Structure-function analysis of USP1: insights into the role of Ser313 phosphorylation site and the effect of cancer-associated mutations on autocleavage

Abstract

BackgroundIn complex with its cofactor UAF1, the USP1 deubiquitinase plays an important role in cellular processes related to cancer, including the response to DNA damage. The USP1/UAF1 complex is emerging as a novel target in cancer therapy, but several aspects of its function and regulation remain to be further clarified. These include the role of the serine 313 phosphorylation site, the relative contribution of different USP1 sequence motifs to UAF1 binding, and the potential effect of cancer-associated mutations on USP1 regulation by autocleavage.MethodsWe have generated a large set of USP1 structural variants, including a catalytically inactive form (C90S), non-phosphorylatable (S313A) and phosphomimetic (S313D) mutants, deletion mutants lacking potential UAF1 binding sites, a mutant (GG/AA) unable to undergo autocleavage at the well-characterized G670/G671 diglycine motif, and four USP1 mutants identified in tumor samples that cluster around this cleavage site (G667A, L669P, K673T and A676T). Using cell-based assays, we have determined the ability of these mutants to bind UAF1, to reverse DNA damage-induced monoubiquitination of PCNA, and to undergo autocleavage.ResultsA non-phosphorylatable S313A mutant of USP1 retained the ability to bind UAF1 and to reverse PCNA ubiquitination in cell-based assays. Regardless of the presence of a phosphomimetic S313D mutation, deletion of USP1 fragment 420–520 disrupted UAF1 binding, as determined using a nuclear relocation assay. The UAF1 binding site in a second UAF1-interacting DUB, USP46, was mapped to a region homologous to USP1(420–520). Regarding USP1 autocleavage, co-expression of the C90S and GG/AA mutants did not result in cleavage, while the cancer-associated mutation L669P was found to reduce cleavage efficiency.ConclusionsUSP1 phosphorylation at S313 is not critical for PCNA deubiquitination, neither for binding to UAF1 in a cellular environment. In this context, USP1 amino acid motif 420–520 is necessary and sufficient for UAF1 binding. This motif, and a homologous amino acid segment that mediates USP46 binding to UAF1, map to the Fingers sub-domain of these DUBs. On the other hand, our results support the view that USP1 autocleavage may occur in cis, and can be altered by a cancer-associated mutation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0311-7) contains supplementary material, which is available to authorized users.

Mutations in the ‘Fingers’ subdomain of the deubiquitinase USP1 modulate its function and activity

“…More importantly, as there is still limited knowledge on the effect that cancer‐related missense mutations may have on DUB function, we extended this analysis to naturally occurring USP1 Fingers mutations that have been detected in tumor samples. Previously, we have identified a cancer mutation (L699P) that hampers USP1 autocleavage . Here, we have identifies a single amino acid change in the Fingers subdomain (S475Y) that abrogates substrate deubiquitination without impairing UAF1 binding or autocleavage.…”

“…The Fingers subdomain appears to be the primary site for ubiquitin binding by USPs. Intriguingly, we have previously shown that the Fingers subdomains of USP1 and USP46 also mediate their binding to UAF1 [26]. Thus, this subdomain may be critical in regulating USP1-UAF1 activity.…”

A conserved two-step binding for the UAF1 regulator to the USP12 deubiquitinating enzyme

“…For such a small interface, even the observed affinity of 4 nM is rather surprising since such strong binding between two proteins is rarely mediated by interfaces having less than 1000 Å 2 buried surface area (Chen et al, 2013). However, previous reports have suggested the importance of the fingers domain for UAF1 binding and activation (Olazabal-Herrero et al, 2015). It is still unclear how S313 phosphorylation which is present in the long insert of USP1 could play a role in UAF1 binding (Villamil et al, 2012b), as the (shorter) corresponding region on USP12 is not involved in UAF1 binding for either of the interfaces.…”

“…It was suggested that UAF1 binding modulates the active site conformation of USP1 resulting in a productive catalytic triad and also that phosphorylation of Ser313 is necessary for its interaction (Villamil et al, 2012a, Villamil et al, 2012b). Other studies have shown that the regions in and around the fingers domain of USP1 might be necessary for UAF1 binding (Olazabal-Herrero et al, 2015). Recently the crystal structure of the USP46-Ub and its complex with UAF1 were determined.…”

Deubiquitinating enzymes in cellular signaling and disease regulation

“…It has been shown that deubiquitinating enzyme of USP family has low‐level activity of linear chain cleavage. One example where DUB cleaves the peptide bond is the USP1 and it undergoes auto proteolysis .…”