Skp1-Cul1-F-box Ubiquitin Ligase (SCF�TrCP)-mediatedDestruction of the Ubiquitin-specific Protease USP37 duringG2-phase Promotes Mitotic Entry*□S

Received for publication, June 10, 2012, and in revised form, September 25, 2012 Published, JBC Papers in Press, October 1, 2012, DOI 10.1074/jbc.M112.390328

Amy C. Burrows, John Prokop, and Matthew K. Summers1

From the Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195

Background: USP37 regulates S-phase progression and is degraded in late M/G1.Results: USP37 undergoes biphasic degradation during G2 and late M/G1.Conclusion: SCF�TrCP and APCCdh1 coordinately regulate USP37 during the cell cycle.Significance: Precise regulation of USP37 activity is required for cell cycle progression.

Ubiquitin-mediated proteolysis is a key regulatory process incell cycle progression. The Skp1-Cul1-F-box (SCF) and ana-phase-promoting complex (APC) ubiquitin ligases targetnumerous components of the cell cycle machinery for destruc-tion. Throughout the cell cycle, these ligases cooperate tomain-tain precise levels of key regulatory proteins, and indirectly,each other. Recently, we have identified the deubiquitinaseUSP37 as a regulator of the cell cycle. USP37 expression is cellcycle-regulated, being expressed in late G1 and ubiquitinated byAPCCdh1 in early G1. Here we report that in addition to destruc-tion at G1, a major fraction of USP37 is degraded at the G2/Mtransition, prior to APC substrates and similar to SCF�TrCP sub-strates. Consistent with this hypothesis, USP37 interacts withcomponents of the SCF in a �TrCP-dependent manner. Inter-actionwith�TrCP and subsequent degradation is phosphoryla-tion-dependent and is mediated by the Polo-like kinase (Plk1).USP37 is stabilized inG2 by depletion of�TrCP aswell as chem-ical or genetic manipulation of Plk1. Similarly, mutation of thephospho-sites abolishes �TrCP binding and renders USP37resistant to Plk1 activity. Expression of this mutant hinders theG2/M transition. Our data demonstrate that tight regulation ofUSP37 levels is required for proper cell cycle progression.

Cell cycle progression requires the regulated, periodic activ-ity of a host of proteins, including cyclins, kinases, and phos-phatases, among others. Regulation occurs on multiple levels,from cell cycle-specific expression to post-translational modi-fication. Two ubiquitin ligase complexes, the anaphase-pro-moting complex (APC)2 and the Skp1-Cul1-F-box (SCF),ensure unidirectional transit of the cell cycle by orchestratingthe orderly ubiquitin-mediated destruction of numerous com-ponents of the cell cycle machinery.

The APC recognizes substrates (e.g. cyclins, securin, Gemi-nin) containing one or more destruction-targeting motifs(degrons), primarily the destruction box (D-box) RXXL (1, 2),and the KEN box (3). The ability of the APC to recognize thesedegrons is conferred, at least in part, by the adaptor/activatorproteins Cdc20 and Cdh1 (4). Rigid control of the APC isachieved by a variety of mechanisms. The expression of Cdc20and Cdh1 as well as their interactions with the APC are cellcycle-dependent with APCCdh1 active primarily in late mitosisthroughG1 andAPCCdc20 active duringmitosis (5, 6). The APCexhibits autonomous regulation by targeting its activators andits cognate E2s for destruction (7, 8). In addition, there are alsoa number of direct inhibitors of the APC. The bulk of APCCdh1

activity is kept in check from G1-M by the inhibitor Emi1.Similar to the APC, SCF ligases are denoted by the substrate-

adapting F-box protein. SCFSkp2 and SCF�TrCP have prominentroles in the cell cycle (9). In contrast to the APC, SCF complexactivity toward substrates is largely mediated by the cellcycle- or stimulus-dependent phosphorylation of substrates.SCF�TrCP, for example, recognizes phosphorylated serines inthe DSGXXS motif in its substrates (e.g. Emi1, Wee1, Claspin,Cdc25A) (9–14). Intriguingly, many of these substrates arephosphorylated by theAPC substrate Plk1 (12, 14–16). There isalso significant cross-talk between theAPCandSCF ligases. Forexample, the ligases act in tandem to regulate the levels of anumber of critical cell cycle regulators, including Cdc25A andClaspin (9, 10, 12, 17–19). In addition, APCCdh1 controls SCFactivity by targeting Skp2, whereas SCF�TrCP regulates APCactivity by targeting Emi1 for destruction (11, 13, 20, 21).Given the critical function of ubiquitin in control of the cell

cycle, deubiquitinating enzymes are expected to play centralroles as well. Indeed, several deubiquitinating enzymes havebeen implicated in the cell cycle. Recently, we have identifiedthe deubiquitinating enzyme USP37 as a regulator of the G1/Stransition (22). USP37 regulates S-phase entry at least in part byenhancing cyclin A stability and accumulation (22). Consistentwith its regulation of this key cell cycle transition, USP37 isrequired for zebrafish development (23). USP37 is regulated bythe oncogenic transcription factor E2F1, and its expression isincreased in several cancers (24–28). Increased USP37 expres-sion is associated with poor prognosis in non-small cell lung

* This work was supported by seed funds provided by the Lerner ResearchInstitute (to M. K. S.).

□S This article contains supplemental Figs. S1–S3.1 To whom correspondence should be addressed: Dept. of Cancer Biology,

Lerner Research Institute, 9500 Euclid Ave., NB40, Cleveland, OH 44195.Tel.: 216-445-2555; E-mail: summerm@ccf.org.

2 The abbreviations used are: APC, anaphase-promoting complex; SCF, Skp1-Cul1-F-box; TrCP, Transducin repeat-Containing Protein; USP, ubiquitin-specific processing protease; Plk1, Polo-like kinase.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 46, pp. 39021–39029, November 9, 2012© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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cancer (29). The phenotypes associated with aberrant USP37activity are unlikely to be explained by its effect on cyclin A andhave prompted us to explore USP37 biology further.Here we report that destruction of USP37 is biphasic. USP37

is destroyed in G2 by the concerted actions of Plk1 andSCF�TrCP, whereas APCCdh1 targets the remaining pool atmitotic exit. By expressing UPS37 mutants that are resistant toSCF�TrCP-mediated ubiquitination, we demonstrate thatdestruction of this pool is required for mitotic entry. Impor-tantly, this destruction event highlights the existence of addi-tional substrates whose destruction is required for the G2/Mtransition.

EXPERIMENTAL PROCEDURES

Cell Culture

HeLa, 293T, U2OS, and T98G cells were obtained fromATCC andmaintained in DMEM supplemented with 10% FBS.HeLa and 293T cells were synchronized as described (30).RO-3306 or nocodazole were added 5 h after release from thy-midine. T98G cells were synchronized by incubation inDMEMwithout FBS for 72 h and stimulated to re-enter the cell cycle bythe addition of 20% FBS. Cells were transfected with TransIT-LT1 (Mirus Bio) or RNAiMAX (Invitrogen) per the manufac-turer’s instructions. Where indicated, cells were treated with100 ng/ml nocodazole, 10 �M RO-3306 (EMD Millipore), 200nM BI2536, and 10 �M MG132 (Boston Biochem).

Antibodies

The USP37 and Emi1 antibodies were described previously(22, 31). Anti-MYC (9E10) was produced at Lerner ResearchInstitute (LRI). Commercial antibodies were as follows: SantaCruz Biotechnology, cyclin B (GNS1), cyclin A (H-432), Skp2(H-435); Sigma, actin (AC-15), Claspin; Thermo Scientific,Cdh1 (DH01); Boston Biochem, UbcH10; Covance, HA.11; BDBiosciences, Aurora B (AIM1), Nek2; Enzo Life Sciences, Plk1(3D8); Cell Signaling, �TrCP (D13F10), pHistone H3, pRb,Cdk1 pY15; Invitrogen, p27, and Securin (pituitary tumor-transforming gene 1); and MBL International, Cdc20.

Plasmids and Recombinant Proteins

USP37 was subcloned into pDEST-CS2-MYC6 and pDEST-GEX-6P1 using Gateway technology (Invitrogen). Mutantswere generated by the QuikChange mutagenesis strategy.Additional plasmids were described previously (13, 16).His6-Ubiquitin was generated by PCR and cloned intopCDNA5/FRT/TO (Invitrogen). Recombinant and in vitrotranslated protein were produced as described (30) except thatUSP37 was produced in wheat germ rather than rabbit reticu-locyte lysate.

Western Blotting and Immunoprecipitation

Cell extracts were generated in EBC buffer (50 mM Tris (pH8.0), 120mMNaCl, 1%Nonidet P-40, 1mMDTT, 25mM �-glyc-erophosphate, 5 mMNaF, 1 mMNaVO4, and leupeptin, pepsta-tin, and chymotrypsin, each at 10 �g/ml. For immunoprecipi-tation, equal amounts of cell lysates were incubated with theindicated antibodies for 2–12 h and washed in EBC buffer

including inhibitors. Immunoprecipitation samples or equalamounts of whole cell lysates were resolved by SDS-PAGE,transferred to PVDF membranes (Millipore) probed with theindicated antibodies, and visualized with the LI-COR Odysseyinfrared imaging system.

Ubiquitination

In Vivo—293T cells were transfected with a 1:1:2 ratio ofHis6-Ub:MYC-USP37:HA-�TrCP between thymidine blocks.10 �M MG132 was added during the last 10–12 h of culture.Lysates were generated as above, except that 2 mM N-ethylma-leimide was added to inactivate deubiquitinating enzymes andDTT was omitted. Equal amounts of lysates were adjusted to1.5% SDS and boiled for 10 min. Lysates were cooled to roomtemperature, and ubiquitinated proteins were purified withNi2�-agarose and processed as above.In Vitro—The in vitro procedure was essentially as described

(32) except that FLAG-�TrCP was used, UbcH3 was the soleE2, and USP37 substrates were translated in vitro.

Flow Cytometry and Immunofluorescence

Analyses were performed as described (30, 33).

RESULTS

USP37 Is Targeted by APCCdh1 in G1—Previously, we dem-onstrated that APCCdh1 targets USP37 for destruction (22).USP37 is modified by Lys11-linked polyubiquitin chains in latemitosis/G1 and is destroyed with similar kinetics to otherAPCCdh1 substrates (22) (supplemental Fig. S1, A and B). How-ever, the requirement for Cdh1 for destruction of USP37 in G1was not determined. We therefore depleted Cdh1 or Cdc20 inthymidine-nocodazole synchronized HeLa cells and followedthe kinetics of USP37 destruction (supplemental Fig. S1C). Asexpected, USP37 levels remained stable in cells depleted ofCdh1, but not Cdc20. To confirm that this was due to directactivity of Cdh1 toward USP37 and not a cell cycle defect, weperformed knockdown experiments in T98G cells synchro-nized in G0 by serum starvation. Cells were transfected withsiRNAs at serum stimulation to prevent additional Cdh1expression. Consistent with our HeLa cell data, depletion ofCdh1 in T98G cells resulted in premature accumulation ofUSP37 and cyclin A (supplemental Fig. S1D).USP37 Is Unstable in G2—During the course of the above

experiments, we observed that the levels of USP37 in nocoda-zole-arrested cells were lower than those in thymidine-arrestedcells. This observation was surprising in light of the resultsabove and our previous study (22) as APCCdh1 is thought to beinactive from G1/S through anaphase. We therefore examinedUSP37 levels inHeLa cells synchronized by a double-thymidineblock as they progressed from S-phase through early G1 (Fig.1A). Cell cycle progression was monitored by flow cytometry(supplemental Fig. S2A). USP37 levels steadily declined as cellsprogressed through G2 (6–8 h) (Fig. 1, A and B, supplementalFig. S2A). The rate of degradation slowed as cells progressedthrough mitosis and into G1 (8–12 h) (Fig. 1, A and B, supple-mental Fig. S2A). We compared the decline of USP37 levelswith substrates of APCCdc20 and APCCdh1 as well as theSCF�TrCP substrate Emi1. Because APC substrates decline

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in two activator-dependent waves, we analyzedUSP37 destruc-tion in two phases and set the level of all proteins to 1 at the firsttime point of each phase (i.e. 6 and 8 h) (Fig. 1,C andD). As cellstransited G2 through the early stages of mitosis, USP37 levelsdeclined by 50% similar to Emi1, whereas the APCCdc20 sub-strates remained stable (Fig. 1C, supplemental Fig. S2A). USP37degradation slowed as APCCdc20 substrates were degraded andthen paralleled the degradation of APCCdh1 substrates (Fig. 1,Cand D, supplemental Fig. S2A). The apparent stabilization ofUSP37 during the period of APCCdc20 substrate destruction isin agreement with the existence of a pool of USP37, whichremains stable in nocodazole (Fig. 1G, supplemental Fig. S1,A–C). The timing of the second wave of destruction is consist-

ent with the Cdh1-dependent destruction of this mitosis-stablepool of USP37 (supplemental Fig. S1C).We confirmed that thiswas not an artifact of thymidine synchronization by examiningUSP37 levels in quiescent T98G cells stimulated to enter thecell cycle by serum addition (Fig. 1E). Flow cytometry analysisindicated that destruction ofUSP37 begins inG2 (supplementalFig. S2B). Similar results were obtained in HeLa cells releasedfrom a nocodazole arrest (Fig. 1F). USP37 levels began todecline prior to APC substrates in both of these populations aswell (Fig. 1, E and F). Together these data indicate that a pool ofUSP37 is degraded in G2. To test this hypothesis, we examinedUSP37 levels in T98G cells treated in late S/G2 with the Cdk1inhibitor (RO-3306) to prevent mitotic entry (34). Indeed,

FIGURE 1. Biphasic destruction of USP37. A, the protein levels of USP37, substrates of APC and SCF ligases, and mitotic markers (phospho-histone H3 Ser-10(pH3); phospho-pRb (pRb)) were monitored throughout the cell cycle in HeLa cells synchronized by a double thymidine block. B–D, quantitation of proteinlevels in A (normalized to actin) for the indicated cell cycle phases and hours after release from the thymidine block. To facilitate comparison of USP37degradation with substrates of both APCCdc20 and APCCdh1, protein levels in C are determined relative to the 6-h time point, whereas in D, they are recalculatedand compared relative to the 8-h time point. A. U., arbitrary units. E, T98G cells were synchronized in G0 by serum starvation and stimulated to re-enter the cellcycle by serum addition. Protein levels were analyzed as in A. F, HeLa cells were arrested in mitosis with a thymidine-nocodazole block. Protein levels wereanalyzed as in A. G, T98G cells, treated as in E, were blocked in G2 with RO-3306 (Cdk1i) at 20 h after stimulation and analyzed as in A. H, HeLa cells weresynchronized by double thymidine, thymidine-RO-3306, or thymidine-nocodazole blocks. Protein levels were analyzed as in A. Quantification of USP37 proteinlevels is presented below the immunoblots.

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USP37 levels declined dramatically in G2 cells (Fig. 1G) as con-firmed by flow cytometry (supplemental Fig. S2C). We con-firmed that USP37 is degraded in G2 by analyzing USP37 pro-tein levels in cells synchronized at G1/S, G2, orM. USP37 levelswere significantly lower in G2 and mitotic HeLa cells than atG1/S (Fig. 1H). Similar results were obtained with MCF-7 andHCT116 cells (supplemental Fig. S2D). Together these dataindicate that USP37 is degraded in a biphasic manner duringthe G2/M and M/G1 transitions in APC-independent andAPCCdh1-dependent events, respectively.USP37 Interacts with the SCF�TrCP—The SCF�TrCP ligase

targets a number of cell cycle regulators, including Emi1,Wee1,Bora, and, Claspin, to promote progression from G2 to mitosis(11–14, 35). The timing of the decline in USP37 levels mirroreddecline in these substrates and suggests that USP37 may be asubstrate of SCF�TrCP. USP37 has been reported to interactwith SCF�TrCP (36). We first confirmed that USP37 interactswith SCF�TrCP. Examination of HA-tagged �TrCP immuno-complexes readily revealed the presence of coexpressed USP37(see supplemental Fig. S3C). We next determined the ability ofepitope-tagged proteins to coprecipitate endogenous interact-ing proteins. USP37-FLAG immunocomplexes containedendogenous Cul1,�TrCP, and Skp1 (Fig. 2A). Similarly, endog-enous USP37 was present in immunocomplexes of tagged Cul1and �TrCP (data not shown). Finally, we determined thatUSP37 was present in immunoprecipitates of endogenous�TrCP (Fig. 2B). From these results, we conclude that USP37 isan SCF�TrCP-interacting protein.USP37 Is Targeted for Destruction by SCF�TrCP in G2—We

next determined whether SCF�TrCP participates in the degra-dation of USP37. We first tested whether perturbing the inter-action of �TrCP with the SCF altered USP37 stability. Coex-pression of USP37with the dominant-negative�TrCP�-F-boxmutant, which cannot interact with Skp1, resulted in increasedUSP37 levels (Fig. 3A). We then tested the ability of �TrCP toinduce destruction of USP37. Expression of �TrCP reducedlevels of exogenous USP37 (Fig. 3B). To confirm that �TrCPwas inducing ubiquitination, we performed an in vivo ubiquiti-nation experiment. The addition ofMG132 stabilizedUSP37 in

the presence of �TrCP and induced the accumulation of ubiq-uitinated forms (Fig. 3C, left panels). We confirmed that thesewere ubiquitin-USP37 conjugates by purifying ubiquitinatedproteins from the denatured lysates and probing for MYC-USP37 (Fig. 3C, right panel). Finally, we tested the requirementfor �TrCP in the degradation of USP37 in G2. Serum-starvedT98G cells were depleted of �TrCP via siRNA oligonucleotidesknown to target both �TrCP1 and �TrCP2 (supplemental Fig.S3A), and USP37 levels were monitored after serum stimula-tion (35, 37). In control siRNA transfected cells, USP37 levelspeaked at 20 h and began to drop at 24 h. In contrast, USP37levels in si�TrCP transfected cells remained stable through theend of the experiment (32 h) (Fig. 3D). As previously reported,�TrCP depletion resulted in the accumulation of cyclins A andB (supplemental Fig. S3B) (11). Together these results indicatethat USP37 is a �TrCP substrate.

To confirm that USP37 is directly regulated by SCF�TrCP, wesought to identify the degronmediating its targeting by �TrCP.The consensus�TrCP recognition sequence isDSGXXS,whereboth serine residues are phosphorylated (9). Several variants ofthis phospho-degron have been identified (Fig. 4B) (9). Exami-nation of the USP37 sequence revealed no consensus degrons,but instead identified multiple sequences resembling nonca-nonical degrons (Fig. 4, A and B). We generated a series of N-and C-terminal deletion mutants to identify sequences thatdirect USP37 degradation and tested their ability to bind�TrCP (Fig. 4A, supplemental Fig. S3C). The deletion panelwasdesigned to maintain individual structural motifs, termedBoxes 1–6, within the catalytic domain of USP familymembersthat have been identified in structural models of USP37 (38).The pattern of �TrCP interaction with these fragments high-lighted a unique insertion between Boxes 4 and 5 of the USP37catalytic domain that contains three ubiquitin-interactingmotifs as well as the APCCdh1-targeting KEN box (Fig. 4A) (22).The ubiquitin-interactingmotif insertion contains three poten-tial �TrCP binding sites, (Fig. 4B). An additional deletionmutant that bisects the ubiquitin-interacting motif insertionfurther highlighted residues 756–888, which contain twopotential binding motifs. We created S 3 A mutants of thelikely phospho-sites within these two motifs (Fig. 4B). USP37S858A, but not the S790A mutant, exhibits weakened bindingto �TrCP (Fig. 4C). Consistent with direct targeting of USP37by SCF�TrCP, USP37 S858A is resistant to �TrCP-driven deg-radation (Fig. 4D).Plk1 Triggers the SCF�TrCP-mediated Destruction of USP37—

The Plk1 kinase is a key regulator of the G2/M transition andfunctions in part by phosphorylating �TrCP recognition sitesin proteins that must be destroyed for mitotic entry/progres-sion (e.g. Emi1,Wee1) (14, 16).We therefore hypothesized thatPlk1 might regulate the destruction of USP37 as well. Indeed,Ser-858 is highly conserved and lies within a consensus Plk1phosphorylation motif, (D/N/E/Y)X(S/T) (supplemental Fig.S3D) (39). We found that USP37 and Plk1 were able to interactin cells, confirming the likelihood that Plk1 plays a role inUPS37 degradation (Fig. 5A). We then asked whether manipu-lating Plk1 activity would affect USP37 levels in cells. Expres-sion of Plk1 reduced levels of coexpressed USP37, whereas thedominant-negative Plk1K82R caused an increase inUSP37 lev-

FIGURE 2. USP37 interacts with SCF�TrCP. A, 293T cells were transfected withconstructs expressing USP37-FLAG. After 48 h, protein lysates were prepared,and FLAG immunocomplexes were probed for the presence of USP37-FLAGand endogenous Skp1, Cul1, and �TrCP. IP, immunoprecipitates; *, antibodyheavy chain. B, endogenous �TrCP was immunoprecipitated from 293Textracts and analyzed for the presence of endogenous USP37.

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els relative to control cells (Fig. 5B). We then asked whetherPlk1 could stimulate �TrCP-mediated destruction of USP37.Indeed, expression of both Plk1 and �TrCP resulted in a strongreduction inUSP37 levels that was rescued by proteasome inhi-bition (Fig. 5C).We then askedwhether Plk1 activity is requiredfor the destruction of USP37. We transfected serum-starvedT98G cells with siRNA targeting Plk1, which prevented theexpression of the kinase upon cell cycle entry. In control popu-lations, USP37 exhibited a steady decline after 24 h, as Plk1levels increased. USP37 levels remained high in the Plk1-de-pleted populations through 32 h (Fig. 5D). We further con-firmed the involvement of Plk1 by inhibiting its function inthymidine-synchronized U2OS cells with the small moleculeBI2536 (supplemental Fig. S3E). Consistent with the siRNAresults, inhibition of Plk1 prevented the degradation of USP37as cells approached mitosis.

The ability of Plk1 to regulate USP37 stability suggests that itshould modulate the interaction of USP37 with �TrCP. Wetested this model with an in vitro pulldown assay. RecombinantGST-USP37 was utilized to capture in vitro translated �TrCP.As expected, in the absence of phosphorylation, USP37 wasunable to interactwith�TrCP (Fig. 5E, lane 2). Surprisingly, theaddition of recombinant Plk1 had little, if any, effect on theability of USP37 to bind �TrCP (Fig. 5E, lanes 3 and 4). How-ever, efficient interaction of Plk1 with its substrates requires apriming phosphorylation event, which is frequently mediatedby cyclin-dependent kinases (40, 41). Cdk2 in complex witheither cyclin A or cyclin E is able to phosphorylate USP37 (22).We reasoned that phosphorylation by these kinases may pro-mote phosphorylation by Plk1 and �TrCP binding. Neithercyclin A-Cdk2 nor cyclin E-Cdk2 induced a strong interactionbetween USP37 and �TrCP (Fig. 5E, lanes 5–8). However, Plk1

FIGURE 3. SCF�TrCP regulates USP37 levels. A, MYC-USP37 was transfected � HA-�TrCP �F-box into 293T cells, and levels of the tagged proteins wereexamined after 48 h. B, MYC-USP37 was transfected � HA-�TrCP into 293T cells synchronized by double thymidine block. C, cells were treated as in B, andHis-ubiquitin was included in the transfection mix. Cells were treated with 10 �M MG132 for the last 12 h. Ubiquitinated proteins were purified by Ni2� affinity.Extracts and pulldowns (PD) were probed for the indicated proteins. D, T98G cells were transfected with control or �TrCP-targeting siRNAs during serumstarvation and stimulated to enter the cell cycle. The indicated proteins were analyzed throughout the cell cycle.

FIGURE 4. Identification of the �TrCP degron. A, schematic representation of USP37. Subdomains of the catalytic domain, KEN-box 3, the ubiquitin-interacting motif (UIM) insertion, and potential �TrCP degrons are indicated. The panel of MYC-USP37 deletion mutants and their ability to bind HA-�TrCP aredepicted. B, the consensus sequences of the �TrCP degron, canonical and noncanonical degrons, and potential USP37 degrons are depicted. USP37 degronmutants are indicated. C, the ability of the USP37 degron mutants to interact with HA-�TrCP was tested as in Fig. 2. Vec, vector; IP, immunoprecipitate; D, theability of �TrCP to induce destruction of USP37 and the S858A mutant was determined as in Fig. 3B.

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in the presence of either cyclin-Cdk2 complex induced a stronginteraction betweenUSP37 and�TrCP (Fig. 5E, lanes 9 and 10).Importantly, the S858A mutation dramatically reduced theability of Plk1 to induce �TrCP binding (Fig. 5F). We then con-firmed that Plk1-mediated, Ser-858-dependent binding to�TrCP was required for ubiquitination by the SCF by perform-ing in vitro ubiquitination assays (Fig. 5G). Indeed, ubiquitina-tion of USP37 S858A by SCF�TrCP was dramatically reduced incomparison with wild type. Taken together, these results iden-tify Plk1 as a novel USP37-interacting and regulatory protein.Destruction of USP37 by SCF�TrCP in G2 Is Required for

Mitotic Entry—We next sought to confirm that SCF�TrCP andPlk1 were mediating destruction of USP37 in G2. We askedwhether �TrCP or Plk1 was required for USP37 destruction inT98G cells stimulated to enter the cell cycle and arrested in G2with RO-3306. In line with results from unperturbed cells (Figs.3D and 5D), depletion of �TrCP or Plk1 or treatment withBI2536 stabilized USP37 in G2-arrested T98G cells (Fig. 6A).We next determined that phosphorylation of Ser-858 isrequired for destruction inG2. USP37 or the S858Amutant wasexpressed during the second thymidine block of HeLa Tet-Oncells. Expression was shut off by releasing cells into doxycy-cline-free media, and protein stability was monitored in cellsarrested in G2 as above. Consistent with our in vitro data, theS858A mutant remained stable, whereas the wild type proteinwas destroyed. Together we interpret these results to confirmthat Plk1 and SCF�TrCP cooperate to trigger destruction ofUSP37 in G2.

To determine the physiological requirement for destructionof this pool of USP37, we determined the cell cycle profile ofasynchronous HeLa cells that were transiently expressingUSP37 or USP37-S858A by flow cytometry. Expression ofUSP37 caused a modest increase in the G2/M population,whereas expression of �TrCP-resistant USP37 caused a 2-fold

increase of the G2/M population (Fig. 6, C andD). Immunoflu-orescence and flow cytometry analyses revealed increasedcyclin B1-positive, nonmitotic cells in USP37 S858A-express-ing cells, but not the mitotic markers MPM-2 and phospho-histone H3 (Ser-10), consistent with an accumulation in G2rather than mitosis (Fig. 6, E–H). Taken together, we concludethat failure to degrade USP37 duringG2 preventsmitotic entry.

DISCUSSION

In this study, we have demonstrated that the deubiquitinaseUSP37 undergoes biphasic degradation during late G2 andmitotic exit/early G1. We confirmed that USP37 is targeted fordestruction at mitotic exit and in G1 by the APCCdh1 ligase asindicated by our previous study (22).Wehave presented severallines of evidence implicating SCF�TrCP and Plk1 as the ligaseand triggering kinase responsible for USP37 destruction in G2:(i) USP37 interacts with Plk1, �TrCP, and components of theSCF in vitro and in vivo; (ii) expression of �TrCP or Plk1 down-regulates USP37 in a proteasome-dependent manner, whereasdominant-negative proteins increase USP37 levels; (iii) �TrCPinducesUSP37ubiquitination invivoand invitro; (iv)phosphor-ylation ofUSP37 by Plk1 promotes binding to�TrCP; (v) loss of�TrCP or Plk1 activity by siRNA or chemical inhibition stabi-lizes USP37; and (vi) mutation of the �TrCP degron stabilizesUSP37. Together these data indicate that phosphorylation byPlk1 leads to SCF�TrCP-mediated ubiquitination and subse-quent destruction of a pool of USP37 during G2.

Our data identify USP37 as a node in the complex circuitryconnecting the APC and SCF ligases throughout the cell cycle.USP37 joins Cdc25A andClaspin as an S-phase regulator that iscoordinately regulated by SCF�TrCP and APCCdh1 (10, 12, 18,19, 37). A significant portion of USP37 remains throughoutmitosis. These observations suggest an improvedmodel for thebiological role of USP37. Inhibition of APCCdh1 promotes

FIGURE 5. Plk1 mediates targeting of USP37 by SCF�TrCP. A, MYC-USP37 and HA-Plk1 K82R were transfected and analyzed as in Fig. 2. IP, immunoprecipitates.B, MYC-USP37 was transfected with HA-Plk1 constructs as in Fig. 3A. Vec, vector. C, 293T cells were transfected with the indicated constructs and treated as inFig. 3, B and C. D, serum-starved T98G cells were stimulated to enter the cell cycle and transfected with the indicated siRNAs. Protein levels were analyzedthroughout the cell cycle. siCTRL, control siRNA; siPlk1, siRNA targeting Plk1. E, recombinant GST-USP37 was incubated with the indicated kinases, captured onGSH-agarose, and tested for the ability to bind in vitro translated HA-�TrCP. Cyc A, cyclin E; Cyc A, cyclin A. F, in vitro translated MYC-USP37 proteins were treatedas in E, immunoprecipitated, and analyzed for interaction with HA-�TrCP. G, MYC-USP37 proteins were treated as in E and mixed with E1, E2, ubiquitin,ubiquitin-aldehyde, and an energy-regenerating system in the presence (�) or absence (�) of SCF�TrCP purified from 293T cells.

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S-phase entry. USP37 then bifurcates into two pools, one ofwhich controls substrates that prevent mitotic entry. Destruc-tion of this pool by SCF�TrCP promotes the G2/M transition.We postulate that the remaining mitotic-stable pool promotesmitotic progression and must be degraded to promote the G1state, similar to many APCCdh1 substrates. Further studies willbe required to define the roles of these pools and how themitotic pool remains stable. Certainly, identification of addi-tional USP37 substrates will be a major step in elucidating thefunction of these pools of USP37.In contrast to USP37, �TrCP-resistant mutants of the APC

inhibitor Emi1 cause accumulation of APC substrates, includ-

ing cyclin A, which must also be degraded for progression pastprometaphase (13, 42, 43). Although USP37 is implicated in itsstability, cyclin A is unlikely to mediate the cell cycle effects wehave observed (22). First, failure to degrade cyclin A results indelay in mitosis rather than G2 (42, 43). Second, cyclin A levelsare not altered when USP37 levels drop in G2-arrested cells.Although we cannot exclude that cyclin A stability is mediatedby the remaining pool of USP37, this is unlikely to be the case ascyclin A is degraded inmitosis despite the presence of this frac-tion of USP37. This is also consistent with the specificity ofUSP37 forAPCCdh1 and the dependence of cyclinAdestructionin G2 and M upon APCCdc20 (22, 44). These data suggest that

FIGURE 6. SCF�TrCP-mediated degradation of USP37 is required for mitotic entry. A, quiescent T98G cells stimulated to enter the cell cycle and treated withRO-3306 at 20 h. USP37 protein levels were analyzed after inhibition of Plk1 with BI2536 at 0 h or by siRNA as in Figs. 3D and 5D. CTRL, control. B, HeLa Tet-Oncells were transfected with the indicated constructs and treated with doxycycline during the second block of a double thymidine synchronization. Cells werethen released in the absence of doxycycline and blocked in G2 by the addition of RO-3306 at 5 h after release. C–H, HeLa cells were transfected with theindicated MYC-tagged USP37 constructs or luciferase (control). After 48 h, the cells were harvested. C, cell cycle status of MYC-positive and -negative cells wasdetermined by flow cytometry. Representative images are shown. D, quantification of mock and MYC� cells from C. E, cells were analyzed by immunofluo-rescence for the presence of the G2 marker cyclin B and the mitotic marker MPM-2. F–G, quantification of cells presented in E. For F, cells with cytoplasmic cyclinB and no evidence of chromatin condensation (as in E) were counted. H, quantification of cells analyzed by flow cytometry for the mitotic marker phospho-histone H3. The average -fold change from two experiments is shown. Error bars represent S.D.

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destruction ofUSP37 inG2 is not prerequisite for destruction ofcyclin A and indicate additional substrates. Intriguingly, thedistinct cell cycle arrests caused by�TrCP-resistant USP37 andEmi1 also suggest that that these inhibitors regulate specificpopulation of APCCdh1.

In summary, the results of our study further confirm the roleof USP37 as a potent cell cycle regulator and underscore theneed for identifying additional substrates of this enzyme to gaina better understanding of its critical functions.

Acknowledgments—We thank Xiaodong Huang and Vishva Dixit forthe USP37 antibody; Janet Houghton and Tapati Mazumdar forassistance with flow cytometry; and Dipali Date, Haifeng Yang, Sau-rav Misra, and Monica Venere for helpful discussions and criticalreading of the manuscript.

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Amy C. Burrows, John Prokop and Matthew K. Summers-phase Promotes Mitotic Entry2Ubiquitin-specific Protease USP37 during G

)-mediated Destruction of theTrCPβSkp1-Cul1-F-box Ubiquitin Ligase (SCF

doi: 10.1074/jbc.M112.390328 originally published online October 1, 20122012, 287:39021-39029.J. Biol. Chem. 

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