Mechanism of CRL4Cdt2, A PCNA-Dependent E3 Ubiquitin Ligase

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  • 10.1101/gad.2068611Access the most recent version at doi: 2011 25: 1568-1582Genes Dev.

    Courtney G. Havens and Johannes C. Walter

    , a PCNA-dependent E3 ubiquitin ligaseCdt2Mechanism of CRL4


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    (98 articles)Post-transcriptional Control (43 articles)Cell Cycle and DNA Replication

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    Copyright 2011 by Cold Spring Harbor Laboratory Press

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    Mechanism of CRL4Cdt2, a PCNA-dependentE3 ubiquitin ligase

    Courtney G. Havens and Johannes C. Walter1

    Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA

    Eukaryotic cell cycle transitions are driven by E3 ubiq-uitin ligases that catalyze the ubiquitylation and destruc-tion of specific protein targets. For example, the anaphase-promoting complex/cyclosome (APC/C) promotes theexit from mitosis via destruction of securin and mitoticcyclins, whereas CRL1Skp2 allows entry into S phase bytargeting the destruction of the cyclin-dependent kinase(CDK) inhibitor p27. Recently, an E3 ubiquitin ligasecalled CRL4Cdt2 has been characterized, which couplesproteolysis to DNA synthesis via an unusual mechanismthat involves display of substrate degrons on the DNApolymerase processivity factor PCNA. Through its de-struction of Cdt1, p21, and Set8, CRL4Cdt2 has emerged asa master regulator that prevents rereplication in S phase.In addition, it also targets other factors such as E2F andDNA polymerase h. In this review, we discuss our currentunderstanding of the molecular mechanism of substraterecognition by CRL4Cdt2 and how this E3 ligase helps tomaintain genome integrity.

    The regulated destruction of proteins is integral to thephysiology of all eukaryotic cells. Thus, cell cycle tran-sitions, the maintenance of genome integrity, signaling,and many other cellular processes involve controlledproteolysis. Regulated proteolysis is carried out by theubiquitinproteasome system. Ubiquitin is attached toproteins destined for destruction via an isopeptide bondbetween the C-terminal glycine of ubiquitin and one ormore lysines of the target. This ubiquitin is thenmodifiedby additional ubiquitins that are connected to a lysine onthe foregoing ubiquitin, thereby forming ubiquitin chains.Most ubiquitin chains target the substrate for destructionby the 26S proteasome. However, some proteins are mono-ubiquitylated or diubiquitylated, while others are poly-ubiquitylated via Lys 63 chains that modulate proteinfunction without causing destruction (Komander 2009;Ye and Rape 2009; Behrends and Harper 2011).The attachment of ubiquitin to substrates is carried out

    by an enzymatic cascade. First, ubiquitin is attached viaa high-energy thioester bond to an E1 ubiquitin-acti-vating enzyme in a reaction that consumes ATP. Next,

    the ubiquitin is transferred from E1 to the cysteine of anE2 ubiquitin-conjugating enzyme. Finally, the E2 in-teracts with an E3 ubiquitin ligase that also binds thesubstrate. The juxtaposition of the substrate and the chargedE2 enzyme leads to ubiquitin transfer to the substrate.The specificity of ubiquitylation is encoded at the level ofsubstrate recognition by the E3 enzymes (Ravid andHochstrasser 2008); however, recently it has becomeclear that E2s can also contribute to processivity andspecificity for ubiquitin chain nucleation and elongation(Jin et al. 2008; Ye and Rape 2009; Rodrigo-Brenni et al.2010; Saha et al. 2011; Wickliffe et al. 2011). Generally,the E3substrate interaction involves the binding ofa substrate receptor subunit of the E3 to a short stretchof amino acids called a degron motif within the sub-strate. The best understood degrons are six to eight aminoacids long, transferable, and necessary and sufficient forbinding to the substrate.There are two major families of multiprotein E3

    ubiquitin ligases in eukaryotes, which are characterizedby the presence of RING or HECT domains (Ardley andRobinson 2005; Deshaies and Joazeiro 2009). The RINGdomain coordinates zinc ions in a protein fold that bindsto an E2-conjugating enzyme. The cullin ring E3 ligases(CRLs) are the largest family of RING ubiquitin ligases. InCRLs, a cullin scaffold binds via its C terminus to theRING domain protein Rbx1, which recruits an E2 (Fig.1A, E2 not shown). The N terminus of the cullin binds toan adaptor (e.g., Skp1), which in turn binds a substratereceptor that contacts the substrate, bringing it close tothe E2 (Fig. 1A). In HECT domain ligases, the E2 enzymetransfers ubiquitin to a cysteine residue in the HECTdomain, fromwhich it is transferred to the substrate (datanot shown).

    Regulation of substrate recognition by E3ubiquitin ligases

    Many substrates of the ubiquitin proteasome system arenot destroyed constitutively, but rather become unstableonly in response to an endogenous or exogenous signal.Often, signaling leads to post-translational modificationof the degron, which triggers binding to the ligase (Ravidand Hochstrasser 2008). For example, the E3 ligase Cul1Skp1FBW7 (CRL1FBW7; also known as SCFFBW7) recog-nizes the Cyclin E degron by this mechanism. FBW7

    [Keywords: Cul4; Ddb1; Cdt2; CRL4; CNA; PIP degron]1Corresponding author.E-mail is online at

    1568 GENES & DEVELOPMENT 25:15681582 2011 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/11;

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  • contains eight WD40 repeats that fold into an eight-bladed b propeller. The bottom surface of the propellerconnects to an F-box motif that interacts with Skp1. Thetop surface of the propeller forms a surface that interactsspecifically with the doubly phosphorylated degron ofCyclin E. Interestingly, some substrates such as Cdc6 andSkp2 can be protected from destruction when theirdegrons are phosphorylated (Mailand and Diffley 2005;Gao et al. 2009). Phosphorylation is not the only post-translational modification that regulates degrons. Forinstance, the E3 ligase CRL2VHL targets HIF1 (hypoxia-inducible factor-1) only when its degron motif is hydrox-ylated, ensuring that HIF1 protein levels increase underhypoxic conditions (Ivan et al. 2001; Jaakkola et al. 2001;Hon et al. 2002; Min et al. 2002). In another example,CRL1Fbx2 recognizes glycosylated proteins that are retro-translocated from the ER into the cytosol and targetsthem for destruction (Yoshida et al. 2002; Mizushimaet al. 2007). Finally, some ubiquitin ligases interactpreferentially with substrates that are sumoylated (Perryet al. 2008). Thus, post-translational modification ofsubstrates represents a common means to couple theactivity of E3 ligases to signaling events that sense intra-or extracellular conditions.Notably, E3substrate interactions are not always de-

    pendent on post-translation modification of the substrate.The anaphase-promoting complex/cyclosome (APC/C),a RING E3 ubiquitin ligase that promotes the exit frommitosis by destroying mitotic cyclins and other proteins,is itself phosphorylated. Phosphorylation of APC/C coresubunits is required to allow activation of the complex

    and the interaction between APC/C and Cdc20 (Kraftet al. 2003), the WD40 substrate receptor that initiallyrecruits APC/C substrates during mitotic exit (Pflegeret al. 2001). Conversely, the other APC/C substratereceptor, Cdh1, is inactive when phosphorylated by CDKs,ensuring that it acts after APC/CCdc20 (Zachariae et al.1998; Jaspersen et al. 1999; Kramer et al. 2000).CRL1TIR1 (SCFTIR1) is another example of a ubiquitin

    ligase that is modified to regulate destruction of a sub-strate, but in this case, the modification is noncovalent.In the absence of the plant hormone auxin, repressorsblock transcription of auxin-responsive factors. However,in the presence of auxin, the repressors are targeted forubiquitin-mediated degradation. Strikingly, auxin bindsdirectly to a pocket in the substrate receptor F-box proteinTIR1, which stabilizes the interaction between CRL1TIR1

    and its transcription repressor substrates, allowing themto be ubiquitylated and destroyed (Mockaitis and Estelle2008; Tan and Zheng 2009).In summary, the interaction of E3 ubiquitin ligases

    with their substrates is regulated in a number of differentways, most of which involve post-translation modifica-tion of the substrate or ligase, or employment of smallmolecule cofactors. In this review, we discuss in detailthe E3 ubiquitin ligase CRL4Cdt2, whose interaction withsubstrates depends on a novel strategy that involves displayof a degron motif on a cell cycle-regulated protein scaf-fold, chromatin-bound PCNA.

    The architecture of CRL4Cdt2

    CRL4 ubiquitin ligases consist of a cullin scaffold (Cul4),an adaptor protein (Ddb1), and a substrate receptor(DCAF Ddb1- and Cul4-associated factor) that bindsdirectly to Ddb1. At least 20 bona fide DCAFs likelyexist in mammalian cells (Angers et al. 2006; He et al.2006; Higa et al. 2006b; Jin et al. 2006; Higa and Zhang2007; OConnell and Harper 2007; Hu et al. 2008; Leeet al. 2008; McCall et al. 2008; Scrima et al. 2008; Choeet al. 2009; Jackson and Xiong 2009; Xu et al. 2010).CRL4Cdt2 contains the DCAF Cdt2 (Cdc10-dependenttranscri


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