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Lysine-targeting specificity in ubiquitin and ubiquitin-like modification pathways

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Francesca Mattiroli1,2 & Titia K Sixma1 Ubiquitin and ubiquitin-like modifications are central to virtually all cellular signaling pathways. They occur primarily on lysine residues of target proteins and stimulate a large number of downstream signals. The diversity of these signals depends on the type, location and dynamics of the modification, but the role of the exact site of modification and the selectivity for specific lysines are poorly understood. Here we review the current literature on lysine specificity in these modifications, and we highlight the known signaling mechanisms and the open questions that pose future challenges to ubiquitin research. Ubiquitin conjugation is a three-step enzymatic process in which E1, E2 and E3 enzymes activate the ubiquitin molecule and ensure specific target modification. Ubiquitin-like (UBL) proteins such as SUMO follow similar pathways with specific sets of E1, E2 and E3 enzymes. Ubiquitin and UBL modifications have a variety of effects on target function, fate or localization, and these effects largely depend on the type, location and dynamics of the modification1. For functional downstream signaling, ubiquitin and UBL modifications are recognized by specific signaling proteins that translate the modification into a cellular effect. These so-called ‘readers’ contain ubiquitinor UBL-binding modules to recognize the modifications, but they frequently have additional features that define specificity. Finally, deconjugating enzymes have a crucial role in pathways dependent on ubiquitin and UBL modifications because their action can revert and modulate the signal arising from these modifications (Box 1). Finding the right target is a critical step in ubiquitin and UBL cascades, and it is precisely regulated, primarily by the E3 enzymes. Part of this process is the identification of the actual lysine or amino group that is to be modified. The level of specificity for a particular lysine varies between different ubiquitination cascades, and the determinants are only beginning to emerge. Frequently, target selection is limited to general recognition of the target protein, without high selectivity for a specific lysine residue. Such general target selectivity is sufficient for those modifications that do not require conjugation to one specific lysine (as seen on targets such as p27, p21 or cyclins). These nonselective lysine (K) modifications include many polyubiquitination signals that lead to proteasomal degradation via K48- or K11-linked polyubiquitin chains. In these cases, the main molecular signaling platform is the polyubiquitin chain itself, and its recognition does not involve the target molecule. In other cases, an additional level of lysine specificity is required. Here, the modification must take place on a specific lysine residue 1Division

of Biochemistry, Cancer Genomics Center, Netherlands Cancer Institute, Amsterdam, The Netherlands. 2Present address: Howard Hughes Medical Institute, Department of Molecular and Radiological Biosciences, Colorado State University, Fort Collins, Colorado, USA. Correspondence should be addressed to T.K.S. ([email protected]). Received 15 November 2013; accepted 13 February 2014; published online 4 April 2014; doi:10.1038/nsmb.2792

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on the target molecule. Substrates such as proliferating cell nuclear antigen (PCNA), histones H2A and H2B and the Fanconi-group proteins FANCD2 and FANCI (Table 1) are modified on one or more selected lysines in defined cellular signaling cascades2–8. In these reactions, the E3 ligases not only bind the target but also orient the ubiquitination machinery to modify a specific site. In some of these cases, the downstream signaling is highly dependent on the location of the modification. Experimental identification of the modified lysines was initially dependent on protein sequencing methods9, but today proteomics techniques are powerful tools for determination of the modification sites, both on targets and within ubiquitin chains10–14. In combination with mutagenesis approaches, these methods facilitate the identification and validation of selective sites, allowing the biological meaning to be unraveled. The recent development of high-affinity antibodies for the diglycine tag that defines ubiquitin modification has enabled large-scale elucidation of ubiquitinated targets15. Differential proteomics analysis in the presence and absence of a selected E3 ligase, such as Parkin, is beginning to define the spectrum of modifications that rely on a single ligase16. This is a powerful approach, although further analysis will be required to show which targets are modified directly by a given ligase. Studies of these modifications and their dynamics under a variety of conditions are now laying the foundation for detailed functional analysis. Here we review current knowledge about lysine specificity in ubiquitin and UBL targeting, focusing on the systems for which a detailed mechanism of modification and downstream signaling has been validated biochemically. We describe what is known about the different mechanisms that confer lysine specificity to ubiquitin and UBL modifications and outline how site specificity is achieved and how it can induce specific cellular signaling. Modes of target recognition Among E3 ligases, different mechanisms are used to achieve target selection. For most E3s, target recognition either involves domains on the E3 ligase distinct from the catalytic domain or involves a binding partner17,18 (Fig. 1a). On the substrates, recognition can be regulated by elements known as degrons, which mediate the interaction with the E3 (ref. 19). In some cases, these regions can be post-translationally

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Box 1  Specificity in deconjugating reactions As in the modification process, the deconjugation process can have specificity for a target as well as for a particular site. In ubiquitin signaling, almost 100 deubiquitinating enzymes (DUBs), distributed over five different classes, have been described to date125. A number of DUBs that target proteins with selectively modified lysines have been described (Table 1). However, it is not clear whether any of these DUBs target only the selective site. In general, there are fewer DUBs than E3 enzymes, and DUBs are thought to have less substrate specificity. How substrate selection occurs for DUB enzymes remains an open question. In most cases, additional domains (for example, DUSP-UBL or TRAF) are responsible for substrate recruitment or DUB localization, and these functions, rather than molecular substrate selection, may have a major role in specificity. Regarding cleavage of ubiquitin chains, JAMM proteases display selectivity for K63-linked ubiquitin chains126, and most members of the ovarian tumor (OTU) family are specific for one or for a subgroup of polyubiquitin-chain types but have low preference for ubiquitinated targets127. This selectivity is achieved by widely different mechanisms among the various OTU family members127,128. For other DUBs, relatively little linkage selectivity has been reported129. SUMOylation can be reverted by the few identified SENP enzymes and the USPL1 protein130. Interestingly, only two SENP homologs have been annotated in yeast, Ulp1 and Ulp2, with complementary cellular localization and functions. SENPs do not display high target selectivity on a molecular level; their apparent target selectivity seems instead to be due to their cellular localization, although they do have specificity for cleaving different SUMO isoforms131. Nedd8 is removed from CRLs by the COP9 signalosome (CSN), whose JAMM protease subunit (CSN5) deconjugates the UBL from the cullin protein132. The molecular details of this reaction are still unknown.

modified (for example, phosphorylated) to modulate the cross-talk between two cellular signaling pathways20–22 (Fig. 1b). In the above reactions, the catalytic domain of the E3 ligase (HECT, RING or RBR) has no direct role in substrate selection, acting merely

to ensure an efficient conjugation reaction. In other E3 ligases, however, the isolated catalytic domain is sufficient to achieve target selection, including lysine specificity, as shown in in vitro reconstitution studies with, for example, RING1B and RNF168 on histone H2A3,23,24 (Fig. 1c). There are also several examples in which the E2 directly recognizes the target site on substrates; this is observed primarily in modifications by the UBL molecule SUMO (Fig. 1d). Lysine specificity on target substrates These diverse modes of target recognition do not always provide a mechanism for lysine selection (Fig. 2a), but they can directly or indirectly lead to lysine-specific modifications. In some cases, a lysine is selected through a detailed and localized biochemical mechanism (for example, in SUMOylation or ubiquitin-chain formation as described below). In other cases, a specific lysine is selected simply through structural constraints that limit the ubiquitination machinery to acting on a given area of the target (Fig. 2b). Finally, in some cases in which the E3 ligase has a large area of action, the presence of only one or a few lysines in this target area can yield selective modification (Fig. 2c). Table 1 summarizes the few E3-dependent site-specific modifications that have been described to date. Here we highlight current knowledge of lysine specificity in each of the different E3 ligase families. There are three major families of E3 ligases, known as homologous to the E6AP carboxyl terminus (HECT), really interesting new gene (RING) and RING between RING (RBR). The HECT ligases form a covalent interaction with ubiquitin through a conserved cysteine and transfer the ubiquitin moiety directly to the lysine residue18. The RING E3s function through a zinc-binding RING domain that binds the ubiquitin-loaded E2 and activates ubiquitin transfer to the target. A large subgroup of RING E3 ligases is composed of cullin–RING ligases (CRLs)25. CRLs are multisubunit complexes in which the elongated cullin protein forms a bridge between a RING-containing subunit (RBX1) and the target-recognition module (F-box protein)26. Finally, the RBR ligases

Table 1  Specific ubiquitin and UBL modifications, and corresponding E3 ligases, readers and deconjugating enzymes Modification Ubiquitin

Organism E3 Human

RNF168

Target

Lysine

Reader

Deubiquitinating enzyme

Refs.

H2A

K13, K15

53BP1

–

3,61–63

Ubiquitin

Human

RING1B or RING1A

H2A

K119

ZRF1

–

4,23,24,59,60,115

Ubiquitin

Human, yeast

RAD18

PCNA

K164

Polη ZRANB3

USP1–UAF1

2,54–58,109,116–118

Ubiquitin

Human

FANCL

FANCD2, FANCI K561, K523

FAN1

USP1–UAF1

6–8,51–53,110–114

Ubiquitin

Human

RNF20–RNF40

H2B

–

USP22 in SAGA

5,49

Yeast

Bre1

Ubiquitin

Human

SCFβ-TrCP

β-catenin

K19, K49

–

–

20,37,38

Ubiquitin

Human

SCFβ-TrCP

IκBα

K21, K22

–

–

20,39,40

Ubiquitin

Yeast

SCFMet30

Met4

K163

Met4

–

41–43

Ubiquitin

Human

TRIM33

SMAD4

K519

–

USP9x

50

Ubiquitin

Yeast

Rsp5

Sna3

K125

–

–

18,65

Yeast

Siz1, Siz2

PCNA

K164, K127

Srs2

Ulp1

2,98,99,120–122

Human

–

PARI

– –

SUMO

K120 K123

Ubp8 in SAGA

SUMO

Human

RanBP2–RanGAP~SUMO–Ubc9 RanGAP

K526

RanBP2

NEDD8

Human

Rbx1–Dcn1

K720 in cullin-1

CAND1 displaced COP9 signalosome 103,104,132

Rub1 (NEDD8 homolog)

Yeast

Hrt1–Dcn1

Cullins

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Recognition by additional domain or binding partners

cyclosome (APC/C), during the cell cycle have been studied in more detail. They target a number of cyclins and cell cycle–control factors (for example, p27) for degradation in a timed manner32,33. Mutation of multiple lysine residues on these substrates is not sufficient to abrogate their ubiquitination, in line with a ubiquitination-zone mechanistic model (Fig. 2a). Nevertheless, some specificity seems to be present in these reactions, as was recently suggested by a proteomic study proposing that the APC/C complex has a site preference for lysines flanking serines on disordered regions34. However, the mechanistic explanation of such specificity remains elusive. Target selection in the CRL family can be dependent on phos­ phorylation of the degron35, and in these situations some reactions can lead to modification of only a few lysines on the substrates. One example is the ubiquitination of Sic1 in yeast by an SCF complex containing the F-box protein Cdc4 (SCFCdc4) during cell-cycle control. The E3-target interaction here is regulated by multiple phosphorylation events, and the subsequent lysine selection is restricted to six lysine residues in the N-terminal domain that can serve as target sites to promote Sic1 degradation21,30,31,36. One single lysine on this domain is sufficient to induce Sic1 degradation, thus suggesting that a single polyubiquitin chain is sufficient to target this protein to the proteasome. Nevertheless, differences in the rate of degradation of Sic1 have been observed for different lysine mutants, indicating that the relative location of the ubiquitin chain can affect target degradation and suggesting that the attachment site of polyubiquitin chains can affect downstream signaling30. In other cases, one or two lysines can be selected for ubiquitination, as is the case for β-catenin and IκBα20,37. β-catenin levels need to be controlled to avoid aberrant Wnt-target activation. Phosphorylated β-catenin is polyubiquitinated by the SCF complex containing the F-box protein β-TrCP (SCFβ-TrCP) on K19 and is subsequently degraded by the proteasome20,37,38. Similarly, during NF-κB signaling, release and activation of the NF-κB transcription factors requires proteasomal degradation of the IκBα protein. This pro­cess also relies on the phosphorylation of IκBα, which recruits the same SCFβ-TrCP complex for polyubiquitination on K21 and K22 of IκBα39,40. Another interesting case is the regulation of the transcriptional activator Met4 in yeast. Met4 differs from the majority of other transcription factors in its regulation because it is not targeted for protein degradation in situations in which its activity is not needed. Met4 is modified by the Met30 E3 ligase at a specific lysine, K163, with a short polyubiquitin chain. Recognition of this chain by Met4’s internal ubiquitin-binding domain blocks chain extension and prevents degradation of Met4 (refs. 41–43). The mechanism that drives site specificity in these CRL-mediated reactions is unknown in most cases. It seems plausible that structural restraints preclude targeting of additional sites, thus limiting the E3’s area of action to a single lysine site (Fig. 2b). Additionally, lysine choice could depend on the availability of lysine residues in the ubiquitination zone (Fig. 2c). It is still unclear whether site

Recognition mediated by post-translational modifications

Target

Target

Ub

Ub

E3–E2

c

E3–E2

d

Recognition mediated by the catalytic domain

Target

Recognition mediated by the E2 enzyme

Target

S

Ub

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E3

E2

Ubc9

E3

Figure 1  Modes of target recognition. (a) The majority of E3s bind their targets via domains distinct from the catalytic core or via binding partners (for example, FANCL ligase on FANCD2–FANCI target). (b) E3-target interactions can be mediated by post-translational modifications of the target (yellow star) (for example, SCFβ-TrCP ligase on IκBα and β-catenin). (c) In a small number of cases, the catalytic core on the E3 is sufficient to provide lysine selection (for example, RING1B and RNF168 ligases on H2A target). (d) E2-target interactions can determine substrate selection. This has mainly been described for SUMO (S) modifications (for example, Ubc9 E2 enzyme on RanGAP target). The light-orange areas on E3–E2 represent catalytic domains (RING–E2 or HECT or RBR). The red circles mark the region of contact between the E3 and the target; on substrates, this region can be called the degron.

function by using a conjugation mechanism that combines RING and HECT activities27. Cullin–RING ligases: targeting a low-specificity ‘ubiquitination zone’. The vast majority of CRL-mediated modifications lead to proteasomal degradation by formation of K48- and K11-linked ubiquitin chains on the targets. CRL complexes recognize their substrates through their F-box subunit, which binds recognition motifs (degrons) on the targets. The RBX1 subunit in CRLs is located ~50 Å away from the F-box substrate-recognition unit and has no apparent role in substrate binding25. Their flexible, elongated structure allows these ligases to cover a large area of action when activated by Nedd8 modification28. These structural features lead to a model in which CRLs, once anchored to the substrate molecule by the F-box subunit, can modify, in a rather nonspecific manner, lysines located in a so-called ubiquitination zone29–31 (Fig. 2a). The reactions of specific CRL complexes, such as Skp, cullin, F box–containing complex (SCF) and anaphase-promoting complex/ Figure 2  E3-mediated lysine specificity. (a) E3 ligases can have low lysine specificity, targeting a large area of action, also called the ubiquitination zone (light-yellow area on the target, for example, SCF ligase on p27 target). (b) E3 ligases can target a single lysine residue via limited structural flexibility that reduces the area of action (for example, SCFβ-TrCP ligase on IκBα or β-catenin target). (c) Specificity otherwise can result from the availability of only one (or a few) lysine(s) in the area of action of the E3 ligase (for example, Rsp5 on Sna3 target).

310

a

b

Broad area of action, low lysine specificity K

K K

K

K K K

Target

K K

K

Ub

E3–E2

K K

K

c

Limited area of action, defined lysine specificity

K

K

K K

K

K

K

Target

Broad area of action, limited lysine availability K

K

K

K

K

Target

Ub

Ub

E3–E2

K

E3–E2

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specificity is required in polyubiquitin-chain formation leading to proteasomal degradation30. RING E3 ligases: different styles of ligase involvement. Many multidomain RING E3 ligases also catalyze polyubiquitination of their substrate proteins without target-lysine specificity. For example, in NF-κB signaling, TRAFs and cIAPs are RING E3s that orchestrate the formation of a variety of polyubiquitin chains on a number of substrates44. It has not been reported whether these ligases have preferences for a specific lysine on their targets, possibly because the polyubiquitin chains serve as signaling platforms in the pathway. A set of E3 RING ligases specifically catalyze multiple monoubi­quitin modifications. A notable example is the c-Cbl E3 ligase, which functions in the internalization of receptor tyrosine kinases (RTKs), modulating their extracellular signaling, endocytosis and subsequent lysosomal degradation. The RING domain of c-Cbl is not sufficient for target modification because motifs on the N-terminal side of the RING domain are required for substrate binding and catalysis45. In this case, the combined effect of the different monoubiquitin modifications is required for receptor internalization46. Targeting is selective for an area rather than for a specific lysine. A variation of this partial specificity is represented by the activity of the RING dimer HDM2–HDMX in the ubiquitination of the tumor suppressor p53 (ref. 47). This modification leads to proteasomal degradation of p53 and is not strictly constrained to a specific lysine but instead involves a group of lysines located on the C-terminal part of p53 (ref. 48). It is unclear at this point whether this partial selectivity for a group of lysines is merely the result of structural constraints on the E3’s function or whether it reflects an important requirement for the localization of downstream signaling. In vivo studies have documented a number of reactions directed to specific lysines, including ubiquitination of histone H2B K120 (K123 in yeast) by the RNF20–RNF40 heterodimer (Bre1 in yeast) in transcriptional control5,49 and SMAD4 modification on K519 by the E3 TRIM33 (also known as ectodermin) during TGF-β signaling50. However, these reactions have not been studied in detailed biochemical reconstitution experiments, and the determinants that control target recognition and site specificity have not yet been resolved. In DNA repair pathways, highly specific lysine modifications have also been reported. The first example involves the Fanconigroup proteins FANCD2 and FANCI (ref. 51). In situations of intercross-linking (ICL) damage to the DNA, the large Fanconi complex is recruited to chromatin, and its subunit FANCL monoubiquitinates the FANCD2–FANCI heterodimer on K561 of FANCD2 and K523 of FANCI6–8. FANCL functions with the E2 enzyme Ube2t, and its isolated RING domain is not sufficient for FANCD2 modification because target recognition is dependent on binding of a different domain on FANCL52,53 (Fig. 1a). Interestingly, in vitro formation of the FANCD2–FANCI complex is required to achieve lysine-specific FANCD2 modification by FANCL, thus suggesting that the target complex has a crucial role in orienting the ubiquitination machinery53. However, reconstitution experiments have not been able to recapitulate an efficient and complete reaction because only FANCD2, and not FANCI, is modified in these reactions. The full Fanconi complex may be required for these additional steps, and post-translational modifications may be important in the reaction51. The best-studied site-selective ubiquitination reaction is that of the replication factor PCNA. PCNA is monoubiquitinated on a single lysine, K164, by the E2–E3 pair RAD6–RAD18, and this can be extended to K63-linked polyubiquitin chains by the E3s HLTF or Shprh (Rad5 in yeast) together with the E2 dimer Ubc13–MMS2

(Ube2n–Ube2v)2,54,55. In vitro and in vivo studies including knockin mice56 have shown that a K164R mutation on PCNA is sufficient to abrogate ubiquitination of PCNA and to inhibit translesion synthesis (TLS) signaling. These results indicate the high specificity of ubiquitin signaling. RAD18 is the E3 responsible for the monoubiquitination reaction on K164. The RAD18 RING domain is not sufficient for PCNA targeting57, thus suggesting that target recognition in this system is achieved by regions outside the RING domain. Interestingly, recent work has shown that the E2 UbcH5C (Ube2d3) alone is sufficient in vitro for specific PCNA ubiquitination on K164 (ref. 58). This raises the possibility that regulation of lysine specificity in this system may involve the PCNA molecule, which may promote modification of K164 over other lysines. Such a mechanism would be independent of the E3 ligase and would suggest a new role for the target molecule in the ubiquitination reaction. Another well-defined case of a lysine-specific modification is the ubiquitination of histone H2A within the nucleosome. H2A is abundantly monoubiquitinated on the C-terminal residue K119 by the RING E3 ligases present in the Polycomb repressive complex 1 (PRC1)4,23,24,59,60. In addition, in response to DNA damage, the N-terminal lysines at positions K13 and K15 in H2A are modified by RNF168 (refs. 3,61). These two modifications are structurally distinct in the context of the nucleosome, and they stimulate different molecular signaling pathways62. In these reactions, the isolated RING domains of the E3s are sufficient to provide lysine specificity in vitro. Moreover, both RING domains function in vitro with the same E2, UbcH5C, thus indicating that lysine specificity is determined by the action of the RING domains of these E3s3,23,63 (Fig. 1c). RING1B targets K119 on H2A only when the histone is assembled into nucleosomes23. The reaction is highly specific, although the adjacent residue, K118, can be targeted in this reaction when K119 is mutated. Biochemical and computational studies have proposed that DNA and possibly histone H4 mediate RING1B-nucleosome interactions to orient the E2~ubiquitin to the H2A K119 site in nucleosomes23. In contrast, the RNF168 RING domain can target H2A on K13 and K15 outside the nucleosomal context, in the dimeric form with H2B3,63. Biochemically, RNF168 does not interact with DNA but directly binds the H2A–H2B dimer. This dimer forms an acidic patch, which is exposed on the nucleosomal surface, located away from the target lysines; the integrity of these charges is required for efficient E3 catalysis63. This represents an additional situation in which the substrate molecule appears to actively participate in the reaction, possibly through long-range interactions distal from the target lysine. HECT ligases: initial observations on lysine-selection mechanism. For the majority of HECT ligases, substrate selection is performed by additional domains or interacting partners that recruit the ligase to the target molecule. Some examples are the HECT E3s Nedd4 and Smurf2, which are involved in receptor internalization 64. Their activity seems to have low lysine specificity and is affected by spatial constraints that limit interaction with domains on the target proteins. Nevertheless, HECT ligases are capable of selecting target lysines in polyubiquitin chains as well as in substrates. For example, in yeast the E3 ligase Rsp5, homolog to human NEDD4, targets the only lysine (K125) present in the cytoplasmic portion of the transmembrane protein Sna3, generating K63-linked polyubiquitin chains that induce sorting of Sna3 into multivesicular bodies65. Substrate recognition takes place via an additional domain on the HECT ligase, but the HECT domain itself selects the target lysine. A recent study provides evidence for a joint function of the N and C lobes of Rsp5 in catalysis and shows

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c

Figure 3  Specificity for amino groups in K63 Ub-chain specificity Linear Ub-chain specificity K11 Ub-chain specificity ubiquitin-chain formation. (a) The E2 dimer Ube2n Ube2s Ubc13–MMS2 (Ube2n–Ube2v) catalyzes HOIP d-Ub d-Ub K63-linked ubiquitin-chain formation. The K11 d-Ub d-Ub d-Ub d-Ub a-Ub K11 Met1 K63 Met1 K63 donor ubiquitin (d-Ub) is covalently attached a-Ub a-Ub a-Ub a-Ub a-Ub to the Ube2n subunit while the back side of Ube2v binds the acceptor ubiquitin (a-Ub) noncovalently to orient the K63 residue toward the catalytic site. (b) The E2 Ube2s catalyzes K11-linked ubiquitin chains by first activating the donor ubiquitin through a hydrophobic interaction (gray circles). A specific charged interaction between the E2 and the acceptor ubiquitin (red and blue circles) orients K11 toward the catalytic site. (c) Formation of linear ubiquitin chains is catalyzed by the RBR E3 ligase HOIP. Its catalytic domain contains a C-terminal extension that orients the acceptor ubiquitin and positions the N-terminal group of Met1 adjacent to the C-terminal end of the donor ubiquitin on the active cysteine. E3-ubiquitin interactions are shown as gray circles.

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that the ligase activity is spatially restricted. Rsp5 can efficiently target lysines only when introduced in sites within a few residues around K125 (ref. 18). This region is reminiscent of the ubiquitination zone of CRLs, except that it has a more constricted area (Fig. 2c). The complete mechanisms of substrate orientation and lysine choice in this family of E3 ligases are not yet fully elucidated, but it seems likely that the relative orientation of the E3 and target has an important role. The early age of RBR ligases. The action of RBR ligases is still relatively obscure because structural and initial biochemical data have become available only recently66–70. To date, no lysine-specific reactions on targets have been identified for RBR E3s, but this may be because autoinhibition of these E3 ligases has precluded full analysis of their specificities in vitro. Substrates of RBR ligases are mostly targeted with polyubiquitin chains and are marked for degradation or used in cellular signaling. Recent studies on the activation of Triad1 and HHARI may pave the way for biochemical analysis of these proteins’ true activity and specificities71. Surprisingly, in vitro targeting of Nemo by the LUBAC complex seems to require the catalytic machinery of both HOIP and HOIL, in a mechanism that is dependent on an unperturbed N terminus in the donor ubiquitin72. Amino-group specificity in ubiquitin-chain formation A particular case of lysine specificity occurs when ubiquitin itself is the target in the formation of polyubiquitin chains. In this process, the donor ubiquitin that forms the thioester with the E2 or E3 enzyme is conjugated to an amino group on the acceptor ubiquitin moiety, which functions as a target in the reaction. As observed by Wenzel and Klevit73, lysine specificity in this context is achieved mainly by the last enzyme that formed a thioester bond with ubiquitin before target modification: either the E2, in RING-dependent reactions, or the E3, in HECT- and RBR-type ligation. A subgroup of E2s participates in specific chain formation by positioning the acceptor ubiquitin in a defined orientation to favor deposition of the donor ubiquitin on the selected lysine. The first example is the E2 E2-25K (Ube2k), which forms K48-specific chains74. The E2 dimer Ubc13–Mms2 forms K63-linked ubiquitin chains in which the back side of the Ubc variant Mms2 provides a noncovalent binding site that orients the acceptor ubiquitin to present the target residue ubiquitin K63 for modification75,76 (Fig. 3a). The E2 Ube2s, which catalyzes formation of K11-linked ubiquitin chains, uses a different approach77,78. In this reaction, Ube2s orients the donor ubiquitin via a noncovalent E2-ubiquitin interaction involving helix αB on the E2 and the Leu8-Ile44-Val70 hydrophobic patch on ubiquitin. K11 specificity is then achieved by binding to the acceptor ubiquitin via electrostatic interactions, as confirmed by an elegant charge-swap experiment using Glu131 on the E2 and Lys6 (K6) on ubiquitin. These interactions bring K11 on the acceptor ubiquitin in proximity to the E2 active site. The final transfer requires a substrate-assisted 312

catalytic step to suppress the pKa of K11, performed by Glu34 on the acceptor ubiquitin78. The interactions between the donor ubi­quitin and E2 are also seen in RING-dependent mechanisms, in which the RING stabilizes the interaction between E2 and ubiquitin79–81, but the K11 positioning and activation seem to be specific for Ube2s (Fig. 3b). Finally, recent studies have identified an E2 enzyme, Ube2w, that specifically targets the N-terminal amino group of proteins82,83. Although mechanistic details are still unclear, the different aminogroup specificity of this enzyme could be explained by the presence of basic residues on Ube2w in place of a region containing a conserved aspartate known to be important in other E2s for deprotonation of the acceptor lysine83. For HECT-type ligation, the type of polyubiquitin chains generated is solely dependent on the E3, not on the E2. In the presence of UbcH5C (Ube2d3), the E3 Nedd4 will catalyze formation of K63linked chains, whereas another HECT E3, E6AP, will form K48-linked polyubiquitins84. This is not surprising because the E2 is unlikely to participate in the reaction: the HECT domain covalently binds the ubiquitin moiety before deposition on the acceptor ubiquitin. Recent data show that the C-terminal part of the C lobe of the HECT domain has a key role in determining chain specificity84,85. The role of the RBR E3 ligases in ubiquitin-chain targeting is not yet fully defined. For linear chain formation (on the N-terminal amino group of Met1 on ubiquitin), it is clear that the RBR protein HOIP is providing the target selectivity within the LUBAC complex. The HOIP RBR and LDD domains are sufficient for linear ubiquitin-chain formation in the absence of HOIL-1L and SHARPIN86,87. Its RING2 domain forms a thioester in a HECT-like manner. The acceptor ubi­quitin is positioned by the RING2 and the unique LDD domain to promote conjugation to the N terminus of ubiquitin70,87 (Fig. 3c). However, other RBRs do not seem to over-rule the E2-dependent chain-forming type, despite their HECT-like mechanism27. Lysine specificity in UBL modifications For the majority of UBL modifications, knowledge of mechanisms of site selection is still limited. Nevertheless, mechanisms of SUMO and Nedd8 modification are slowly beginning to be revealed. SUMO: E2-driven lysine selection. SUMOylation controls a wide range of cellular signaling pathways. SUMO exists in human cells in three isoforms: SUMO1, SUMO2 and SUMO3. In cells, protein modification with these UBL isoforms occurs at very low levels, and this hampers experimental analysis in endogenous conditions and limits understanding of SUMOylation dynamics. SUMO E3 ligases mainly function via an SP-RING domain, similarly to the ubiquitin E3 RING domain, by providing a bridge between the SUMO E2 enzyme Ubc9 and the substrate. As mentioned above, SUMOylation occurs often on lysines within specific consensus sites. In these cases, the consensus sequence

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Figure 4  Modes of recognition by readers. (a) Different chain types are recognized by specific readers, owing to their unique quaternary structures. (b) Specific modified targets are recognized by a combination of the ubiquitin- or UBL-binding domain and target-specific interaction motifs (for example, FAN1 nuclease or TLS polymerase Polη). Ub/UBL, ubiquitin or UBL. (c) Polyubiquitinated proteins are recognized by a specific ubiquitin interaction involving additional composite motifs that select the target and the polyubiquitin-chain type (for example, DNA helicase ZRANB3).

a Ub

Recognition of ubiquitin chains Ub

Ub

b

Ub

K K

Reader Ub

Ub Ub

Ub

Target

K

K

K Ub/ UBL

Recognition of polyubiquitinated targets

K

K K

K

K K

K

Target

Ub Ub Ub

Reader

ψKX(D/E), where ψ is a large hydrophobic residue and X is any amino acid, is directly bound by the SUMO E2 enzyme Ubc9 (refs. 88–90; Fig. 1d). Because this consensus site includes the targeted lysine, the substrate-E2-E3 interaction directs lysine selection to this site (Fig. 3d). Ubc9 can also selectively SUMOylate substrates in a phosphorylation-dependent manner. These target molecules contain a PDSM consensus motif (ψKXEXXSP) that accommodates the phosphorylation site and the target lysine for Ubc9 (refs. 91–93). The specificity in these reactions is provided by the E2 enzyme, even though E3 ligases may be involved in the reaction (Fig. 1d). One example of this type of modification involves a component of the nuclear pore complex, RanGAP. This protein is abundantly modified with SUMO1 on the consensus site K526 (ref. 94). This modification is mediated by the E3 ligase RanBP2, which promotes RanGAP modification by binding the Ubc9–SUMO complex in a manner that is neither RING like nor HECT like95,96. These molecules then form an E3 ligase complex required for nuclear pore–associated SUMOylation of other substrates97. In other cases, SUMOylation of a nonconsensus lysine has been reported. An example is the SUMOylation of the replication factor PCNA2 to prevent recombination during replication98. In yeast, K127 and K164 on PCNA can be SUMOylated: K127 is located in a Ubc9 consensus site and is modified even in the absence of E3 ligase, whereas modification of K164 requires the presence of the SUMO E3 ligase Siz1. E3 target-specific interactions have been mapped between the PINIT domain on Siz1 and the MEH loop on PCNA, ~25 Å away from the target lysine K164 (ref. 99). PCNA SUMOylation in human cells has also been reported, although the E3 ligase responsible has not yet been identified. It will be interesting to investigate whether K164 modification with SUMO is driven by a molecular mechanism similar to that of its ubiquitination2,58 (described above); in other words, does PCNA present its K164 for modification, or are the E3s (and E2) selecting this lysine? The SUMOylation of a nonconsensus lysine in E2-25K (Ube2k) seems to be dependent on the secondary structure of the target at the location of the lysine. Ubc9 modifies exclusively K14 on this target protein, despite this residue’s position between two consensus-site lysines, K10 and K18, where it sits in an α-helix. In contrast, in an unfolded peptide with the same sequence, the modification takes place at the consensus sites100. A recent report has highlighted the generation of non­specific SUMOylation signals after DNA damage101. This study substantiated previous observations that the SUMOylation machinery can target several proteins synchronously and often at multiple sites, thus leading to the modification of a group of proteins rather than specific targets 101,102. This ‘group modification’ acts as a ‘glue’ to promote protein-protein interaction, thereby enhancing DNA repair. Interestingly, these observations suggest that E3-substrate

c

Recognition of ubiquitinated targets

Reader

Ub

Reader

recognition in this system is driven not by specific proteinprotein interactions but rather by local proximity within the cell. The small numbers of SUMO E3 ligases identified and the regulation of target modification by the Ubc9 consensus sequence strongly support this view of nonspecificity for at least a subset of SUMO modifications. Nedd8. Nedd8 is a UBL molecule with key roles in the regulation of CRL function. Nedd8 conjugation on the cullin subunit of CRLs is necessary for activity of the E3 complex. Nedd8 conjugation is catalyzed in vivo by the RING-containing CRL subunit RBX and is enhanced by the DCN1 protein and the Nedd8-specific E2 UbcH12. Site-selective neddylation of cullins occurs by a two-step reaction wherein a canonical RING reaction, catalyzed by the RBX subunit of the CRL, is assisted by the DCN1 protein, which gives specificity for Nedd8 over ubiquitin conjugation and induces the RBX1–UbcH12~Nedd8 complex to adopt a catalytically competent orientation103,104. This reaction occurs on a specific site, K720 on cullin-1, and on structural homologous residues on the other cullin proteins. The importance of specific readers of ubiquitin and UBL signals For functional downstream signaling, ubiquitin and UBL modifications need to be recognized by specific signaling proteins that translate the modification into a cellular effect. These molecules are often called readers, and they contain ubiquitin- or UBL-binding modules to recognize the modifications. Some readers recognize different types of ubiquitin chains. In fact, each type of chain can assemble in a specific quaternary structure, according to the lysine linkage that it contains105–107. This creates diverse molecular platforms that differentiate the signal arising from the different types of chain. A number of chain-specific interacting domains have been described to date107, and their binding to poly­ ubiquitin chains is largely independent of the substrate to which those chains are conjugated (Fig. 4a). Similarly, proteins that are capable of recognizing multiple monoubiquitin modifications, such as EPS15 in RTK internalization pathways, mainly rely on binding to the ubiquitin moieties rather than to specific targets108. In addition, several reader proteins have been described for specific modifications (Table 1). These can simultaneously interact with the ubiquitin moiety and the target molecule by the joint action of two or more motifs (Fig. 4b). In this way, they ensure selective recognition of ubiquitin modifications to drive downstream signaling. For example, the TLS polymerases, such as polymerase η (Polη), contain a conserved ubiquitin-binding domain (UBZ) and a PCNAinteracting peptide (PIP) that allow selective dual binding to the ubiquitinated form of PCNA109. This induces the molecular switch between replicative polymerases and TLS polymerases in situations of DNA damage. However, fusion of ubiquitin to the C terminus of

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RE V IE W PCNA seems to rescue the DNA damage–avoidance phenotypes of K164 mutants, thus indicating that the dual affinity is not very selective for the relative orientation of the ubiquitin and the PIPbinding region on the substrate109. Similarly, the FAN1 nuclease contains a UBZ domain and specifically binds ubiquitinated FANCD2–FANCI dimers on DNA to promote ICL repair110–114. In the case of H2A ubiquitination, binders specific to ubiquitinated K119 have been difficult to identify. A recent study has proposed the zuotin domain–containing protein Zrf1 as the reader in Polycomb signaling115, although further biochemical validation is still pending. Interestingly, in DNA-damage signaling, the K13–15 ubiquitin modification is recognized directly by the 53BP1 protein, which specifically binds ubiquitinated K15 on H2A with its ubiquitination-dependent recruitment (UDR) motif and simultaneously binds methylated Lys20 on histone H4 via a Tudor domain62. An interesting case of specific ubiquitin-signal reading comes from the recent identification of the DNA helicase ZRANB3 as a specific reader of K63-linked polyubiquitination on PCNA 116–118. ZRANB3 contains two PCNA-interacting domains—the PIP and APIM motifs—as well as an NZF zinc finger that preferentially binds K63-linked polyubiquitin chains (Fig. 4c). With the concerted action of these three motifs, ZRANB3 preferentially associates with the K63polyubiquitinated form of PCNA and may participate in the subsequent template-switch mechanism116–118. SUMO modifications are read by proteins containing SUMOinteracting motifs (SIMs)119. These domains are found on a large number of proteins participating in virtually every cellular pathway. Specific SUMO modifications are also recognized by proteins containing SIM domains in tandem with target-specific interaction motifs. Readers of SUMOylated PCNA have been identified in yeast (Srs2) and humans (PARI); these are DNA helicases that contain a SIM and a PIP motif and function in suppressing unwanted DNA recombination at the replication fork98,120–122. A new SUMO-interaction mode has recently been identified, which involves a SUBA domain that allosterically regulates the activity of dipeptidyl peptidase-9 (DPP9)123. In the case of Nedd8-conjugated CRLs, no specific readers have been identified, but Nedd8 modification is required to displace the CAND1 protein from the CRL to activate the ligase function. CAND1 allows the exchange of F-box units to CRLs and, when bound, renders the complex incompetent for catalysis28. Readers are key players in ubiquitin and UBL signaling, and their recognition of modified targets is vital for further signaling. It is clear that geometry can play a major part in the recognition of specific ubiquitin and UBL signaling. The composite action of multiple domains requires a precise location of the ubiquitin or UBL moiety with respect to the target.

therefore becomes part of the modification process. Similarly, the target molecule has a central role in downstream signaling and the deconjugation process124. The molecular organization of specific readers supports the idea that spatial constraints in ubiquitin or UBL modifications are important to allow the modification to be recognized in combination with a surface on the substrate. This was also observed for degradative signals, for example on Sic1 (ref. 30). Nevertheless, some flexibility can be accommodated in these systems; for example, Srs2 can recognize K164- and K127-SUMOylated PCNA molecules equally well98,121. The complexity of lysine specificity in different ubiquitin and UBL reactions reflects the great diversity of signals arising from these modifications. Generalization of reaction mechanisms may obscure understanding of the details that drive each specific ubiquitin or UBL signaling cascade.

Conclusions Understanding of mechanisms that drive lysine specificity in ubi­quitin and UBL modifications is still limited. This is not unexpected, because the choice of target lysines ranges broadly from completely nonspecific to highly selective. It is apparent that in some pathways, such as SUMO modification and ubiquitin-chain formation, the specificity largely depends on the action of E2 enzymes. In contrast, in the majority of other systems, lysine specificity seems to involve E3 interactions. Whether lysine specificity is a determinant or a mere consequence of such interactions still remains to be elucidated for the individual modifications. It is becoming clear that lysine choice may also actively involve the target molecule itself, as seen in H2A or PCNA 63. The target

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Acknowledgments We thank R. Klevit for discussion and M. Uckelmann, D. Sahtoe and A. Murachelli for critical reading of the manuscript. Funding is from European Research Council advanced grant 249997 and Netherlands Organisation for Scientific Research-Chemical Sciences (NWO-CW) TOP grant 714.012.001 and Cancer Genomics Centre (CGC.nl). COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. Reprints and permissions information is available online at http://www.nature.com/ reprints/index.html.

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VOLUME 21  NUMBER 4  APRIL 2014  nature structural & molecular biology