title: DNA-binding regulates site-specific ubiquitination of IRF-1.
abstract: Understanding the determinants for site-specific ubiquitination by E3 ligase components of the ubiquitin machinery is proving to be a challenge. In the present study we investigate the role of an E3 ligase docking site (Mf2 domain) in an intrinsically disordered domain of IRF-1 [IFN (interferon) regulatory factor-1], a short-lived IFNγ-regulated transcription factor, in ubiquitination of the protein. Ubiquitin modification of full-length IRF-1 by E3 ligases such as CHIP [C-terminus of the Hsc (heat-shock cognate) 70-interacting protein] and MDM2 (murine double minute 2), which dock to the Mf2 domain, was specific for lysine residues found predominantly in loop structures that extend from the DNA-binding domain, whereas no modification was detected in the more conformationally flexible C-terminal half of the protein. The E3 docking site was not available when IRF-1 was in its DNA-bound conformation and cognate DNA-binding sequences strongly suppressed ubiquitination, highlighting a strict relationship between ligase binding and site-specific modification at residues in the DNA-binding domain. Hyperubiquitination of a non-DNA-binding mutant supports a mechanism where an active DNA-bound pool of IRF-1 is protected from polyubiquitination and degradation.
authors.affiliation: Cell Signalling Unit, Edinburgh Cancer Research Centre, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Crewe Rd South, Edinburgh EH4 2XR, UK.
authors.forename: Vivien
authors.initials: V
authors.lastname: Landré
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Pion
authors.forename: Vikram
authors.initials: V
authors.lastname: Narayan
authors.forename: Dimitris P
authors.initials: DP
authors.lastname: Xirodimas
authors.forename: Kathryn L
authors.initials: KL
authors.lastname: Ball
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D004247:DNA / Q000378:metabolism*; D006801:Humans; D050856:Interferon Regulatory Factor-1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008239:Lysine / Q000737:chemistry; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D051736:Proto-Oncogene Proteins c-mdm2 / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D054875:Ubiquitination
chemical_list: C494257:IRF1 protein, human; D050856:Interferon Regulatory Factor-1; D050505:Mutant Proteins; D011994:Recombinant Proteins; D004247:DNA; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; C467728:STUB1 protein, human; D044767:Ubiquitin-Protein Ligases; D008239:Lysine