Biao Ma
Ludwig Institute for Cancer Research, University of Oxford, Old Road Campus Research Building, Off-Roosevelt Drive, Oxford OX3 7DQ, UK
Roman Fischer
TDI Mass Spectrometry Laboratory, Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7FZ, UK
Stefan M.V. Freund
Division of Protein and Nucleic Acid Chemistry, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK
David Komander
Division of Protein and Nucleic Acid Chemistry, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK; The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville VIC 3052, Australia; Department for Medical Biology, University of Melbourne, Melbourne VIC 3000, Australia; Corresponding author
Mads Gyrd-Hansen
Ludwig Institute for Cancer Research, University of Oxford, Old Road Campus Research Building, Off-Roosevelt Drive, Oxford OX3 7DQ, UK; LEO Foundation Skin Immunology Research Center, Department of Immunology and Microbiology, University of Copenhagen, Maersk Tower, Blegdamsvej 3B, 2200 Copenhagen, Denmark; Corresponding author
Summary: Non-degradative ubiquitin chains and phosphorylation events govern signaling responses by innate immune receptors. The deubiquitinase CYLD in complex with SPATA2 is recruited to receptor signaling complexes by the ubiquitin ligase LUBAC and regulates Met1- and Lys63-linked polyubiquitin and receptor signaling outcomes. Here, we investigate the molecular determinants of CYLD activity. We reveal that two CAP-Gly domains in CYLD are ubiquitin-binding domains and demonstrate a requirement of CAP-Gly3 for CYLD activity and regulation of immune receptor signaling. Moreover, we identify a phosphorylation switch outside of the catalytic USP domain, which activates CYLD toward Lys63-linked polyubiquitin. The phosphorylated residue Ser568 is a novel tumor necrosis factor (TNF)-regulated phosphorylation site in CYLD and works in concert with Ser418 to enable CYLD-mediated deubiquitination and immune receptor signaling. We propose that phosphorylated CYLD, together with SPATA2 and LUBAC, functions as a ubiquitin-editing complex that balances Lys63- and Met1-linked polyubiquitin at receptor signaling complexes to promote LUBAC signaling.
