title: UBXN7 docks on neddylated cullin complexes using its UIM motif and causes HIF1α accumulation.
abstract: BACKGROUND
  The proteins from the UBA-UBX family interact with ubiquitylated proteins via their UBA domain and with p97 via their UBX domain, thereby acting as substrate-binding adaptors for the p97 ATPase. In particular, human UBXN7 (also known as UBXD7) mediates p97 interaction with the transcription factor HIF1α that is actively ubiquitylated in normoxic cells by a CUL2-based E3 ligase, CRL2. Mass spectrometry analysis of UBA-UBX protein immunoprecipitates showed that they interact with a multitude of E3 ubiquitin-ligases. Conspicuously, UBXN7 was most proficient in interacting with cullin-RING ligase subunits. We therefore set out to determine whether UBXN7 interaction with cullins was direct or mediated by its ubiquitylated targets bound to the UBA domain.
  
  
  RESULTS
  We show that UBXN7 interaction with cullins is independent of ubiquitin- and substrate-binding. Instead, it relies on the UIM motif in UBXN7 that directly engages the NEDD8 modification on cullins. To understand the functional consequences of UBXN7 interaction with neddylated cullins, we focused on HIF1α, a CUL2 substrate that uses UBXD7/p97 as a ubiquitin-receptor on its way to proteasome-mediated degradation. We find that UBXN7 over-expression converts CUL2 to its neddylated form and causes the accumulation of non-ubiquitylated HIF1α. Both of these effects are strictly UIM-dependent and occur only when UBXN7 contains an intact UIM motif. We also show that HIF1α carrying long ubiquitin-chains can recruit alternative ubiquitin-receptors, lacking p97's ATP-dependent segregase activity.
  
  
  CONCLUSIONS
  Our study shows that independently of its function as a ubiquitin-binding adaptor for p97, UBXN7 directly interacts with neddylated cullins and causes the accumulation of the CUL2 substrate HIF1α. We propose that by sequestering CUL2 in its neddylated form, UBXN7 negatively regulates the ubiquitin-ligase activity of CRL2 and this might prevent recruitment of ubiquitin-receptors other than p97 to nuclear HIF1α.
authors.affiliation: Scottish Institute for Cell Signalling, College of Life Sciences, University of Dundee, UK.
authors.forename: Susanne
authors.initials: S
authors.lastname: Bandau
authors.forename: Axel
authors.initials: A
authors.lastname: Knebel
authors.forename: Zoe O
authors.initials: ZO
authors.lastname: Gage
authors.forename: Nicola T
authors.initials: NT
authors.lastname: Wood
authors.forename: Gabriela
authors.initials: G
authors.lastname: Alexandru
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000251:Adenosine Triphosphatases / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D044842:Cullin Proteins / Q000378:metabolism*; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism*; D053858:Metabolic Networks and Pathways; D008969:Molecular Sequence Data; D000075687:NEDD8 Protein; D009687:Nuclear Proteins / Q000378:metabolism; D016133:Polymerase Chain Reaction; D054730:Protein Interaction Domains and Motifs; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D014452:Ubiquitins / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C105153:CUL2 protein, human; D002352:Carrier Proteins; D044842:Cullin Proteins; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D000075687:NEDD8 Protein; C082860:NEDD8 protein, human; D009687:Nuclear Proteins; C531895:UBXN7 protein, human; D014452:Ubiquitins; D044767:Ubiquitin-Protein Ligases; D000251:Adenosine Triphosphatases; C404942:p97 ATPase