title: Synthesis of Isomeric Phosphoubiquitin Chains Reveals that Phosphorylation Controls Deubiquitinase Activity and Specificity.
abstract: Ubiquitin is post-translationally modified by phosphorylation at several sites, but the consequences of these modifications are largely unknown. Here, we synthesize multi-milligram quantities of ubiquitin phosphorylated at serine 20, serine 57, and serine 65 via genetic code expansion. We use these phosphoubiquitins for the enzymatic assembly of 20 isomeric phosphoubiquitin dimers, with different sites of isopeptide linkage and/or phosphorylation. We discover that phosphorylation of serine 20 on ubiquitin converts UBE3C from a dual-specificity E3 ligase into a ligase that primarily synthesizes K48 chains. We profile the activity of 31 deubiquitinases on the isomeric phosphoubiquitin dimers in 837 reactions, and we discover that phosphorylation at distinct sites in ubiquitin can activate or repress cleavage of a particular linkage by deubiquitinases and that phosphorylation at a single site in ubiquitin can control the specificity of deubiquitinases for distinct ubiquitin linkages.
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, CB2 OQH Cambridge, UK.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Huguenin-Dezot
authors.affiliation: Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, DD1 5EH Dundee, UK.
authors.forename: Virginia
authors.initials: V
authors.lastname: De Cesare
authors.affiliation: Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, DD1 5EH Dundee, UK.
authors.forename: Julien
authors.initials: J
authors.lastname: Peltier
authors.affiliation: Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, DD1 5EH Dundee, UK.
authors.forename: Axel
authors.initials: A
authors.lastname: Knebel
authors.affiliation: Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, DD1 5EH Dundee, UK.
authors.forename: Yosua Adi
authors.initials: YA
authors.lastname: Kristaryianto
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, CB2 OQH Cambridge, UK.
authors.forename: Daniel T
authors.initials: DT
authors.lastname: Rogerson
authors.affiliation: Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, DD1 5EH Dundee, UK.
authors.forename: Yogesh
authors.initials: Y
authors.lastname: Kulathu
authors.affiliation: Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, DD1 5EH Dundee, UK. Electronic address: m.trost@dundee.ac.uk.
authors.forename: Matthias
authors.initials: M
authors.lastname: Trost
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, CB2 OQH Cambridge, UK. Electronic address: chin@mrc-lmb.cam.ac.uk.
authors.forename: Jason W
authors.initials: JW
authors.lastname: Chin
mesh_terms: D001665:Binding Sites / Q000235:genetics; D000072017:Deubiquitinating Enzymes / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D010766:Phosphorylation / Q000235:genetics*; D012694:Serine / Q000235:genetics / Q000378:metabolism; D025801:Ubiquitin / Q000235:genetics* / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism
chemical_list: D025801:Ubiquitin; D012694:Serine; D044767:Ubiquitin-Protein Ligases; D000072017:Deubiquitinating Enzymes