title: Rad23 interaction with the proteasome is regulated by phosphorylation of its ubiquitin-like (UbL) domain.
abstract: Rad23 was identified as a DNA repair protein, although a role in protein degradation has been described. The protein degradation function of Rad23 contributes to cell cycle progression, stress response, endoplasmic reticulum proteolysis, and DNA repair. Rad23 binds the proteasome through a UbL (ubiquitin-like) domain and contains UBA (ubiquitin-associated) motifs that bind multiubiquitin chains. These domains allow Rad23 to function as a substrate shuttle-factor. This property is shared by structurally similar proteins (Dsk2 and Ddi1) and is conserved among the human and mouse counterparts of Rad23. Despite much effort, the regulation of Rad23 interactions with ubiquitinated substrates and the proteasome is unknown. We report here that Rad23 is extensively phosphorylated in vivo and in vitro. Serine residues in UbL are phosphorylated and influence Rad23 interaction with proteasomes. Replacement of these serine residues with acidic residues, to mimic phosphorylation, reduced proteasome binding. We reported that when UbL is overexpressed, it can compete with Rad23 for proteasome interaction and can inhibit substrate turnover. This effect is not observed with UbL containing acidic substitutions, consistent with results that phosphorylation inhibits interaction with the proteasome. Loss of both Rad23 and Rpn10 caused pleiotropic defects that were suppressed by overexpressing either Rad23 or Rpn10. Rad23 bearing a UbL domain with acidic substitutions failed to suppress rad23Δ rpn10Δ, confirming the importance of regulated Rad23/proteasome binding. Strikingly, threonine 75 in human HR23B also regulates interaction with the proteasome, suggesting that phosphorylation is a conserved mechanism for controlling Rad23/proteasome interaction.
authors.affiliation: Institute of Biomedical Sciences, National Chung Hsing University, Taichung 40227, Taiwan.
authors.forename: Ruei-Yue
authors.initials: RY
authors.lastname: Liang
authors.affiliation: Department of Pharmacology, Robert Wood Johnson Medical School, Rutgers University, Piscataway, NJ 08854, USA.
authors.forename: Li
authors.initials: L
authors.lastname: Chen
authors.affiliation: Institute of Biomedical Sciences, National Chung Hsing University, Taichung 40227, Taiwan.
authors.forename: Bo-Ting
authors.initials: BT
authors.lastname: Ko
authors.affiliation: Institute of Biomedical Sciences, National Chung Hsing University, Taichung 40227, Taiwan.
authors.forename: Yu-Han
authors.initials: YH
authors.lastname: Shen
authors.affiliation: Institute of Biomedical Sciences, National Chung Hsing University, Taichung 40227, Taiwan.
authors.forename: Yen-Te
authors.initials: YT
authors.lastname: Li
authors.affiliation: Institute of Biomedical Sciences, National Chung Hsing University, Taichung 40227, Taiwan.
authors.forename: Bo-Rong
authors.initials: BR
authors.lastname: Chen
authors.affiliation: Institute of Biomedical Sciences, National Chung Hsing University, Taichung 40227, Taiwan.
authors.forename: Kuan-Ting
authors.initials: KT
authors.lastname: Lin
authors.affiliation: Department of Pharmacology, Robert Wood Johnson Medical School, Rutgers University, Piscataway, NJ 08854, USA. Electronic address: maduraki@rwjms.rutgers.edu.
authors.forename: Kiran
authors.initials: K
authors.lastname: Madura
authors.affiliation: Institute of Biomedical Sciences, National Chung Hsing University, Taichung 40227, Taiwan. Electronic address: smchuang@dragon.nchu.edu.tw.
authors.forename: Show-Mei
authors.initials: SM
authors.lastname: Chuang
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000235:genetics; D045744:Cell Line, Tumor; D004260:DNA Repair; D045643:DNA Repair Enzymes / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D009154:Mutation; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000378:metabolism*; D059748:Proteolysis; D034622:RNA Interference; D016601:RNA-Binding Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D025801:Ubiquitin / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; C498506:PSMD4 protein, human; C513287:RAD23B protein, human; D016601:RNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; C086985:RAD23A protein, human; D012694:Serine; D046988:Proteasome Endopeptidase Complex; D045643:DNA Repair Enzymes
keywords: Rad23; UbL; degradation; phosphorylation; proteasome