title: Serine phosphorylation is critical for the activation of ubiquitin-specific protease 1 and its interaction with WD40-repeat protein UAF1.
abstract: Deubiquitinating enzymes (DUBs) are important for the normal function of a number of cellular processes, including transcriptional regulation, cell cycle control, and DNA damage response. The enzymatic activity of DUB is regulated by different mechanisms. DUBs in several different families are post-translationally modified by phosphorylation. Large-scale phosphoproteomic studies of human DUBs revealed that a majority of ubiquitin-specific proteases (USPs) are phosphorylated. USP1 is a prototypical DUB that requires a specific interaction with a WD40-repeat protein, UAF1, for its catalytic activity. In this study, we show that Ser313 phosphorylation in USP1 is required for its interaction with UAF1 and for the stimulation of USP1's activity. In contrast, two other known USP1 serine phosphorylations (Ser42 and Ser67) are dispensable with respect to the activity of the USP1/UAF1 complex. An S313D phosphomimetic mutation in USP1 can substitute for Ser313 phosphorylation in promoting the formation of the USP1/UAF1 complex. We further demonstrated that CDK1 is responsible for Ser313 phosphorylation, and protein phosphatase treatment of USP1 can lead to inactivation of USP1/UAF1. An inserted domain in USP1 (amino acids 235-408) was found to interact with UAF1, and this interaction is mediated by Ser313 phosphorylation. Our findings revealed an intriguing mechanism of regulating USP1 activity that combines phosphorylation of a key serine residue in USP1 and the specific interaction of USP1 with a WD40-repeat protein UAF1. The pSer313-dependent formation of the USP1/UAF1 complex points to a new approach for inhibiting USP1 activity by disrupting the interaction between the UAF1's WD40-repeat domain and the Ser313-containing phosphopeptide in USP1.
authors.affiliation: Department of Chemistry and Biochemistry, 214A Drake Hall, University of Delaware, Newark, Delaware 19716, United States.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Villamil
authors.forename: Qin
authors.initials: Q
authors.lastname: Liang
authors.forename: Junjun
authors.initials: J
authors.lastname: Chen
authors.forename: Yong Seok
authors.initials: YS
authors.lastname: Choi
authors.forename: Shuyu
authors.initials: S
authors.lastname: Hou
authors.forename: Kelvin H
authors.initials: KH
authors.lastname: Lee
authors.forename: Zhihao
authors.initials: Z
authors.lastname: Zhuang
mesh_terms: D029681:Arabidopsis Proteins; D010450:Endopeptidases / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation; D012694:Serine / Q000378:metabolism; D064570:Ubiquitin-Specific Proteases
chemical_list: D029681:Arabidopsis Proteins; D010450:Endopeptidases; D009687:Nuclear Proteins; D012694:Serine; D064570:Ubiquitin-Specific Proteases; C526165:WDR48 protein, human; C579228:USP1 protein, human