Ubiquitin-protein ligases

P. A. Robinson* and H. C. Ardley

Molecular Medicine Unit, University of Leeds, Clinical Sciences Building, St. James's University Hospital, Leeds, LS9 7TF, UK

* Author for correspondence (e-mail: p.a.robinson{at}leeds.ac.uk)

Post-translational covalent tagging of proteins with the 76-residue protein ubiquitin (Ub) serves many functions. Polyubiquitylated proteins are directed to the large multi-component, multi-catalytic protease the 26S proteasome. The ubiquitin-26S proteasome (UPS) pathway is the major mechanism by which eukaryotic cells target normal and misfolded cytosolic or membrane proteins for degradation. UPS-mediated degradation of proteins is essential for many cellular processes, including apoptosis, MHC class I antigen presentation, the cell cycle and intracellular signalling. By contrast, mono-ubiquitylation of proteins has nondegradative cellular functions. These include transcriptional activation, methylation of histones, endocytosis and endosomal sorting (Conaway et al., 2002Go; Sun and Allis, 2002Go; Raiborg et al., 2003Go).Go



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Protein ubiquitylation is an energy-dependent, four-step pathway that operates in all eukaryotic cell types (Hershko and Ciechanover, 1998Go; Pickart, 2001Go). In the first step, which requires ATP, ubiquitin is bound by a thioester linkage through its C-terminal glycine residue to a ubiquitin-activating enzyme (E1). Ubiquitin is then transferred first to one of a number of ubiquitin-conjugating enzymes (E2s) by trans-thiol esterification and then to an {epsilon}-amino group of a lysine residue in a target protein (T), which is generally facilitated by a ubiquitin-protein ligase (E3). The conjugated ubiquitin itself may then serve as a ubiquitylation substrate and repeated ubiquitylation leads to the formation of a polyubiquitin chain. In general, ubiquitin K48 acts as the receptor for conjugation, and a chain of at least four ubiquitin molecules is required to activate proteasomal degradation. However, other lysine residues (e.g. K11) can also accept ubiquitin moieties. Protein targeting specificity normally relies on the unique interaction between a particular combination of an E2 (of which there are relatively few), an E3 (of which there are many) and target protein. However, there are rare instances in which an E2 (as defined by the presence of characteristic E2 sequence motifs), such as RAD6, can promote ubiquitylation in the absence of an E3 (Hoege et al., 2002Go; Sun and Allis, 2002Go).

A number of ubiquitin-like molecules (e.g. SUMO or NEDD8) are also covalently tagged to proteins (Muller et al., 2001Go). However, substrate SUMOylation and NEDDylation require their own unique combinations of E1, E2 and E3. The addition of these tags probably serves a different function to ubiquitylation. For example, the modification of cullins by NEDD8 may regulate binding of E2s to SCF-type E3 complexes, thereby enhancing their activity (Kawakami et al., 2001Go; Ohh et al., 2002Go).

In many cases, what drives the processivity of ubiquitin chain elongation has not been determined. It is unlikely from a steric and conformational perspective that the initial recognition between E2, E3 and target protein – which results in the first ubiquitylation conjugation – can then regulate chain extension while the enzymes remain bound to substrate. The presence of one or more additional factors, such as ubiquitylation factor E4 (Ufd2), may be required. Alternatively, a second E3 (E3B) or dissociated components of the original E2-E3 complex, may catalyse chain extension.

Three classes of E3 have now been identified: the HECT (homologous to E6-AP C-terminus), the RING (really interesting new gene) finger, and U-box domain types. HECT domain family members directly catalyse the final attachment of ubiquitin to substrate proteins. Interaction between an E2 and the HECT E3 results in the conjugation of a ubiquitin moiety to a characteristic cysteine residue in the ~350-residue HECT domain. Subsequently, the HECT E3 interacts with target protein, which results in transfer of ubiquitin. The prototypical HECT E3 is E6-AP. Loss of E6-AP expression in humans results in the neurodevelopmental disease Angelman Syndrome. By contrast, RING-finger and U-box E3s do not have a direct catalytic role in protein ubiquitylation but act as `facilitators of interaction' between an E2 and target protein. The result is a direct transfer of ubiquitin from the E2 to the target protein. RING finger and U-box domains are structurally related (Hatakeyama et al., 2001Go).

RING finger E3s can be loosely categorised into two types. The first type is based upon a single key component. Probably the most studied of this group is the N-recognin ligase that regulates the N-end rule pathway (Varshavsky et al., 1998Go). This ligase selects proteins for degradation on the basis of the amino acid residue at their N-terminus. Other E3s of this type include CBL and IAPs (inhibitor of apoptosis proteins). CBL promotes the ubiquitylation and endocytosis of receptor tyrosine kinases, including the epidermal growth factor receptor (EGFR) (Honda and Yasuda, 2000Go; Tsygankov et al., 2001Go). The mechanism underlying the subsequent decision process that decides between degradation or recycling to the plasma membrane, and the additional components required, is the subject of intensive research effort at present. IAPs are E3s that modulate caspase activity and therefore represent key regulators of cellular apoptosis. In addition to the RING domain that regulates interaction with E2s, two other characteristic domains are found within IAPs, the BIR (baculovirus IAP repeat) and CARD (caspase recruitment domain) domains. The BIR domain regulates interaction with IAP E3 antagonists including Grim, Hid and Reaper that possess an IAP-binding motif (IBM).

The second type of E3s are multicomponent complexes. The modular SCF (Skp1/Cullin/F-box/Rbx1/2) family of E3s are probably the largest group (Deshaies, 1999Go; Zheng et al., 2002Go). The central core component is one of the cullin (Cul) family members.

The RING box 1 protein, Rbx1 (also named ROC1), regulates interaction between an E2 and cullin. The interaction between the various other components of this complex is defined by characteristic signature sequences. For example, the F-box sequence found within the target-interacting protein regulates interaction with SKP1 [multiprotein complex image adapted from (Zheng et al., 2002Go)]. Many hundreds of F-box proteins are found in protein databases. Consequently, there is the potential for formation of many different SCF complexes that each have a different targeting specificity.

The von-Hippel Lindau (VHL) tumour suppressor protein E3 complex represents a variant SCF-type complex. It has Rbx1 and Cul2A at its core. However, SKP1 is replaced by the elongin B/C complex that regulates interaction with VHL [and other proteins, including RNA polymerase II elongation factor A and the SOCS (suppressors of cytokine signalling) box proteins]. Its targeting specificity highlights the simple but extremely effective way by which a cell's microenvironment can influence the level of protein ubiquitylation through post-translational modification (Min et al., 2002Go; Kim and Kaelin, 2003Go; Pugh and Ratcliffe, 2003Go). Under normoxic conditions, hydroxylation of a key proline residue in hypoxia inducing factor (HIF)-1{alpha} or HIF-2{alpha} maintains VHL interaction and promotes rapid UPS-mediated turnover. By contrast, under hypoxic conditions, this proline residue is not hydroxylated and VHL interaction is lost. The consequence is stabilisation of HIF-1{alpha}/HIF-2{alpha}. Interaction with its constitutively expressed binding partner HIF-1ß to form the HIF-1{alpha}/HIF-2{alpha}–HIF-1ß heterodimeric transcription-factor complex then leads to transcription of downstream genes, such as that encoding vascular endothelial growth factor (VEGF). In cancer, loss of VHL activity through mutation or chromosomal loss similarly results in increased HIF-1{alpha}/HIF-2{alpha} levels.

Proteasomal hydrolysis of ubiquitylated proteins results in the production of small peptides that can then be further degraded or processed for MHC class I antigen presentation as they pass through the endoplasmic reticulum (ER) carried by the transporter associated with antigen processing (TAP) protein complex (Shastri et al., 2002Go). Further trimming of the peptides by ERAAP (ER aminopeptidase associated with antigen processing) may then occur. However, there are rare examples in which a ubiquitylated protein is only partially hydrolysed, such as the generation of the active p50 NF-{kappa}B protein from its larger p105 precursor (Ciechanover et al., 2001Go).

The requirement for the formation of polyubiquitin chains might be part of a proof-reading mechanism, because a large number of de-ubiquitylating enzymes (DUBs) are also found within cells. However, remarkably little is known how the different components of the UPS and DUBs interact to regulate the levels of ubiquitylation of individual substrates and why a cell may possess so many different DUBs. These might be present to prevent the formation of a branched structure sufficiently large to activate proteasomal destruction. However, an added complication is the discovery that the DUB UCH-L1 is also an E3.

Modulating the levels of key checkpoint proteins within the cell cycle is an important function of the UPS. The anaphase-promoting complex (APC) is a large multiprotein-E3 complex (Peters, 2002Go) whose substrates include mitotic cyclins and securin. Although this complex is built up of many components, not all appear to be involved directly in the ubiquitylation of its substrates. The APC2, APC10 and APC11 subunits, which are characterised by cullin, DOC and RING finger domains, respectively, appear to be key regulators of substrate ubiquitylation. By contrast, key regulatory proteins at the G1-S checkpoint are regulated by SCF complexes. For example, the SCFSKP2 complex promotes the degradation of the cyclin dependent kinase (CDK) inhibitor p27KIP1. This degradation step is regulated by phosphorylation of p27KIP1 and interaction with the CDK subunit 1 (Cks1) protein. Interestingly, APC regulates the levels of Skp2 and Cks1, keeping their levels low during G1.

Not every target protein within a cell has its own unique combination of E2 and E3. Substrate selection is often determined by post-translational modification (Karin and Ben-Neriah, 2000Go; Yamanaka et al., 2003Go). For example, the E3 SCFTrCP ligase mediates the phosphorylation-dependent ubiquitylation of both I{kappa}B{alpha} and ß-catenin. The I{kappa}B kinase complex controls phosphorylation and hence ubiquitylation of I{kappa}B{alpha}; by contrast a complex of APCp (adenomatous polyposis coli tumour suppressor protein), axin and glycogen synthase kinase 3ß (GSK3ß) promotes ubiquitylation of ß-catenin (Karin and Ben-Neriah, 2000Go; Salic et al., 2000Go). Therefore, the decision to ubiquitylate lies with the activity level of the appropriate kinase. By contrast, the processing of Smad7 by Smurf (Smad ubiquitylation regulatory factor) E3s is regulated by the competition between ubiquitylation and acetylation of specific lysine residues (Gronroos et al., 2002Go).

The surface expression of many normal and misfolded membrane proteins such as the cystic fibrosis transmembrane conductance regulator protein (CFTR) is controlled by ubiquitylation (Ward et al., 1995Go). The degradation of misfolded proteins relies on their recognition by chaperones in the lumen of the ER. They are then retrotranslocated by the SEC61-SEC62/63 complex to the cytoplasm, where they are ubiquitylated by the transmembrane E3 Hrd1p/Der3p and the E2 Ubc7. This E2 is anchored to membranes by Cue1p. The membrane E2 Ubc6 is also likely to play a role in this process. Hrd1p/Der3p can undergo auto-ubiquitylation but is stabilised by association with Hrd3p. Polyubiquitylated proteins exit with the aid of the AAA-ATPase Cdc48(p97)/-Ufd1-Npl complex. The surface levels of the epithelial Na+ channel ENaC is also regulated by ubiquitylation, which is catalysed by the HECT E3 NEDD4-2 (Debonneville et al., 2001Go). The interaction involves the WW domains of NEDD4-2 and the PPXY motifs in ENaC. Negative regulation of NEDD4-2 activity is through phosphorylation by the kinase SGK-1. This causes NEDD4-2 to disassociate from ENaC, resulting in increased surface levels of ENaC.

Protein ubiquitylation regulates transcription – for example, through histone modification (Sun and Allis, 2002Go) – and also plays important roles in signalling [Transcription image adapted from (Hicke, 2001Go)]. In fact, further details of its roles in Wnt, Notch and TGF-ß signalling can be found in previous Cell Science at a Glance articles (Hülsken and Behrens, 2002Go; Kopan, 2002Go; Moustakas, 2002Go). In addition, it is a stress responsive pathway. Consequently, synthesis of E3s to supra-physiological levels or their presence at abnormal subcellular locations might activate E3 autoubiquitylation or ubiquitylation of other proteins that are not normal physiological substrates. Auto-ubiquitylation might represent a protective self-regulatory mechanism by which E3s target themselves for degradation when expressed at levels above their endogenous binding partners. An unfortunate consequence of abnormal ubiquitylation may be the formation of protein aggregates or inclusions. Ubiquitylated inclusions are key pathological features of liver disease (Mallory bodies) and many neurodegenerative diseases, including Alzheimer's disease (paired helical filaments and amyloid plaques), Parkinson's disease (Lewy bodies) and a number of trinucleotide repeat expansion diseases, such as Huntington's disease. Their tagging with ubiquitin might reflect attempts by the cell to remove them, because they are also often characterised by the presence of components of the UPS (e.g. the E3 parkin) and the chaperone system (e.g. Hsp70). Interestingly, the U-box E3 and co-chaperone CHIP (carboxyl terminus of Hsc70-interacting protein) targets an aggresome-promoting mutant form of alphaB-crystallin (alphaBR120G) for UPS-mediated degradation.

Viruses such as HPV, Ebola and HIV have adapted the process of protein ubiquitylation to promote their survival and propagation. For example, E6-AP is co-opted by the E6 protein in HPV16-infected cells to target the ubiquitylation and degradation of p53. However, p53 is not the normal substrate for E6-AP. The RING finger E3 MDM2 normally regulates the ubiquitylation of p53. Ebola and HIV co-opt components of the endosomal sorting pathway such as TSG101 (an E2 variant) to aid their budding from infected cells (Martin-Serrano et al., 2003Go; Raiborg et al., 2003Go).

Although many aspects of protein ubiquitylation have been elucidated, some key features still need to be resolved. In particular, the activity and substrates of many putative E3s observed in in vitro systems needs to be confirmed in vivo when they are expressed at endogenous levels. Understanding the molecular interactions that underpin ubiquitin-mediated protein degradation will offer a tremendous opportunity to develop novel therapeutic approaches based upon modulating protein levels for the treatment of diseases such as cancer, neurodegenerative disorders and viral infections.


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Many additional references can be found in the Table of ubiquitin-protein ligases at www.leeds.ac.uk/medicine/res_school/mol_med/res_robinson.htm.


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