Dossier on ubiquitination

In a review published in 2004 [1] and that still repays reading today, Cecile Pickart traced the evolution of research on ubiquitination from its origins in the proteasomal degradation of proteins through the revelation that it has a central role in cell cycle regulation and the recognition of regulatory roles for ubiquitin in intracellular membrane transport, cell signalling, transcription, translation, and DNA repair.

Pickart’s article marked the expansion of ubiquitination from what most regarded as a niche preoccupation, with implications only for housekeeping protein turnover and the destruction of damaged ribosomal products, to seize the attention and excite the imagination of researchers in every area of cell biology. Comparisons to phosphorylation are rife – specific ubiquitin ligases promote ubiquitina tion and deubiquitinating enzymes terminate its effects of ubiquitination just as ubiquitination just as kinases and phosphatases induce and terminate the effects of phosphorylation – though ubiquitination, unlike phosphorylation, can operate irreversibly, by delivering its targets to the proteasome: hence its vital role in the progression of the cell cycle.

It was already clear in 2004 that the number of ubiquitinating and deubiquitinating enzymes was very large, and that ubiquitin tags can be attached to proteins either as monomers or as poly-ubiquitin chains. But it had only recently been discovered that there are at least seven different kinds of poly-ubiquitin chains, and how the diversity of poly-ubiquitin signals is generated and interpreted in cells was in large part territory still to be explored.

In a series of articles the first three of which are published this month, we review what is now known about some of the central issues in research on ubiquitination, revisiting the questions of how ubiquitin signals are conjugated to and removed from specific targets, and how they are recognized and contribute to the regulation of central processes in cells.

Where are we now?Ubiquitin is a protein of 76 amino acids whose structure is shown in Figure 1. It is attached to a lysine in its target proteins either as a monomer or as a poly-ubiquitin chain each monomer of which is linked through its carboxy-terminal glycine to (usually) a lysine in the preceding ubiquitin in the chain. Three enzymes, known generically as E1, E2 and E3, act in series to catalyze ubiquitination (Figure 2). The E1 is the ubiquitin-activating enzyme, to which ubiquitin becomes attached in an ATP-dependent reaction through a reactive thioester bond. E2 is the

Ubiquitin ligases and beyondIvan Dikic1,* and Miranda Robertson2,*

E D I TO R I A L Open Access

*Correspondence: [email protected], [email protected] 1Institute of Biochemistry II, Medical Faculty of the Goethe University, University Hospital Building 75, Theodor-Stern-Kai 7, 60528 Frankfurt am Main, Germany 2BMC Biology, BioMed Central, 236 Gray’s Inn Road, London WC1X 8HL, UK

© 2012 Dikic and Robertson; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and

Figure 1. The structure of ubiquitin. Ubiquitin is a small, compact protein characterized by a b-grasp fold. The seven lysines that can be linked to the terminal glycine of another ubiquitin molecule to form poly-ubiquitin chains are colored red. The green shading indicates the hydrophobic patch through which ubiquitin interacts with specific ubiquitin-binding proteins. Image created by Masato Akatsu, Frankfurt University.

Dikic and Robertson BMC Biology 2012, 10:22 http://www.biomedcentral.com/1741-7007/10/22

ubiquitin-conjugating enzyme, to which the ubiquitin is transferred from the E1; and E3 is the ubiquitin ligase, which binds the target protein and directly or indirectly catalyzes its ligation to the ubiquitin. The E3 therefore determines the substrate specificity of ubiquitination, and the diversity of the cellular functions of ubiquitination is reflected in the existence of some hundreds of different mammalian E3s, compared with a few dozen E2s and two E1s.

The E2 conjugating enzymes have special significance in determining the type of ubiquitin chain assembled. There are seven lysines in ubiquitin, and poly-ubiquitin chains can be assembled through linkage to any of the seven: the distinct chains are known as K6, K11, K27, K29, K33, K48 and K63 chains, depending upon the lysine through which the monomers are linked. In most cases (the so-called RING E3 ligases – see below – with are by far the most numerous) it is the E2 that decides which type of chain is made. This generalization, and the generalization that ubiquitination involves linkage through a lysine fell victim in 2006 to the discovery that ubiquitin chains can be formed through linkage between the carboxy-terminal glycine of one ubiquitin and the amino-terminal methionine of another, to form so-called linear ubiquitin chains; and in this case it is the E3 (a complex known as LUBAC) that determines the linkage (see [2]). What we now know of the mechanism of linear ubiquitin chain assembly and the function of linear ubiquitin chains in immune signaling is discussed by Henning Walczak, Kazuhiro Iwai and Ivan Dikic [2] in one of the three inaugural reviews published this month.

A third generalization has succumbed to research described in the second article, from Dawn Wenzel and Rachel Klevit [3]. E3 ubiquitin ligases have until recently been classified as belonging to one of two structurally and functionally distinct families: the HECT ligases, and the RING/Ubox ligases. The mechanisms of these ligases

are lucidly outlined by Wenzel and Klevit and illustrated schematically in their Figure 1. Briefly, whereas in the case of the HECT ligases, the ubiquitin is transferred from the E2 to the E3, which then directly catalyzes its attachment to the substrate, in the RING ligases, the ubiquitin is transferred from the E2 to the substrate bound by the E3 (Figure 3). The RBR ligases, which are the topic of the article by Wenzel and Klevit, contain a

Figure 2. Three enzymes act in sequence to ubiquitinate targets. The E1 enzyme is the activating enzyme, to which ubiquitin is attached in an ATP-dependent reaction by a thioester bond (shown in red). The E2 enzyme is the conjugating enzyme, to which the ubiquitin is transferred from the E1. The E3 is the ubiquitin ligase, which directly or indirectly catalyzes the transfer of the ubiquitin to the target protein (the substrate), with the formation of an isopeptide bond (shown in black).

Figure 3. HECT and RING ligases act by different mechanisms. HECT E3 ligases (top) directly catalyze the attachment of ubiquitin to the substrate, whereas in the case of RING ligases (bottom) the ubiquitin is transferred from the E2 which, with the substrate, is bound to the E3. Exactly how the catalytic action of E2 is facilitated by the RING E3 is not known. Reactive thioester bonds are shown in red; the isopeptide bond with the target (substrate) protein is shown in black.

Dikic and Robertson BMC Biology 2012, 10:22http://www.biomedcentral.com/1741-7007/10/22

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RING domain that is structurally similar to that of other RING-type E3s, and had been regarded as a subclass of RING ligases; but it transpires that the RBR ligases behave more like the HECT family of E3s, and directly transfer the ubiquitin to the target protein. The active enzyme in the LUBAC complex belongs to the RBR subclass of E3s, helping to explain its eccentric behaviour. These fresh insights however raise again outstanding issues of how the different domains of these ligases contribute to their catalytic action and the type of chain assembled by them, and show how astonishingly little is still known about some of the fundamental mechanisms of ubiquitination.

Beyond ligasesThe world beyond the assembly of ubiquitin chains and the attachment of ubiquitin to cellular targets now encompasses all of cell biology. Ubiquitin chains of different linkages have distinct structural properties the principles of whose recognition by ubiquitin-binding proteins have yet to be fully explored. In particular, it is not known how the ubiquitin-binding modules that recognize mono-ubiquitins and the different poly-ubiquitin chains achieve the specificity for different ubiquitin species, and how this is translated into physiological responses. Recent studies have provided initial data about the mechanisms of deconjugation and functions of deubiquitinating enzymes, but many questions on the specificity of deubiquitinating enzymes and their roles in cell-specific functions remain open.

In the third review published this month, Simona Polo reaches into the territory of cell biology and explores the contribution of ubiquitination to the regulation of cell signaling by endocytosis [4], drawing parallels with the regulation at many levels of signaling pathways by phosphorylation. The critical issue here is how ligand-induced signaling regulates the activity of the E3 ligases that ubiquitinate receptors to initiate endocytic sorting for subsequent receptor degradation in the lysosome. Another important contribution of ubiquitination to the regulation of endocytosis is the ubiquitination of endocytic adaptor molecules through a process called coupled mono-ubiquitination that can be either E3-dependent and E3-independent.

Later articles in the series will confront some of the many open issues in research on ubiquitination, and extend the exploration of its contributions to fundamental cell biological processes.

Published: 15 March 2012

References1. Pickart CM: Back to the future with ubiquitin. Cell 2004, 116:181-190.2. Walczak H, Iwai K, Dikic I: Generation and physiological roles of linear

ubiquitin chains. BMC Biol 2012, 10:23.3. Wenzel D, Klevit R: Following Ariadne’s thread: a new perspective on RBR

ubiquitin ligases. BMC Biol 2012, 10:24.4. Polo S: Signaling-mediated control of ubiquitin ligases in endocytosis.

BMC Biol 2012, 10:25.

Dikic and Robertson BMC Biology 2012, 10:22 http://www.biomedcentral.com/1741-7007/10/22

doi:10.1186/1741-7007-10-22Cite this article as: Dikic I and Robertson M: Ubiquitin ligases and beyond. BMC Biology 2012, 10:22.

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Ubiquitination was first recognized for its function in tagging proteins for destruction by the proteasome [1-5], but is now known to be one of the major types of post-translational modifications necessary for proper functioning of signaling cascades [6-8]. The attachment

of ubiquitin molecules to their targets occurs through reactions mediated by proteins of three classes, acting in sequence: a ubiquitin-activating enzyme, E1, which contains an active-site cysteine to which the carboxy-terminal glycine of ubiquitin becomes attached through a reactive thioester bond; a ubiquitin-conjugating enzyme, the E2, to which the ubiquitin is transferred by an analogous reaction; and a ubiquitin ligase, the E3, which catalyzes the attachment of the ubiquitin to a lysine in the target protein [4,5,9-11]. Seven of the 76 amino acids of ubiquitin are lysines, which can themselves be targeted by ubiquitination to generate polyubiquitin chains of different linkage types depending on which lysine residue acts as the acceptor site for the incoming ubiquitin [12-14]. In an exception to this pattern, linear ubiquitin chains can be generated by the formation of a peptide bond between the carboxy-terminal glycine of the incoming and the amino-terminal methionine residue of the preceding ubiquitin molecule [15]. Recent research has established the identity and composition of an E3 ubiquitin ligase that generates linear ubiquitin chains, and has shown that these chains play an important part in several innate and adaptive immune signaling pathways, including the one triggered by tumor necrosis factor (TNF) [16-21]. Here we review what is known about the process by which linear ubiquitin chains are assembled, and how they contribute to TNF receptor 1 (TNFR1) signaling.

LUBAC and the assembly of linear ubiquitin chainsThe assembly of linear ubiquitin chains is unusual in three ways. First, as we have already mentioned, the linkage does not involve any of the lysine residues in the ubiquitin molecule, but occurs between the amino-terminal methionine of one ubiquitin and the carboxy-terminal glycine of the next in the chain. For this reason, linear ubiquitin chains are also known as M1-linked chains. The second unusual feature of linear ubiquitin chain assembly is that it is the E3 that determines the nature of the linkage in these chains [15] – a decision that is normally the prerogative of the E2, at least in reactions

AbstractUbiquitination now ranks with phosphorylation as one of the best-studied post-translational modifications of proteins with broad regulatory roles across all of biology. Ubiquitination usually involves the addition of ubiquitin chains to target protein molecules, and these may be of eight different types, seven of which involve the linkage of one of the seven internal lysine (K) residues in one ubiquitin molecule to the carboxy-terminal diglycine of the next. In the eighth, the so-called linear ubiquitin chains, the linkage is between the amino-terminal amino group of methionine on a ubiquitin that is conjugated with a target protein and the carboxy-terminal carboxy group of the incoming ubiquitin. Physiological roles are well established for K48-linked chains, which are essential for signaling proteasomal degradation of proteins, and for K63-linked chains, which play a part in recruitment of DNA repair enzymes, cell signaling and endocytosis. We focus here on linear ubiquitin chains, how they are assembled, and how three different avenues of research have indicated physiological roles for linear ubiquitination in innate and adaptive immunity and suppression of inflammation.

Generation and physiological roles of linear ubiquitin chainsHenning Walczak1,*, Kazuhiro Iwai2,* and Ivan Dikic3,*

R E V I E W Open Access

*Correspondence: [email protected], [email protected], [email protected] 1Tumour Immunology Unit, Department of Medicine, Imperial College London, 10N5 Commonwealth Building, Du Cane Road, London W12 0NN, UK 2Department of Biophysics and Biochemistry, Graduate School of Medicine and Cell Biology and Metabolism Group, Graduate School of Frontier Biosciences, Osaka University 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan 3Institute of Biochemistry II, Medical Faculty of the Goethe University, University Hospital Building 75, Theodor-Stern-Kai 7, 60528 Frankfurt am Main, Germany

© 2012 Walczak et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Walczak et al. BMC Biology 2012, 10:23 http://www.biomedcentral.com/1741-7007/10/23

involving RING-class E3s [22]. The linear ubiquitin chain E3 is now known to be composed of three proteins. The first two of these – the heme-oxidized IRP2 ubiquitin ligase-1 (HOIL-1, also known as HOIL-1L and RBCK1) and the HOIL-1-interacting protein (HOIP, also known as RNF31) – were identified as part of this multi-component E3 by Kirisako et al. [15], who also coined the term linear ubiquitin chain assembly complex (LUBAC) for this novel type of E3.

Subsequent research, however, revealed that LUBAC also contains a third component, SHARPIN (SHANK-associated RH domain interacting protein), whose carboxy-terminal region has high sequence similarity with the amino-terminal part of HOIL-1 [19-21]. The structural features of the three components of LUBAC and their interactions are schematically illustrated in Figure 1. All three contain ubiquitin-binding domains whereby they may bind to ubiquitin or to one another through ubiquitin-like (UBL) domains. HOIP is the central architectural component of the tripartite LUBAC, binding to both HOIL-1 and SHARPIN through their respective UBL domains. The stoichiometry of the three components that make up the 600 kDa LUBAC is currently unknown and it is also possible that complexes consisting of only two of the three factors exist [15]. In addition, it appears that in different cell types varying

amounts of HOIL-1, HOIP and SHARPIN are present independently of the other LUBAC components. It is therefore possible that these proteins may also serve functions that are independent of LUBAC activity [19-21].

Several lines of evidence indicate that LUBAC generates exclusively linear ubiquitin chains: (i) LUBAC can generate ubiquitin chains with lysine-less (K0) ubiquitin in vitro [15,18,21]; (ii) LUBAC is unable to generate ubiquitin chains from amino-terminally tagged ubiquitin [15,19]; and (iii) mass spectrometric analysis of polyubiquitin chains generated in vitro by LUBAC reveals linear ubiquitin linkages [15].

Where is the ubiquitin ligase activity of LUBAC and how is it activated?There are two classes of E3s: RING (really interesting new gene) or U-box-type E3s catalyze the E2-mediated transfer of ubiquitin to target proteins [23,24], whereas in the case of HECT (homologous with E6-associated protein C-terminus)-type E3s ubiquitin is first transferred to the E3 by the formation of a thioester bond, and then from the E3 to the substrate. Both HOIL-1 and HOIP contain a RING-in-between-RING (IBR)-RING (RBR) domain (Figure 1), and hence form part of the RBR subclass of RING-E3s, so in principle either HOIL-1 or

Figure 1. Schematic representation of the LUBAC components, SHARPIN, HOIP and HOIL-1. There is significant sequence homology (45% identity) between the carboxyl terminus of SHARPIN and the amino terminus of HOIL-1, each of which contains a UBL and an NZF motif. HOIP is the catalytic subunit of the tripartite LUBAC with SHARPIN and HOIL-1 as accessory factors that bind via their respective UBL domains to the NZF2 and UBA domains of HOIP, respectively. HOIP, SHARPIN and HOIL-1 also bind to ubiquitin chains through NZF-mediated interactions. The functions of the ZnF domain of HOIP and the coiled-coil domain of SHARPIN are currently unknown. The RBR domain of HOIP, but not of HOIL-1, is responsible for linear ubiquitin chain generation by LUBAC. Arrows indicate confirmed interactions between the proteins. Abbreviations: ZnF, zinc finger; NZF, Npl4 zinc finger; UBL, ubiquitin-like domain; UBA, ubiquitin-associated domain; IBR, in-between RING domain; RBR, RING-IBR-RING domain.

UBL

NZF

Coiled-coil Sharpin

RING1 RING2 IBR NZF

UBL HOIL-1

HOIP ZnF UBA RING1 RING2 IBR NZF1

NZF2

Ubiquitin binding

Ubiquitin binding

Ubiquitin binding


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HOIP could account for the ubiquitin ligase activity of LUBAC. However, the combination of recombinant SHARPIN and HOIL-1 cannot generate linear ubiquitin chains in vitro, whereas recombinant HOIP together with HOIL-1 or SHARPIN (or of course both) can; moreover, overexpression of these combinations is also capable of activating NF-kB, one of the key transcription factors activated by TNF (see below) [19-21].

This is in line with experiments showing that, despite the fact that HOIL-1 and HOIP both contain an RBR domain (Figure 1), it is the RBR of HOIP that mediates the formation of the linear ubiquitin linkage in these different complexes because the intact RBR of HOIP, but not of HOIL-1, is required for LUBAC activity [15]. Indeed, despite its containing an apparently complete RBR domain [25,26], no linear ubiquitination activity has so far been detected for recombinant wild-type HOIL-1 in ubiquitination assays in vitro. It is possible, however, that interactions with partners other than HOIP and SHARPIN, or perhaps post-translational modification, may induce its activation.

If HOIP is the active E3 in LUBAC, what is the contribution of HOIL-1 and SHARPIN? The answer to this question and to the question of HOIL-1 E3 activity may lie in a mechanism recently reported for Parkin, another RBR-containing E3, which closely resembles HOIL-1 in domain structure [27,28]. Parkin is auto-inhibited by its UBL and this auto-inhibition may be relieved by binding to a co-factor or a substrate [29]. The zinc finger and the UBL domains of HOIL-1 and SHARPIN are crucial for activation of the linear-ubiquitin-generating activity of HOIP [16], and it may be that the binding of SHARPIN and/or HOIL-1 to HOIP relieves an auto-inhibition in HOIP in a way that is analogous to the activation of Parkin by binding to a partner (K Rittinger and B Stieglitz, personal communication). No qualitative differences have yet been discovered in the potential of SHARPIN and HOIL-1 to unleash the linear-ubiquitin-generating capacity of HOIP, although they seem likely to exist. It is tempting to speculate that SHARPIN and HOIL-1 may direct the linear ubiquitination activity of HOIP to different targets.

It remains to be determined whether there are binding partners for HOIL-1 other than HOIP and SHARPIN, and, if so, whether this results in HOIL-1-mediated generation of linear or other ubiquitin chain linkages. Recent results from Rachel Klevit and colleagues on Parkin and another RBR-domain-containing protein, human homologue of Ariadne (HHARI), may hint at the mechanism whereby LUBAC promotes the formation of ubiquitin chains. They showed that HHARI, and possibly also Parkin, functions as an HECT-like E3 ligase, through a conserved cysteine residue in the second RING domain, RING2, that accepts a charged ubiquitin in a thioester

intermediate before transferring the bound ubiquitin to a substrate [30]. This insight into mechanism, however, cannot explain the specific generation of linear ubiquitin linkages by HOIP, because Parkin is known to generate K48- and K63-linked chains [31,32].

Clearly we are only just beginning to explore the biochemistry of linear ubiquitin chain formation by LUBAC, and much remains to be discovered about the specificity of this complex in the exclusive generation of linear ubiquitin chains, and the exact actions of the different components within the protein complex.

Linear ubiquitination in the TNF receptor pathwayUbiquitination by K63- and K48-linked chains was already known, before the discovery of linear ubiquitin chains, to play an important part in the activation of NF-kB, arguably the most crucial output of TNFR1 signaling. Activation of the TNFR1 pathway occurs when trimeric TNF crosslinks three TNFR1 monomers to initiate formation of the TNFR1 signaling complex (TNF-RSC). As schematically illustrated in Figure 2, TNFR1 activation results in the induction of gene activation by NF-kB and mitogen-activated protein kinases (MAPKs) and, depending on the strength of these gene-activatory signals, also in cell death, which can be either apoptotic (non-inflammatory) or necroptotic (inflammatory).

NF-kB is a central transcriptional regulator in the induction of immune response genes that, in the absence of activating signals, is located in the cytoplasm. Activation of NF-kB occurs through the action of a kinase complex, referred to as the IkB kinase (IKK) complex, which consists of two catalytic subunits, IKKa (IKK1) and IKKb (IKK2), and a critical regulatory subunit called NEMO (IKKg). This complex is required to phosphorylate the inhibitor of NF-kB (IkB), thereby inducing its degradation and releasing NF-kB to relocate to the nucleus and bind to the promoters of immune genes. The IKK complex is recruited to the TNF-RSC through NEMO, and this results in activation of the kinase activity of this complex. MAPKs are activated as a result of recruitment of the TAB/TAK complex into the TNF-RSC. Whilst the TAB/TAK complex is currently thought to be recruited exlusively to K63-linked chains within the TNF-RSC, the IKK complex can be recruited to this complex via linear chains and, albeit with lesser affinity, also via K63- and K11-linked chains [33].

LUBAC activity was first implicated in signaling from TNFR1 when TNF-mediated NF-kB activation was shown to be impaired in primary hepatocytes from HOIL-1 knockout mice, and LUBAC was shown to form part of the signaling complex that forms on binding of TNF by the receptor, and moreover to be crucial both to the stability of the TNF-RSC and in determining the outcome of TNF signaling [16-18]. How LUBAC


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recruitment to the TNF-RSC influences signaling outcome is not known in detail, but it is known that NEMO, which is the regulatory component of the kinase complex that activates NF-kB, recognizes linear ubiquitin chains through its specialized ubiquitin-binding domain, UBAN (ubiquitin-binding domain present in ABINs and NEMO) [17,34]. The UBAN motif is known also to recognize ubiquitin chains with other linkages – in particular K63 chains, which are also present on components of the TNF-RSC, including on RIP1 [19]; but the UBAN of NEMO binds linear di-ubiquitin with a

different topology and about 100-fold higher affinity than it does K63-linked di-ubiquitin. This suggests that the promotion of NF-kB activation by LUBAC following TNF stimulation may be due to linear ubiquitination of a component of the signaling complex whereby NEMO is recruited to, or retained in, the complex more effectively.

LUBAC also linearly ubiquitinates NEMO itself in the native TNF-RSC [19]. TNF-induced linear ubiquitination of NEMO preferentially occurs on K285 and K309, and in cells expressing a NEMO K285R/K309R mutant, NF-kB activation induced by LUBAC overexpression or by

Figure 2. Model of TNFR1 signaling with and without LUBAC activity. Binding of trimeric TNF crosslinks the extracellular domains of three TNFR1 molecules and induces the formation of the TNF-RSC (also referred to as complex I). The tripartite LUBAC (ochre) is recruited to the TNF-RSC in a TRADD-, TRAF2- and cIAP-dependent manner (left panel) [16,19]. LUBAC activity in the TNF-RSC results in linear ubiquitination of RIP1 and NEMO [19] and enables the NF-kB and MAPK pathways to be activated to their full physiological extent. After a delay, and probably as a consequence of deubiquitination events at the membrane-bound TNF-RSC, the composition of the complex changes, and a second complex, complex II, appears in the cytosol [45]. Complex II (not shown) recruits FADD and caspase- 8, which are responsible for the induction of apoptosis, and includes RIP1 and RIP3, which mediate necroptosis. In the presence of LUBAC, however, the induction of cell death is prevented, probably by both stabilization of complex I by linear ubiquitination and the actions of genes induced by the NF-kB and MAPK pathways [16]. In the absence of SHARPIN (right panel), the other two LUBAC components are also drastically diminished, TNF-induced gene activation is attenuated and the TNF-RSC is destabilised, resulting in enhanced complex II formation and, consequently, cell death induction by apoptosis and necroptosis. Note that we have drawn the ubiquitin chains as diubiquitins. The actual length of the individual ubiquitin chains attached to components of the TNF-RSC – or indeed to components of any other signaling complex – is currently unknown.

MAPK

Gene induction

LUBAC present LUBAC absent

NF-kB

IKKb IKKb

TNF

TNF-R1 TR

ADD

TRAF2

cIAP1/2

K63

linear

K11 K48

Cell death

IKKb IKKb

TNF

TNF-R1

TRAD

D

TRAF2

cIAP1/2

K63 K11 K48

MAPK

Gene induction

NF-kB

Cell death

Inflammation


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stimulation with IL-1b was reduced [18]. The mechanism of linear-ubiquitination-induced NF-kB activation has not been solved, but current data indicate that binding of NEMO to linearly linked ubiquitin induces a conformational change in the helical structure of NEMO that may promote the kinase activity of the IKK complex [17,35]. Alternatively, recognition of linear chains by NEMO conjugated to the NEMO molecules of other IKK complexes could bring the kinase domains of the respective IKK complexes into close proximity, thereby enabling trans-autophosphorylation [17], a process similar to the one that occurs between receptor tyrosine kinases when activated by ligand-induced dimerization.

Together, these findings indicate a functional role for linear ubiquitination in full gene activation by the signaling pathways triggered by TNF in vivo. In the absence of LUBAC components the TNF-RSC still forms and activation of NF-kB still occurs, albeit at significantly reduced levels [20,21]. Experiments with HOIP-deficient cells will be needed to strictly corroborate these findings, but it is likely that the NF-kB activation that still occurs in the absence of LUBAC is mediated by K63- and/or K11-linked chains, which are also present in the native TNF-RSC [19] and can also bind or be attached to NEMO [33,36-39].

Absence of LUBAC components also renders cells sensitive to TNF-induced cell death [16,20]. Intriguigingly, this cell death is not only apoptotic [19,20] but also necroptotic [19]. Importantly, this is also true of primary keratinocytes obtained from young, non-diseased cpdm mice. These mice, which are genetically deficient in SHARPIN and thus lack functional LUBAC complexes [19], have played a central part in the discovery of the physiological function of LUBAC. They present with stark immune system developmental abnormalities, and develop a chronic multi-organ inflammatory syndrome with strong manifestation in the skin (hence the name of this mutation: chronic proliferative dermatitis (cpdm)) at about 4 to 6 weeks of age [40]. The inflammatory syndrome that characterizes cpdm mice is apparently paradoxical, because it is generally thought that aberrantly high TNF-induced gene activation is the source of inflammation induced by this cytokine. Our finding that TNF stimulation results in aberrant death of cpdm-derived cells, and that this cell death has both an apoptotic and a necroptotic (and thus inflammatory) component [19,41,42], suggested a different explanation: namely, that the inflammation in cpdm mice could be due to inflammatory cell death consequent on the absence of SHARPIN-requiring LUBAC activity. To investigate this possibility, we crossed cpdm mice with TNF-deficient mice, and were able to show that even partial genetic ablation of TNF prevented the formation of inflammatory lesions in cpdm mice,

indicating that TNF-induced cell death is indeed causative for the inflammatory phenotype that characterizes these mice [19]. It is possible that secondary necrosis, which can occur as a consequence of apoptosis, may also contribute to inflammation in cpdm mice.

Hence, linear ubiquitination is implicated in two different physiological processes: the development of the immune system and the prevention of chronic inflammation, where the latter effect is achieved through interference with TNF-induced cell death. Whether the aberrant cell death in the absence of LUBAC is due to reduced gene-inducing capacity of TNF, to a more direct effect of absence of linear ubiquitin chains from the signaling complexes induced by TNF, or perhaps to a combination of both these effects remains to be established. Our current suggestion for the contribution of LUBAC to these pathways is schematically illustrated in Figure 2.

What next?The discovery of linear ubiquitin chains and their specific ligase complex (LUBAC) has sparked considerable interest in the physiological roles of these cellular signals. Rapid progress in the delineation of protein assemblies involved in conjugation and recognition of linear ubiquitination in vivo have provided a platform for addressing new challenges in the field. Among them are proteomic studies of the linear ubiquitinome – the set of linearly ubiquitinated proteins in cells; analysis at atomic resolution of protein complexes implicated both in conjugation and recognition functions; and the possibility of finding novel regulatory components of LUBAC by identification of regulatory principles of LUBAC functions and of novel linear ubiquitin binding domains (LUBIDs). Interestingly, the new LUBIDs include the zinc finger (ZF) domain of HOIL-1, which has recently been shown to recognize specifically linear ubiquitin chains [43]. One of the greatest challenges, however, will be to understand how the different types of ubiquitin linkages cooperate to achieve the exact physiologically required signaling output, and how this is regulated at the level of the receptor signaling complexes. Identifying the individual ubiquitination events that occur in the TNF-RSC and determining their respective physiological roles is likely to provide valuable insight into biochemistry and function of different types of ubiquitinations, including linear ubiquitination [44].


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20. Ikeda F, Deribe YL, Skånland SS, Stieglitz B, Grabbe C, Franz-Wachtel M, van Wijk SJ, Goswami P, Nagy V, Terzic J, Tokunaga F, Androulidaki A, Nakagawa T, Pasparakis M, Iwai K, Sundberg JP, Schaefer L, Rittinger K, Macek B, Dikic I: SHARPIN forms a linear ubiquitin ligase complex regulating NF-kappaB activity and apoptosis. Nature 2011, 471:637-641.

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doi:10.1186/1741-7007-10-23Cite this article as: Walczak H, et al.: Generation and physiological roles of linear ubiquitin chains. BMC Biology 2012, 10:23.


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Ubiquitination modifies proteins in a variety of ways, the significance of which we only partially comprehend. Ubiquitin can be attached: as an individual moiety to a single or multiple lysine residues of substrate (mono- or multiple monoubiquitination); as chains of ubiquitin moieties that are interlinked through any one of the seven lysine residues of ubiquitin (for example K48- or K63-linked chains); or as branched chains, to name but a few [1]. The cell interprets each of these modifications as a distinct signal. The first described role of ubiquitination as mediating protein degradation through targeting to the proteasome has now been complemented with numerous other functions [2]. For example, the signal encoded by K63-linked chains can mediate functions as diverse as receptor endocytosis [3,4], activation of protein kinases in the NF-κB pathway and the initiation of error-free DNA repair [2].

Signal transduction from transmembrane cell surface receptors to nuclear transcription factors is regulated at multiple levels by protein ubiquitination. The covalent attachment of one, or often more, ubiquitin moieties has emerged as the principal mechanism for termination of

signaling, by targeting the receptor for endocytosis and, ultimately, degradation in the lysosome [3]. This device controls a vast array of mammalian signaling receptors, such as receptor tyrosine kinases, G-protein coupled receptors (GPCRs), growth hormone receptors, the major histocompatibility complex I, NOTCH, various channels and transporters, and cytokine and interferon receptors [3]. Receptors that are internalized after activation are directed first into the endosomes of the endocytic pathway, and then into multivesicular bodies (MVBs), which undergo a process of maturation that ends with fusion with the lysosome and delivery of the contents for degradation. Ubiquitination of the receptor provides the crucial signal for entering this pathway [3,5-7].

Subsequent delivery of membrane receptors to the lysosome requires accurate recognition of the ubiquitinated cargo by endosomal adaptors and sorting proteins. For the EGF receptor, for example, these are EPS15, epsin and ESCRTs (endosomal sorting complexes required for transport), which contain one or more ubiquitin-binding domains (UBDs) [8]. EPS15 and epsins act at the initial steps of internalization, serving to recruit the enzymes required for ubiquitination of downstream components of the endocytic pathway. ESCRTs act sequentially at various points of the degradative route, sorting the ubiquitinated cargo at the endosomal membrane for inclusion into the intraluminal vesicle of the MVB. ESCRT-0, composed of the two interacting proteins HRS and STAM, is the first in this process. Three additional complexes, ESCRT-I, ESCRTII and ESCRT-III, are then involved in the generation of inward vesicle budding, which is required for MVB maturation (for reviews see [5,9]). The ubiquitin-binding ‘route controllers’ that operate in this way to ferry the internalized receptor inexorably towards a degradative fate in lysosomes are also inducibly ubiquitinated [10,11] (Figure 1).

The inducibility of the system illustrates the dynamic nature of ubiquitin-based endocytic regulation. Indeed, over the past 15 years, studies from different laboratories

AbstractUbiquitin-dependent regulation of endocytosis plays an important part in the control of signal transduction, and a critical issue in the understanding of signal transduction therefore relates to regulation of ubiquitination in the endocytic pathway. We discuss here what is known of the mechanisms by which signaling controls the activity of the ubiquitin ligases that specifically recognize the targets of ubiquitination on the endocytic pathway, and suggest alternative mechanisms that deserve experimental investigation.

Signaling-mediated control of ubiquitin ligases in endocytosisSimona Polo1,2


Correspondence: [email protected] 1IFOM, Fondazione Istituto FIRC di Oncologia Molecolare, Via Adamello 16, 20139, Milan, Italy. 2Dipartimento di Medicina, Chirurgia ed Odontoiatria, Università degli Studi di Milano, Via di Rudinì 8, 20122 Milan, Italy.

© 2012 Polo; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Polo BMC Biology 2012, 10:25http://www.biomedcentral.com/1741-7007/10/25

have revealed a critical role for ubiquitination in receptor down-regulation, a process that is essential for the spatial and temporal resolution of receptor signaling [3].

The central component of this regulatory pathway is the E3 ligase that attaches the ubiquitin molecules to the receptor, or to the ubiquitin-binding endocytic adaptors (Figure 1). A hierarchical set of three types of enzyme is

required for substrate ubiquitination: ubiquitin-activating (E1), ubiquitin-conjugating (E2) and ubiquitin-protein ligase (E3) enzymes. In mammals two E1-activating enzyme transfer ubiquitin to roughly three dozen E2s, which function together with several hundred different E3 ubiquitin ligases to ubiquitinate thousands of substrates. In the endocytic pathway distinct E3 enzymes generally catalyze the ubiquitination of the cell-surface receptors and the endosomal sorting proteins [3]. Therefore the endocytic sorting of a given target generally involves more than one E3 ubiquitin protein ligase.

Here we focus on how signaling controls the activity of the E3 ligases that ubiquitinate receptors and endocytic adaptors. Although E3 ligases have largely been considered to be constitutively active and regulated only at the level of target binding, it has recently become evident that they are subject to regulation by either E3 or substrate phosphorylation, or by exploitation of adaptor proteins that facilitate E3 activity. We discuss here the various ways of regulating E3 ligases in the context of endocytosis.

Ligand-induced E3 ligases for receptor ubiquitinationProtein phosphorylation, which is well known to function in recruitment of the ubiquitination machinery to the substrate [12], may also act directly to regulate the activity of distinct ubiquitin ligases.

The best-characterized circuitry involves the E3 ligase Cbl, which is responsible for the ubiquitination of several receptor tyrosine kinases (RTKs) [3]. The mammalian Cbl protein family consists of the three homologs c-Cbl, Cbl-b, and Cbl-3, all of which associate with a wide variety of signaling proteins [13]. Two highly conserved amino-terminal domains contribute strongly to E3 regulatory function. First, the amino-terminal tyrosine kinase binding (TKB) domain of Cbl recognizes phosphotyrosine residues and allows Cbl to interact directly with activated RTKs at the plasma membrane (Figure 2). Second, the RING finger domain recruits ubiquitin-loaded E2s, whose interaction with Cbl results in the ubiquitination and subsequent degradation of the associated RTK. In the case of the epidermal growth factor receptor (EGFR) and the hepatocyte growth factor receptor MET, the molecular mechanism of receptor ubiquitination has been investigated in detail. In both cases, Cbl binds directly to phosphotyrosine (pY)-sites on the activated receptor through its TKB [14,15], as well as indirectly through its constitutive partner GRB2, which is recruited to receptors via other pY sites [7,16-18]. Both direct and indirect interactions of Cbl with the EGFR or MET are required for full ubiquitination of these receptors (Figure 2). Once bound, the ligase is phosphorylated and consequently activated [19]. Two

Figure 1. Receptor internalization and the role of ubiquitin. The example shown here is that of the EGF receptor. An activated EGFR is ubiquitinated at the plasma membrane and recruits the endocytic adaptor proteins EPS15 and EPSIN-1. These are ubiquitinated, in turn, and direct the internalized receptor to the endosomal pathway where it binds the sorting protein HRS. This is also ubiquitinated and directs progression of the ubiquitinated receptors towards lysosomal degradation through the ESCRT complexes. For simplicity, the EGF receptor is depicted as monoubiquitinated: in reality, it is both multimono- and polyubiquitinated. MVB, multivesicular body.

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structural studies have now shed light on the mechanism of phosphorylation-induced activation of c-Cbl and Cbl-b [20,21]. In the absence of substrate binding, the TKB and RING domains form a compact structure that masks the E2 binding site. Binding of the TKB to the substrate induces a first rotation of the linker region, allowing phosphorylation of tyrosine 371 (363 in Cbl-b). This phosphorylation event induces a complete rotation of the linker region that unmasks the RING E2 binding surface and activates the ligase [20,21].

Another class of E3 ligases, the HECT NEDD4 family [22], whose regulation has been extensively studied, also regulates endocytosis and sorting of numerous signaling receptors [3,5-7]. These enzymes present a conserved modular organization with an amino-terminal C2 domain that is crucial for membrane localization, between two and four WW domains capable of recognizing substrates and adaptor proteins through PY motifs, and a carboxy-terminal catalytic HECT domain. In contrast to RING-based ligases in which the RING is an allosteric activator of the E2, HECT-containing E3s have intrinsic catalytic activity and directly ubiquitinate their targets. In humans, there are nine members of this family: NEDD4 (also known as NEDD4-1), NEDD4L (also known as NEDD4-2), ITCH (also known as AIP4),

WWP1, WWP2, SMURF1, SMURF2, NEDL1 (also known as HECW1) and NEDL2 (also known as HECW2). Rsp5 is the unique, essential member of the NEDD4 family in Saccharomyces cerevisiae. In normal conditions most of them appear to be in an inactive state because of an intramolecular inhibitory interaction between the carboxy-terminal HECT and the amino-terminal C2 domain (in the case of SMURF2, NEDD4 and WWP2 [23]) or the WW domains (in the case of ITCH [24]). Activation of this class of enzyme can occur in various ways that are briefly described below.

ITCH is the E3 ligase for the chemokine receptor CXCR4 [25]. The ubiquitin moiety on CXCR4 serves as a signal on endosomes for entry into the degradative pathway and long-term attenuation or downregulation of signaling [25]. ITCH can interact directly with CXCR4 through a non-canonical WW domain-mediated interaction involving serine residues within the carboxy-terminal tail of CXCR4. These serine residues are phosphorylated upon agonist activation, and are critical for mediating agonist-promoted binding of ITCH and the subsequent ubiquitination and degradation of CXCR4 [26] (Figure 3a). Also in this case, the ligase appears to be regulated by phosphorylation. ITCH phosphorylation is activated by JNK1 [24], and is thought to lead to

Figure 2. EGFR ubiquitination by Cbl. Upon EGF-dependent receptor activation, the GRB2-Cbl complex binds to the receptor through interactions of: i) the SH2 domain of GRB2 with pY1045 of EGFR, and ii) the TKB domain of Cbl (either c-Cbl or Cbl-b) with pY1068 or pY1086. This substrate interaction may either stabilize or select for a partially open Cbl conformation (see bottom panel and main text). EGFR-bound Cbl becomes phosphorylated on a critical tyrosine, leading to full rotation of the linker region. This, in turn, exposes the RING domain for ubiquitin-charged E2 binding, resulting in the allosteric activation of the E2 by Cbl and ubiquitination of the EGFR. Note that, to simplify the picture, Cbl bound to one receptor molecule is depicted to ubiquitinate the other molecule of the dimer. No available data suggest that this is indeed the case. For simplicity, the EGF receptor is depicted as monoubiquitinated: in reality, it is both multimono- and polyubiquitinated.

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conformational changes that disrupt the inhibitory intramolecular interactions between its WW and the HECT domains.

In the case of SMURF2, autoinhibition of the HECT domain by the C2 domain helps in maintaining the steady-state levels of this E3 ligase and can be relieved by adaptor-mediated substrate targeting [23]. SMURF1 and SMURF2 bind to TGF-β family receptors through the inhibitory Smad proteins, SMAD6 and SMAD7, to induce their ubiquitin-dependent degradation. Wiesner et al. [23] demonstrated that intramolecular interactions between the C2 and HECT inhibit SMURF2 catalytic activity interfering with ubiquitin thioester formation. This in cis autoinhibition can be relieved by binding of

the amino-terminal domain (NTD) of the adaptor protein SMAD7 to the E3 HECT domain (Figure 3b). The SMAD7 NTD further enhances the catalytic activity of the SMURF2 ligase by recruiting the E2 UbcH7 to the HECT domain [27]. By releasing C2-mediated autoinhibition, stimulating E2 binding, and recruiting SMURF targets, SMAD7 functions at multiple levels to control E3 activity and ensure specificity in SMURF-catalyzed ubiquitination.

Recently, a role for a UBD present on the N-lobe of the HECT domain of NEDD4 and Rsp5 has been identified [28,29]. The ability of the HECT domain to bind non-covalently to the distal ubiquitin at the growing end of the polyubiquitin chain on the substrate allows enzyme

Figure 3. Activation of E3 ubiquitin ligases through recruitment to activated receptors. (a) Ubiquitination of CXCR4 by ITCH. ITCH activity is inhibited as a result of the intramolecular interaction between the WW domain and the carboxy-terminal catalytic HECT domain. Upon agonist-mediated activation, CXCR4 becomes phosphorylated at Ser324 and Ser325 by an unknown kinase. This leads to the recruitment of the ITCH, through its WW domain, and consequent release of the inhibitory intramolecular interaction, allowing ubiquitination of the receptor. (b) Ubiquitination of the TGF-β receptor by the SMURF2-SMAD7 complex. SMURF2 activity is inhibited as a result of the intramolecular interaction between the amino-terminal C2 and the carboxy-terminal catalytic HECT domain. The interaction with SMAD7 NTD displaces the C2 domain of SMURF2 from the HECT domain and activates the ligase. The activated SMURF2-SMAD7 complex associates with activated TGF-β receptor complexes at the membrane via the displaced C2 domain, causing receptor ubiquitination.

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processivity [28]. It is tempting to attribute an inhibitory role of the C2 binding for this critical feature of these enzymes. Accessibility of the UBD may be restored in response to upstream signaling events capable of inducing phosphorylation and/or ubiquitination of critical sites in the C2 or in the HECT domain, leading to full ligase activation. While this hypothesis needs to be experimentally verified, we notice that ubiquitination of NEDD4 is a critical event for the coupled monoubiquitination of EPS15 ([30] and below).

In some cases, such as for the epithelial Na+ channel (ENaC), receptor:ligase interaction – and consequent receptor ubiquitination – is the default pathway, with phosphorylation negatively regulating ligase activity. NEDD4-2 binds constitutively to ENaC PPxY-containing motifs and catalyzes its ubiquitination, internalization and lysosomal targeting. This prevents Na+ overload in epithelial cells and is necessary for the maintenance of salt and fluid balance in the body. To increase ENaC abundance at the surface and enhance epithelial Na+ absorption, NEDD4-2 is phosphorylated by various kinases, including protein kinase A (PKA), serum- and glucocorticoid-inducible kinase (SGK), and IκB kinase (IKK)β (Figure 4a). Phosphorylation induces binding of

14-3-3 proteins, which prevents NEDD4-2 from binding to ENaC [31-33].

Regulation of receptor ubiquitination by adaptor proteinsFinally, specific binding proteins can regulate the process of ubiquitination by acting as adaptors to recruit E3 proteins to receptors that lack a direct binding motif for the ligase (reviewed in [34]). The best evidence for this mechanism so far comes from membrane transport directed by the yeast HECT E3 ligase Rsp5, which is the unique homolog of the mammalian NEDD4 family proteins. While in humans there are nine members of the NEDD4 family, yeast Rsp5 is sufficient on its own to control most membrane traffic ubiquitination events at the plasma membrane and at other biomembranes [35]. Cooperation with adaptors such as Bul1/Bul2, Bsd2/Tre1/Tre2, or Ear1/Ssh4 enables Rsp5 to cope with this large number of substrates [35], and the discovery of the yeast family of ARR-related proteins (ARTs), which direct Rsp5 activity to various plasma membrane receptors [36-38], shows that the adaptor mechanism is even more extensive than previously thought (Figure 4b). Does receptor signaling regulate these HECT adaptor proteins?

Figure 4. Regulation of channels and transporters by ubiquitination. (a) ENaC ubiquitination by NEDD4-2. NEDD4-2 binds to PY motifs on the epithelial Na+ channel ENaC and catalyzes its ubiquitination. This induces ENaC endocytosis and lysosomal targeting, resulting in fewer channels at the cell surface. To increase Na+ transport, NEDD4-2 is phosphorylated by kinases, including PKA, SGK, and IKKβ, in turn activated by various signaling pathways. Phosphorylation of NEDD4-2 induces binding of 14-3-3 dimers (not shown), which prevents NEDD4-2 from binding to ENaC. As a result, endocytosis of ENaC is inhibited, and increased ENaC presence at the surface enhances epithelial Na+ absorption. (b) Rsp5 ubiquitinates permeases and transporters. In yeast, arrestin-related endocytic adaptors (ARTs) and the E3 ubiquitin ligase Rsp5 are recruited to the plasma membrane in response to environmental stimuli that trigger the endocytosis of proteins such as permeases and transporters (for example, the arginine transporter Can1). Through their PY motifs, ARTs bind to the WW domain of Rsp5 and mediate ubiquitination of cargo. ARTs are also ubiquitinated by Rsp5, an event required for endocytosis.

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Two recent papers provide evidence that it does [39,40]. MacGurn et al. [40] demonstrated that signaling from TORC1, which is a central regulator of cell growth in response to amino acid availability, regulates Rsp5 targeting and endocytosis of nutrient transporters at the plasma membrane. The effector mechanism that regulates endocytosis involves a TORC1-Npr1 negative kinase signaling cascade that tunes the phosphoinhibition of the ubiquitin ligase adaptor Art1 [40]. Leon and colleagues [39] identified the α-arrestin Rod1/Art4 as a direct target of the glucose signaling pathway. Glucose promotes Rod1/Art4 dephosphorylation and its subsequent release from a phospho-dependent interaction with 14-3-3 proteins. This allows Rsp5-mediated Rod1 ubiquitination, a prerequisite for transporter endocytosis [39]. It is conceivable that other signaling pathways may similarly regulate the activity of other ART family proteins.

Does this mechanism also operate in mammals? And if yes, how? These important gaps in our understanding need to be filled. Of note, arrestin domain containing protein 3 (ARRDC3) was recently shown to interact with NEDD4 and to be an essential adaptor for β2-adrenergic receptor (β2AR) ubiquitination [41].

Ligand-induced E3 ligases for adaptor ubiquitinationThe adaptors we have just discussed function to recruit E3 ligases to receptors. We now return to the endocytic adaptors that are recruited to the ubiquitinated receptors and direct their subsequent endocytosis. As with direct receptor ubiquitination, the ubiquitination of endocytic adaptors plays a critical role in endocytosis. The arrestin (ARR) family of proteins is able to direct internalization of GPCR cargo. Signaling from activated GPCRs is terminated when GPCRs are phosphorylated by GPCR kinases (GRKs), leading to the recruitment of ARR, which binds to AP-2 and clathrin, structural components of the vesicles formed at the plasma membrane causing the whole complex to be internalized. Agonist-stimulated ubiquitination of ARR mediated by the E3 ligase murine double minute (MDM2), an important negative regulator of p53, is critical for rapid receptor internalization [42]. MDM2-ARR binding is constitutive and does not persist after receptor activation, suggesting that ubiquitin modi-fication might cause a conformational change on ARR required to promote internalization. GPCRs themselves can also be ubiquitinated, most probably by NEDD4, an event required for cargo degradation but not internalization [43]. Thus by analogy with the ‘phosphorylation code’ on the receptor carboxy tail, ubiquitin modifications on both adaptors and receptors result in a ‘ubiquitination code’ that fine-tunes signal strength, localization, and cellular functions of GPCR (Figure 5a).

ARR is not the sole example of an endocytic adaptor subjected to ubiquitin modification. Several components of the downstream endocytic machinery are monoubiquitinated upon RTK activation [10,11,44,45]. In most cases, these adaptors are ubiquitin receptors that are ubiquitinated by the E3 ligase NEDD4. The presence of a UBD is required for monoubiquitination of the UBD-harboring adaptor, in a process termed coupled monoubiquitination whose molecular workings have been elucidated using the endocytic proteins EPS15 and EPSIN-1 as models [30,46] (Figure 5b). By contrast, the mechanism by which NEDD4 recruitment is induced by the activated EGFR remains to be clarified.

What is the role of adaptor ubiquitination? Monoubiquitination might permit the formation of several tiers of ubiquitination-dependent interactions in the endosome, by allowing binding of ubiquitinated cargo (through UBDs) and recruiting another layer of ubiquitin receptors through the monoubiquitin signal. The result would be signal amplification and progression of ubiquitinated cargoes along the endocytic pathway.

Monoubiquitination of ubiquitin receptors may also result in an intra-molecular interaction between their UBDs and monoubiquitinated residues, with resulting dissociation from the ubiquitinated cargo [47,48]. These two possibilities are not mutually exclusive and both mechanisms may be involved in the regulation of endocytic processes, possibly by acting at distinct steps and/or regulating different endocytic adaptors.

Is the ubiquitination cascade like the phosphorylation cascade?It is important to realize that the power of ubiquitin stems from its capacity to act as a protein-protein interaction module that targets substrates to a plethora of downstream effectors. In order to realize this network, complex molecular machines generate and recognize signal diversity based on ubiquitin-binding modules. The network is fine-tuned by ubiquitinating (E3 ligases) and deubiquitinating enzymes (DUBs) that balance the absolute levels of protein ubiquitination, as well as the abundance and localization of adaptors that contain docking sites for specific ubiquitinated proteins (UBDs) [49].

One such network pivots around EGFR. EGF stimulation promotes ubiquitination of EGFR and of EGFR endocytic adapters, providing a striking example of concerted regulation of signaling, ultimately regulating the route of EGFR internalization [50,51]. Our own approach to understanding the complex interplay between EGFR-induced signaling circuitries has been the recent elucidation of the EGF-induced ‘ubiproteome’ [52]. This work has uncovered an extensive ubiquitin-based signaling network that impinges on a wide array of signaling circuitries and various aspects of cellular


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physiology including DNA repair, nuclear transport, mRNA processing, metabolic pathways, and ribosome biogenesis. Interestingly, many ubiquitin machinery enzymes were detected in the EGF-induced ubiproteome. Regardless of the initial triggering mechanism (which necessarily involves the kinase activity of the EGFR), the ubiquitin signal seems to be rapidly transmitted to, and amplified through, the ubiquitin machinery. Just as in the phosphorylation cascade, in which critical enzymes such as kinases and phosphatases are often activated by phosphorylation, ubiquitinating enzymes appear to be

regulated by ubiquitination. Moreover, a comparison of the EGF-induced ubi- and pY-proteomes revealed a significant overlap and identified many highly connected ‘hub’ proteins that are both phosphorylated and ubiquitinated [52]. These data suggest that two complementary and interlinked enzymatic cascades drive the flow of information from receptors to downstream signaling molecules: kinases/phosphatases and E3 ligases/DUBs. In essence, these two post-translational-modification-based networks can be conceptualized as two overlapping, diffusely interconnected, matrices

Figure 5. Ubiquitination of adaptors. (a) Agonist induces rapid ubiquitination of GPCR-recruited ARR by MDM2, a process required for receptor internalization. Once internalized, GPCRs can be dephosphorylated and rapidly recycled to the plasma membrane through a mechanism that involves the sorting proteins EBP50 and NSF. (b) Activated EGFR is ubiquitinated at the plasma membrane by Cbl and recruits the UBD-containing endocytic adaptors EPS15 and EPSIN-1 at the plasma membrane, and subsequently HRS at the endosomal membrane. These adaptors, in turn, are ubiquitinated by NEDD4 through a process known as coupled monoubiquitination. This directs progression of the ubiquitinated receptors toward lysosomal degradation through the ESCRT complexes. A similar mechanism can be envisioned for other RTKs.

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through which external signals are transduced and interpreted by the cell. To understand how this is achieved, a multidisciplinary approach is required. No single ‘omics’ analysis can fully unravel the complexities of the system. Complete understanding will be achieved only by integrating information from high-resolution molecular investigations, ‘omics’ approaches and ‘top-down’ systems-based modeling.

AcknowledgementsResearch in the Polo laboratory is supported by grants from the Associazione Italiana per la Ricerca sul Cancro, the Italian University Research Program for the Development of Research of National Interest and of Health (PRIN), the Association of International Cancer Research, the CARIPLO Foundation, and the EMBO Young Investigator Program.


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doi:10.1186/1741-7007-10-25Cite this article as: Polo S: Signaling-mediated control of ubiquitin ligases in endocytosis. BMC Biology 2012, 10:25.

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E3 ligases: historically a two-party systemUbiquitination is the process by which proteins are selectively targeted for a variety of cellular fates. This post-translational modification is carried out by a trio of enzymes: an E1 ubiquitin-activating enzyme, an E2 ubiquitin-conjugating enzyme, and an E3 ubiquitin ligase. In most cases, E3 ubiquitin ligases assume the role of transferring activated ubiquitin from a restricted cohort of E2s to specific substrates. In a given genome, putative E3s greatly outnumber E2s, underscoring their role in substrate selection. For example, in humans, there are over 600 E3 ubiquitin ligases and fewer than 40 E2s [1]. On the basis of their mechanism and structure, E3 ligases have historically been classified into two families, the HECT- and RING/UBOX-type ligases (Figure 1). Recently we determined that Ariadne, the defining member of a subclass of RING-containing E3 ligases known as RING-between-RINGs (RBRs), blurs the line between RING and HECT-type E3s.

Eukaryotic E3 ubiquitin ligases are generally identified by the presence of either a HECT or a RING domain. The features of each type of domain are well defined and are readily predictable by primary sequence analysis. RINGs are characterized by a regular spacing of conserved cysteines and histidines which bind two Zn2+ ions that stabilize the overall structure of this domain, allowing for recognition and activation of E2 Ub-conjugating enzymes [2]. HECT domains are identified on the basis of their similarity to the founding member of the family, E6AP. In contrast to RING domains, which can occur at any position within a given protein, all known HECT domains are found at the carboxy-terminal end of their respective proteins. The HECT domain has a bilobal structure: the lobe at the amino-terminal end of the domain (the N-lobe) serves as the E2-binding domain, and the lobe at the carboxyl terminus (the C-lobe) contains the catalytic cysteine.

There are two general mechanisms by which the ultimate substrate-ubiquitin isopeptide adduct is formed. An essential difference between the two mechanisms is the location of activated ubiquitin at the final transfer step (Figure 1a). In reactions involving RING/UBOX-type ligases, the ubiquitin is attached to an E2 to form an E2~Ub thioester conjugate, and the E3 binds both substrate and the E2~Ub simultaneously to promote the aminolysis reaction in which ubiquitin is transferred to a lysine on a substrate. By an as yet undetermined mechanism, E3 binding enhances the reactivity of the E2~Ub thioester bond to allow for aminolysis [3,4]. Catalytic residues have not been identified for RING/UBOX type ligases and are presumed not to exist. In reactions involving HECT E3s, ubiquitin is transferred from an E2~Ub to form an E3~Ub thioester conjugate and the final transfer step occurs directly from the E3 active site to a substrate lysine. Thus, the two mechanisms differ in terms of the identity of the active site that is responsible for the aminolysis: it is the E2 active site in RING/UBOX-catalyzed reactions and it is the E3 active site in HECT-catalyzed reactions. Substrates may be mono-ubiquitinated at one or more sites; or poly-ubiquitin chains may be attached to them. Poly-ubiquitin

AbstractUbiquitin signaling pathways rely on E3 ligases for effecting the final transfer of ubiquitin from E2 ubiquitin conjugating enzymes to a protein target. Here we re-evaluate the hybrid RING/HECT mechanism used by the E3 family RING-between-RINGs (RBRs) to transfer ubiquitin to substrates. We place RBRs into the context of current knowledge of HECT and RING E3s. Although not as abundant as the other types of E3s (there are only slightly more than a dozen RBR E3s in the human genome), RBRs are conserved in all eukaryotes and play important roles in biology. Re-evaluation of RBR ligases as RING/HECT E3s provokes new questions and challenges the field.

Following Ariadne’s thread: a new perspective on RBR ubiquitin ligasesDawn M Wenzel1 and Rachel E Klevit2,*


*Correspondence: [email protected] 2 Department of Biochemistry, University of Washington, Seattle, Washington 98195, USA. Full list of author information is available at the end of the article

© 2012 Wenzel and Klevit; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Wenzel and Klevit BMC Biology 2012, 10:24 http://www.biomedcentral.com/1741-7007/10/24

chains may be of eight known topologies determined by their distinct linkages, and named K48, K63, K11, K27, K29, K33, and K6 chains according to the lysine residue through which the ubiquitins are linked to one another; or linear chains when the linkage is between the carboxyl terminus of one ubiquitin and the amino terminus of the next

Because the E2 active site is responsible for aminolysis in RING/UBOX-catalyzed reactions, it follows that the product produced by RING/UBOX-type ligases, be it mono-ubiquitination or a poly-ubiquitin chain of a specific topology, is determined in large part by the identity of the E2 involved in the reaction. A RING-type

Figure 1. RING and HECT-type mechanisms of ubiquitin transfer. (a) On the left, a RING E3 ligase (blue) is shown bound to a ubiquitin-conjugated E2, from which the ubiquitin is transferred to a lysine on the substrate. On the right, a HECT E3 ligase (orange) is shown bound to a ubiquitin-conjugated E2, from which ubiquitin is first transferred to the active-site cysteine of the E3, and is then transferred to a lysine on the substrate bound to the E3 (lower panel). (b) Proposed mechanism for RBR ubiquitin transfer. RBR ligases combine features of both RING- and HECT-type ligases. The ubiquitin-conjugated E2 binds to the RING1 domain of the RBR E3 ligase. The ubiquitin is then transferred from the E2 to the E3 RING2 domain from which it is transferred to the substrate.

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E3 that can bind a diverse set of E2s has the potential to produce several distinct types of ubiquitination products. This phenomenon has been demonstrated for a growing number of RING-type ligases such as BRCA1/BARD1 and the APC, which can produce poly-ubiquitin of K63 and K48 linkages, as well as mono-ubiquitin, depending on the E2 [5,6]. In contrast, it is the identity of the HECT-type E3 itself that determines the type of ubiquitin modifications conferred on substrates by this class of E3 ligases, and residues in the active-site-containing C-lobe have been shown to determine the type of ubiquitination signal generated by HECT-type E3s [7].

RBR E3s: Reaching across the aisleRBR E3s were originally identified by virtue of their RING domains, but the presence of two additional domains, IBR and RING2, define them as a subclass [8,9]. RBR-type ligases are defined by a trio of closely spaced domains: 1) a canonical amino-terminal RING domain, dubbed RING1, 2) an in-between RING domain (IBR), and 3) a domain named RING2 [10]. As in the case of canonical RING domains, RBR domains are not limited to any particular location within the proteins that contain them. Although RING1 domain sequences follow the cysteine and histidine patterns typical of RINGs from other E3 ligase families, the IBR and RING2 domain do not, and these two domains are unique to RBR-type proteins. RBR E3 ligases are found throughout eukaryotes with two members in yeast and thirteen in human [10].

Despite their strong persistence throughout evolution, most RBR E3s are not well understood, and their substrates and E2 partners are poorly defined. Members of this E3 family mediate diverse processes that include the regulation of translation and the activation of NF-κB signaling, among others [11,12]. The most studied RBR E3 is Parkin, because of its association with Parkinson’s disease [13]. The list of Parkin substrates continues to grow, although it is not clear which are critical for understanding the death of dopaminergic neurons, the hallmark of Parkinson’s disease. The first substrates reported for Parkin were α-synuclein, Pael-R, and CDCrel-1, all of which accumulate in patients with heritable Parkinson’s disease [14,15,16]. These substrates suggest a role for Parkin in the clearance of misfolded or aggregation-prone proteins. Additionally, Parkin is recruited to mitochondria where it is thought to regulate turnover of damaged mitochondria. Proteins that regulate mitochondrial morphology, mitofusin-1 and mitofusin-2, are substrates of Parkin ubiquitination [17,18].

Several RBR ligases have roles in regulating immune signaling. Overexpression of RNF216 (TRIAD3, ZIN) enhances the degradation of specific Toll-like receptors, with co-ordinate down-regulation of Toll-like receptor

signaling [19]. RNF216 may function to attenuate the host response to viral invasion, as RNF216 ubiquitinates TRAF3, a potentiator of the anti-viral response, and targets it for proteasomal degradation [20]. The RBR proteins Rbck1 (HOIL-1) and RNF31 (HOIP) form a heterodimeric E3 ligase that function together with the protein SHARPIN to generate linear ubiquitin chains [21,22]. Linearly-linked poly-ubiquitin chains specifically recruit components of the NF-κB signaling pathway including NEMO to promote the phosphorylation and subsequent degradation of inhibitors of NF-κB.

Yeast contain two RBR proteins, orthologs for Ariadne (ARIH1) and RNF14 (ARA54). It has been suggested that the extreme conservation of RBR genes from the earliest eukaryotes to human may indicate that these proteins assume a housekeeping role [23]. Human RNF14 was first identified through a yeast two hybrid screen for proteins that bind to the androgen receptor, and is thought to be a co-activator of androgen receptor function [24]. The yeast ortholog to RNF14 may have a function in the regulation of translation termination [25]. The Drosophila protein Ariadne, which was the first member of the RBR family to be described, is important for the development of the fly nervous system: neuronal differentiation is disrupted in Ariadne mutants because of a failure in axon guidance [9]. Ariadne was named after the Greek goddess Ariadne, whose thread was said to help guide Theseus out of the Minotaur’s labyrinth.

Ariadne and other RBR-type E3s have been presumed to be RING-like in both structure and mechanism because of the similarities between RING1 and canonical RING domains. Unexpectedly, we discovered that the human RBR E3 HHARI (human homolog of Ariadne) functions in a way that is analogous to that of a HECT-type ligase, in that it forms an obligate thioester bond with ubiquitin before it is transferred to the substrate [3] (Figure 1b). A cysteine in the RING2 domain that is highly conserved among all RBR-type E3s functions like a HECT active site cysteine: when this cysteine is mutated to serine, an oxy-ester-linked HHARI~Ub intermediate can be trapped. Mutation of the analogous cysteine residue (C431) in RING2 of Parkin disrupts ubiquitin transfer mediated by this RBR E3, suggesting that this mode of ubiquitin transfer is a general feature of RBR E3s. Because they contain both a RING-type E2-binding domain at their amino-terminal end and an active-site-containing domain at their carboxy-terminal end, we proposed that RBRs can be thought of as RING-HECT hybrids.

Domain and structural analysis of RBRsTo date, structural insights into the RBR ligase supradomain are limited to structures solved for individual component domains: there are two structures


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of IBR domains, and one structure each of a RING1 and RING2 domain [26,27] [PDB ID 1WIM, PDB ID 2CT7]. The dearth of structural information is probably due to the difficulty of expressing and purifying these proteins in large amounts. With such a small sampling, it is unclear whether these structures will be representative of the entire family of RBR ligases. However, the sequence similarity of these domains among human members of the RBR ligase family suggests we may expect to see similar structures.

Though limited, the available structures do provide some functional insights. The structure of the RNF144 RING1 is similar to that of other RING-type E3s such as BRCA1, with the E2-binding interface readily apparent (PDB ID 1WIM, Figure 2). E2-E3 disrupting mutations that are structurally analogous to those in bona fide RING-type E3s abrogate the binding of the E2 UbcH7 to HHARI [5,28,29]. Although the E2-E3 binding interactions of RBR E3 ligases seem to be analogous to those of the RING E3 ligases, the functional consequence of the interaction is not. Whereas binding of the BRCA1/BARD1 RING domain leads to a significant enhancement of the thioester reactivity of the E2~Ub conjugate (in this case UbcH5c~Ub) towards lysine, no enhancement of E2 reactivity was detected for a construct composed of the

RING1 and IBR domains of HHARI [3]. Thus, although the RING1 domain of HHARI serves as an E2-binding domain, it lacks the catalytic capacity of other canonical RING domains. Whether this is a general feature of the RING1 domains of RBRs remains to be tested experimentally.

The function of the IBR domains of RBR ligases is even less clear. The IBR domains of Parkin and RNF31 (HOIP) are structurally sparse outside of the two Zn2+ binding-centers (Figure 3) [26]. Shaw and colleagues noted that the amino- and carboxy-termini of the IBR domain are close to one another and may therefore serve to bring the RING1 and RING2 domains together [26]. Such a function would be analogous to that of the flexible linker found between the amino-lobe and the carboxy-lobe in HECT-type E3s, which allows conformational changes essential for E3 activity. If so, one would predict that mutations that either reduce the flexibility of the putative linker region or change the orientation of the two RING domains would disrupt ubiquitin transfer, as reported for the HECT ligase WWP1 [30]. However, the structural conservation of the IBRs in terms of Zn2+-binding residues, number of amino acids, and the fact that this domain is found almost exclusively in RBR E3s, suggest that the domain is unlikely to serve merely as an elaborate linker.

Figure 2. RING1 of RBRs maintains features characteristic of canonical RINGs. (a) Structures of RING domains are displayed with Zn2+ coordinating residues as yellow sticks and Zn2+ ions displayed as grey spheres. A conserved hydrophobic residue important for E2 binding is shown as orange sticks. The structures are (from left to right) the E3 ligase CNOT4 (blue) bound to the E2 UbcH5b (purple) (PDB ID 1UR6) (the E2 active site is shown as yellow spheres); the heterodimeric RING E3 ligase BRCA1 (blue)/BARD1 (green) (PDB ID 1JM7); TRAF6 (PDB ID 3HCT); RING1 of the RBR E3 RNF144 (PDB ID 1WIM). (b) Multiple sequence alignment of the RING domains of CNOT4, BRCA1, TRAF6, and RNF144. Coloring in the multiple sequence alignment corresponds with the colors in the structures, highlighting residues important for Zn2+ coordination and E2 binding. Sequences were aligned using CLUSTALW and manually adjusted based on structure [44].

CNOT4: CPLCMEPLEIDDINFFP-CTCGYQICRFCWHRIRT---DEN---G--LCP--ACBRCA1: CPICLELIK-EP--VSTKCDHIF--CKFCMLKLLN---QKK---GPSQCP--LCTRAF6: CPICLMALR-EA--VQTPCGHRF--CKACIIKSIR---D-----AGHKCP--VDRNF144: CKLCLGEYPVEQMTTIAGCQCIF--CTLCLKQUVELLIK-EGLETAISCPDAAC

(a)

(b)


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Our finding that Parkin RING2 residue C431 is an active site cysteine clarifies several observations about Parkin activity, while raising new questions about the structure and mechanisms of this family of ligases. The Parkin mutation C431F gives rise to a loss-of-function phenotype and is associated with juvenile onset Parkinson’s disease, a phenotype that was earlier attributed to structural destabilization, under the presumption that C431 was involved in Zn2+ binding [31]. More recent experiments, however, in which the conserved cysteines of Parkin were systematically mutated to alanine, showed that most mutants were insoluble and mislocalized, except the C431A mutant, suggesting that this residue is not part of the Zn2+ coordination network of Parkin [32]. The only RING2 structure to date is of HHARI RING2 [27] (Figure 4). Unlike the RING1 domain, whose structure led to the notion that the mechanism of the RBR ligases would be like that of the canonical RING ligases, the HHARI RING2 domain structure looks nothing like a canonical E2-binding RING: we discuss below the implications of this structural dissimilarity. Despite the presence of six well conserved cysteines, the structure binds only one Zn2+ ion, leaving two cysteine residues unliganded [27]. We have identified one of the free cysteines, C357, as the active site cysteine of HHARI. The single Zn2+-binding site in HHARI RING2 contrasts with electrospray mass

spectrometry data that predict two Zn2+ ions for Parkin RING2 [33]. The coordination of Zn2+ residues in Parkin may well differ from that of HHARI, and how this is accomplished remains to be determined structurally. Rankin and colleagues propose a Parkin RING2 model with two Zn2+-binding sites, composed of residues C418, C421, C441, C436 and C446, C449, H461, C457 [34]. This model leaves C431 free to form a thioester bond with ubiquitin (Figure 4).

RBR isopeptide catalysisThe observation that RBR ligases function through a HECT-like mechanism implies that there are additional residues near the active site cysteine that facilitate isopeptide catalysis with a substrate lysine. However, if studies with HECT E3s are any indication, these may be difficult to identify. Aside from the active site cysteine, few catalytic residues have been identified within HECT domain ligases. Studies involving a bacterial HECT-like E3, SopA, identified a conserved motif among HECT domain active sites surrounding the active site cysteine, LXXShTCfXn (where upper case letters indicate invariant residues, lower case letters indicate conserved residues and X indicates a variable position) [35]. Mutation of the conserved leucine or threonine to an alanine decreases activity [35]. Removal of a highly conserved phenylalanine in the carboxy-terminal tail of

Figure 3. Conservation of the IBR domain. (a) Structures of IBR domains solved to date from RNF31 (left) (PDB ID 2CT7) and Parkin (right) (PDB ID 2JMO). (b) Multiple sequence alignment of the IBR domain from human RBR ligases. Residue numbers are shown at the beginning of the alignment. Sequences were aligned using CLUSTALW [44]. Swiss-Prot numbers for sequences used in multiple sequence alignments are as follows: Cullin-9: Q81WT3, Parkin: O60260, ANKIB1: Q9P2G1, ARIH1: Q9Y4X5, ARIH2: O95376, RBCK1: Q9BYM8, RNF144A: P50876, RNF144B:Q7Z419, RNF19A:Q9NV58, RNF19B: Q6ZMZ0, RNF216: Q9NWF9, RNF14: Q9UBS8, and RNF31: Q96EP0.

RNF144B_[IBR] QLYQRLKFEREVH--LDPYRTWCPVA-DCQTVCPVASSDPGQPVLVECP------------SCH--LKFCSCC-KDAW---HAEVSCRNF144A_[IBR] QRYKKLQFEREVL--FDPCRTWCPAS-TCQAVCQLQDVGLQTPQPVQCK------------ACR--MEFCSTC-KASW---HPGQGCCullin-9_[IBR] SKYEKALLRGYVE--SCSNLTWCTNPQGCDRILCRQGLGCGT---TCS-------------KCG--WASCFNCSFPEA---HYPASCANKIB1_[IBR] KRYLQFDIKAFVE--NNPAIKWCPTP-GCDRAVRLTKQGSNT---SGSDTLSFPLLRAPAVDCGKGHLFCWEC-LGEA---HEPCDCARIH2_[IBR] -KYRRYLFRDYVE--SHYQLQLCPGA-DCPMVIRVQEPRARR---VQCN------------RCN--EVFCFKC-RQMY---HAPTDCARIH1[IBR] LKYQHLITNSFVE--CNRLLKWCPAP-DCHHVVKVQYPDAKP---VRC-------------KCG--RQFCFNC-GENW---HDPVKCRNF19B_[IBR] HKYEEFMLRRYLA--SDPDCRWCPAP-DCGYAVIAYGCASCP--KLTCER----------EGCQ--TEFCYHC-KQIW---HPNQTCRNF19A_[IBR] EKYEEFMLRRWLV--ADPDCRWCPAP-DCGYAVIAFGCASCP--KLTCGR----------EGCG--TEFCYHC-KQIW---HPNQTCRNF14_[IBR] ARYDRLLLQSSLD--LMADVVYCPRP--CCQLPVMQEPGCTMG---ICS------------SCN--FAFCTLC-RLTY---HGVSPCParkin_IBR NRYQQYGAEECVL--QMGG-VLCPRP-GCG-AGLLPEPDQRK---VTCEG-------GNGLGCG--FAFCREC-KEAY---HEG-ECRNF31_[IBR1_deg ALFHKKLTEGVLM--RDPKFLWCAQ---CSFGFIYEREQLEA----TCP------------QCH--QTFCVRC-KRQWEEQHRGRSCRBCK1[HOIL-1]_I ------ENRSAFS--YH-----CKTP-DCKGWCFFEDDVNEF----TCP------------VCF--HVNCLLC--KAI---HEQMNCRNF216_[IBR] YKYYERKAEEEVAAAYADELVRCPS---CSFPALLDSDVKRF----SCPN----------PHCR--KETCRKC-QGLWK-EHNGLTC

- -l----v---- ----wCp-p--C-----v---- - ---------C-----fC--C-k--w---H----C

RNF144B _101RNF144A _91

Cullin-9 _2140ANK1B1 _402ARIH2 _208ARIH1 _256RNF19B _186RNF19A _199RNF14 _289Parkin _313RNF31 _779RBCK1 _362RNF216 _583

** * * * * * *

RNF31 Parkin

(b)

(a)


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HECT domain ligases abolishes the ability of HECT E3s to modify lysines on substrates, but does not interfere with transthiolation from E2 to the HECT active site cysteine [36]. Whether the conserved phenylalanine in HECT domains plays a direct role in catalysis remains to be seen. It is not clear if RBR-type E3s will use a similar mechanism for function. The lack of similarity between HECT E3 C-lobes and RBR RING2s at the sequence level suggests it will be challenging to identify residues that are important for isopeptide catalysis on the basis of our current knowledge. Residues around the RBR active site cysteine do not follow the LXXShTCfXn motif, and as the role of the carboxy-terminal phenylalanine in HECT-type ligases is not well understood, it is impossible to know if RBR-type ligases have residues that fulfill a similar function. The possibility remains that residues in other domains of the RBR may contribute to the activity of RING2. Structural and biochemical characterization of RING2 in context of the RING1 and IBR domains may be key to future progress in this respect.

Like HECT ligases, the ubiquitination products generated by RBRs are independent of E2 identity and are likely to be an intrinsic property of each RBR E3. To date, polyubiquitin chains with linear, Lys48, and Lys63 linkages, multiple mono-ubiquitination, and mono-ubiquitination have all been reported as products of RBR ligases [37,38,39,40,41]. Although Parkin itself has been reported to form many types of products, a recent study suggests that Parkin products can be influenced by the fusion of artificial molecular tags, or truncation of constructs, so previous results should be viewed in this

light [42]. The RBR proteins RBCK1 (HOIL-1) and RNF31 (HOIP) form a complex known as LUBAC (Linear Ubiquitin Assembly Complex) and produce linear polyubiquitin chains with the human E2s UbcH7, UbcH5, and Ube2k [37]. To date, no HECT-type or canonical RING-type E3s have been reported to build linear poly-ubiquitin chains, suggesting that this property may be distinctive of RBR E3s. Future studies aimed at understanding the determinants within RBRs that specify the type of product generated are needed to further our understanding of RBR mechanism and function.

Unanswered questionsThe distinct features of the RBRs indicate that attempts to extrapolate from knowledge of the other better characterized classes of E3 ligases are likely to fail. This opens a host of unanswered questions regarding RBR function. Which E2s work to transfer ubiquitin to RBR E3s and what are the determinants and features of this step? To date, the cohort of E2s identified as working with RBR-type ligases most often includes the E2s UbcH7 and UbcH8 [10]. UbcH7 (and probably UbcH8 by analogy) can only transfer ubiquitin via a transthiolation reaction and are therefore specialized E2s for HECT-type transfer mechanisms [3]. Even though it is a RING domain (RING1) of RBRs that binds E2s directly, the RBR version of the domain appears to bind E2s with higher affinity than do canonical E3 RING domains: complexes of UbcH7 and RING1s can be identified by pull-down and co-immunoprecipitation experiments [28]. What are the determinants and consequences of stronger E2

Figure 4. Comparison of HHARI RING2 with Parkin RING2. (a) Multiple sequence alignment of HHARI RING2 and Parkin RING2. Zn2+-liganding residues determined structurally for HHARI RING2 are denoted in yellow. Potential additional Zn2+ coordinating residues in Parkin as proposed by Rankin et al. [34] are highlighted in red. The active site cysteine is denoted by a double dagger. (b) The structure of HHARI RING2 displays Zn2+-liganding residues as yellow sticks. The active site cysteine is shown as orange sticks (PDB ID 1WD2). Sequences were aligned using CLUSTALW [44].

Parkin_418 CPRCHVPVEKNGGCMHMKCPQPQCRLEWCWNCGCEWNRVCMGDHWFDV- ARIH1_ 344 CPKCHVTIEKDGGCNHMVCRNQNCKAEFCWVCLGPWEP--HGSAWYNCN

‡(a)

(b)


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binding and what features of UbcH7 and UbcH8 makes them preferred RBR-binding partners? How do RBR ligases recognize and bind substrates? Substrate binding has been reported for almost every domain from RING1 to RING2 and in regions outside the RBR domain [10]. Given the variety of substrate binding modes documented for other families of E3 ligases, it seems unlikely that RBR ligases will have a common mechanism for substrate binding. The RBR ligase Parkin has been found to associate with SCF-type ubiquitin ligases, raising the possibility that adapter proteins may provide substrate specificity for a subset of these E3s [43]. Future studies informed by the new insights regarding the distinctive mode of action for RBR E3s will no doubt shed light on these and other questions surrounding RBR function.

AcknowledgementsWe thank P Brzovic, J Pruneda, and K Dove for critical reading and comments. We acknowledge support from the National Institute of General Medical Sciences in the form of 5R01 GM088055 (REK).

Author details1 Department of Biochemistry, University of Utah School of Medicine, 15 N. Medical Drive East, Salt Lake City, UT 84112-5650, USA. 2 Department of Biochemistry, University of Washington, Seattle, Washington 98195, USA.


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doi:10.1186/1741-7007-10-24Cite this article as: Wenzel DM, Klevit RE: Following Ariadne’s thread: a new perspective on RBR ubiquitin ligases. BMC Biology 2012, 10:24.

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