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 USP1/UAF1 complex points to a new approach of inhibiting USP1 activity by disrupting the interaction between the UAF1’s WD40-repeat domain and the Ser313-containing phosphopeptide in USP1.

A selective USP1–UAF1 inhibitor links deubiquitination to DNA damage responses

“…2). Mapping the peptides to a modeled USP1 structure revealed that they are located outside the USP1 catalytic site 27 . The peptides comprising residues 412–448, 451–481, 524–563 and 553–566 are in the finger and palm domains that form the ubiquitin-binding site.…”

Deubiquitylases From Genes to Organism

“…Simpler instances of allosteric regulation are found with the increase in k cat following interaction of USP1, USP12, and USP46 with UAF1 (WDR48) (40, 41). However, the interaction between USP1 and UAF1 is itself regulated by CDK1-mediated serine phosphorylation of USP1 (267). The COOHterminal region of USP7 contains 5 Ubiquitin-like (Ubl) domains organized into 2-1-2 Ubl units, the last pair of which (HUBL-45) activate USP7 by 100-fold (66,71 (171,218).…”

Layers of DUB regulation

“…Activation by phosphorylation also takes place during the cell cycle, where USP37 is modified by CDK-2 to directly stimulate DUB activity [74]. In an analogous manner, USP1 phosphorylation was suggested to be required for complex formation with the activator UAF-1 [75].…”