title: Conjugation of complex polyubiquitin chains to WRNIP1.
abstract: Werner helicase interacting protein 1 (WRNIP1) is a ubiquitin-binding protein that undergoes extensive post-translational modification including ubiquitination, sumoylation, and phosphorylation. These post-translational modifications are expected to regulate the function of WRNIP1 in the DNA damage response. In this study, we use a denaturing tandem affinity purification technique along with mass spectrometry to show that, unlike most ubiquitin-binding proteins, WRNIP1 is polyubiquitinated. WRNIP1 polyubiquitination is reminiscent of the well-characterized phenomenon of the coupled monoubiquitination of ubiquitin-binding proteins in that this polyubiquitination is dependent on the presence of an intact ubiquitin-binding domain. The polyubiquitin chains conjugated to WRNIP1 are linked through lysines 11, 48, and 63. This study presents the first evidence for the conjugation of K11-K48-K63 polyubiquitin chains to a specific substrate in vivo. Polyubiquitination is likely to regulate WRNIP1's function in the DNA damage response, as UV radiation induces the hyperubiquitination of WRNIP1. Polyubiquitination with noncanonical intraubiquitin linkages may represent a unique mode of regulation of UBZ domain-containing proteins.
authors.affiliation: Watson School of Biological Sciences, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA. bishr@mskcc.org
authors.forename: Rebecca A
authors.initials: RA
authors.lastname: Bish
authors.forename: Oliver I
authors.initials: OI
authors.lastname: Fregoso
authors.forename: Antonella
authors.initials: A
authors.lastname: Piccini
authors.forename: Michael P
authors.initials: MP
authors.lastname: Myers
mesh_terms: D000074183:ATPases Associated with Diverse Cellular Activities; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D002853:Chromatography, Liquid; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D051379:Mice; D025821:Polyubiquitin / Q000378:metabolism*; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D054875:Ubiquitination; D014466:Ultraviolet Rays
chemical_list: D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D025821:Polyubiquitin; C430237:WRNIP1 protein, human; D000074183:ATPases Associated with Diverse Cellular Activities