Elusive recognition determinants for ubiquitination
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Review Elusive recognition determinants for ubiquitination Amit Banerjee 1 * and Rebecca C. Wade 2,3 * 1 Institute of Environmental Health Sciences, Wayne State University, 2727 Second Avenue, Detroit, MI 48201, USA 2 European Media Laboratory, Schloss-Wolfsbrunnenweg 33, 69118 Heidelberg, Germany 3 European Molecular Biology Laboratory, 69012 Heidelberg, Germany How are proteins recognized as substrates for ubiquitination? Here we summarize insights from recent experiments that address this issue. These highlight the diversity and complexity of determinants for substrate recognition, and raise many questions for further investigation. Copyright # 2002 John Wiley & Sons, Ltd. Keywords: ubiquitination; protein degradation; substrate recognition; sequence motif; protein domain; phosphorylation Despite the prevalence of the ubiquitination-mediated protein degradation pathway and its intense investigation, the features by which proteins are recognized as targets for ubiquitination still remain elusive. Ubiquitination is a post- translational modification by which ubiquitin is attached covalently to a lysine residue on the target protein. Consequently, it might be expected that ubiquitination sites would be recognized in a similar way to other (ubiquitous) sites of post-translational modification such as glycosylation or phosphorylation in which the modified residue occurs within a defined short sequence motif. However, it is becoming clear that there are no such ‘ubiquitination motifs’. Instead, proposed recognition determinants for ubiquitination vary in complexity from N-end rule ‘de- grons’, consisting simply of the N-terminal residue and a lysine further along in the sequence, to sizeable ‘domains’ of residues, sometimes discontiguous, that may be struc- tured and contain several lysine residues with different probabilities of being ubiquitinated. So what are the determinants of substrate recognition for ubiquitination? To answer this question, it is worth first considering that the ubiquitination machinery itself is actually many machineries. Ubiquitination is the process by which an isopeptide bond is formed between the C- terminus of ubiquitin and a lysyl -amino group in the substrate protein (reviewed in Hochstrasser, 1996; Hershko and Ciechanover, 1998). Additional ubiquitins are added to form a multiubiquitin chain. The multiubiquitinated protein is then bound specifically by the 26S proteasome, which degrades it in an ATP-dependent manner. The ubiquitina- tion machinery consists of a set of enzymes: E1 ubiquitin- activating enzyme, E2 ubiquitin-conjugating enzymes and E3 ubiquitin-protein ligases. There are usually multiple proteins in an E3 complex and they provide spatial and temporal specificity to the substrate ubiquitination process. Each organism has many E2s and E3s (many machineries) enabling ubiquitination to be substrate-specific and result in different protein substrate fates. The following four examples of experimentally proven targets of the ubiquitination machinery illustrate some of the salient features identified for the recognition mechanisms for ubiquitination. A protein ‘domain’ can be essential Bohmann and coworkers (Treier et al., 1994) have suggested a novel route to oncogenesis by demonstrating that c-Jun, but not its transforming counterpart, the retro- viral v-Jun oncogene product, can be efficiently multi- ubiquitinated, thereby imparting v-Jun with a longer half- life than c-Jun. The escape of v-Jun from multiubiquitina- tion is due to deletion of the d-domain, a stretch of ca 30 amino acids that is present in c-Jun but not in v-Jun. In many instances (see reviews cited above), phosphorylation of a protein substrate is a necessary prelude to its subsequent ubiquitination. There is evidence suggesting that the c-Jun d-domain helps regulate the transcriptional activating capacity of c-Jun by modulating the amino-terminal phosphorylation of this protein (Adler et al., 1994). These results raise the question whether the d-domain sequence determines the c-Jun protein half-life by virtue of it regulating the amino-terminal c-Jun phosphorylation that is essential for its subsequent ubiquitination or by forming the domain structure that is recognized by the ubiquitination machinery. JOURNAL OF MOLECULAR RECOGNITION J. Mol. Recognit. 2002; 15: 3–5 DOI:10.1002/jmr.561 Copyright # 2002 John Wiley & Sons, Ltd. *Correspondence to: R. C. Wade, European Media Laboratory, Schloss- Wolfsbrunnenweg 33, 69118 Heidelberg, Germany. E-mail: Rebecca.wade @eml.villa-bosch.de or A. Banerjee, Institute of Environmental Health Sciences, Wayne State University, 2727 Second Avenue, Detroit, MI 48201, USA. E-mail: [email protected] Contract/grant sponsor: NIH (USPHS); contract/grant number: GM59467.
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Transcript of Elusive recognition determinants for ubiquitination
ReviewElusive recognition determinants forubiquitination
Amit Banerjee1* and Rebecca C. Wade2,3*1Institute of Environmental Health Sciences, Wayne State University, 2727 Second Avenue, Detroit, MI 48201, USA2European Media Laboratory, Schloss-Wolfsbrunnenweg 33, 69118 Heidelberg, Germany3European Molecular Biology Laboratory, 69012 Heidelberg, Germany
How are proteins recognized as substrates for ubiquitination? Here we summarize insights from recentexperiments that address this issue. These highlight the diversity and complexity of determinants forsubstrate recognition, and raise many questions for further investigation. Copyright � 2002 John Wiley &Sons, Ltd.
Keywords: ubiquitination; protein degradation; substrate recognition; sequence motif; protein domain;phosphorylation
Despite the prevalence of the ubiquitination-mediatedprotein degradation pathway and its intense investigation,the features by which proteins are recognized as targets forubiquitination still remain elusive. Ubiquitination is a post-translational modification by which ubiquitin is attachedcovalently to a lysine residue on the target protein.Consequently, it might be expected that ubiquitination siteswould be recognized in a similar way to other (ubiquitous)sites of post-translational modification such as glycosylationor phosphorylation in which the modified residue occurswithin a defined short sequence motif. However, it isbecoming clear that there are no such ‘ubiquitinationmotifs’. Instead, proposed recognition determinants forubiquitination vary in complexity from N-end rule ‘de-grons’, consisting simply of the N-terminal residue and alysine further along in the sequence, to sizeable ‘domains’of residues, sometimes discontiguous, that may be struc-tured and contain several lysine residues with differentprobabilities of being ubiquitinated.
So what are the determinants of substrate recognition forubiquitination? To answer this question, it is worth firstconsidering that the ubiquitination machinery itself isactually many machineries. Ubiquitination is the processby which an isopeptide bond is formed between the C-terminus of ubiquitin and a lysyl �-amino group in thesubstrate protein (reviewed in Hochstrasser, 1996; Hershkoand Ciechanover, 1998). Additional ubiquitins are added toform a multiubiquitin chain. The multiubiquitinated proteinis then bound specifically by the 26S proteasome, whichdegrades it in an ATP-dependent manner. The ubiquitina-
tion machinery consists of a set of enzymes: E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzymes andE3 ubiquitin-protein ligases. There are usually multipleproteins in an E3 complex and they provide spatial andtemporal specificity to the substrate ubiquitination process.Each organism has many E2s and E3s (many machineries)enabling ubiquitination to be substrate-specific and result indifferent protein substrate fates.
The following four examples of experimentally proventargets of the ubiquitination machinery illustrate some of thesalient features identified for the recognition mechanismsfor ubiquitination.
A protein ‘domain’ can be essential
Bohmann and coworkers (Treier et al., 1994) havesuggested a novel route to oncogenesis by demonstratingthat c-Jun, but not its transforming counterpart, the retro-viral v-Jun oncogene product, can be efficiently multi-ubiquitinated, thereby imparting v-Jun with a longer half-life than c-Jun. The escape of v-Jun from multiubiquitina-tion is due to deletion of the �-domain, a stretch of ca 30amino acids that is present in c-Jun but not in v-Jun. In manyinstances (see reviews cited above), phosphorylation of aprotein substrate is a necessary prelude to its subsequentubiquitination. There is evidence suggesting that the c-Jun�-domain helps regulate the transcriptional activatingcapacity of c-Jun by modulating the amino-terminalphosphorylation of this protein (Adler et al., 1994). Theseresults raise the question whether the �-domain sequencedetermines the c-Jun protein half-life by virtue of itregulating the amino-terminal c-Jun phosphorylation thatis essential for its subsequent ubiquitination or by formingthe domain structure that is recognized by the ubiquitinationmachinery.
JOURNAL OF MOLECULAR RECOGNITIONJ. Mol. Recognit. 2002; 15: 3–5DOI:10.1002/jmr.561
Copyright � 2002 John Wiley & Sons, Ltd.
*Correspondence to: R. C. Wade, European Media Laboratory, Schloss-Wolfsbrunnenweg 33, 69118 Heidelberg, Germany. E-mail: [email protected] or A. Banerjee, Institute of Environmental HealthSciences, Wayne State University, 2727 Second Avenue, Detroit, MI 48201,USA. E-mail: [email protected]/grant sponsor: NIH (USPHS); contract/grant number: GM59467.
More than one lysine in a ‘ubiquitination domain’ canbe a ubiquitin acceptor
The bacterially expressed and purified yeast Cdc34 proteinhas been shown to be able to catalyze its own autoubiqui-tination via an intramolecular transfer of its thiol ester-linked ubiquitin to a lysine (Banerjee et al., 1993). In thisprocess, multiple ubiquitin moieties are added to Cdc34, andthe major ubiquitin linkage site is located within the C-terminal ca 40 residues of Cdc34. The four lysyl residues inthis region of Cdc34 were substituted by arginine eithersingly or in combination to produce a set of Cdc34 mutants.Analysis of these mutants for autoubiquitination revealedthat the multiubiquitin chain can be formed on any one ofthese four lysines, and the Cdc34 conjugates contain a singlemultiubiquitin chain. This demonstrated that mutagenesis ofone lysyl residue in the ‘ubiquitination domain’ would stillenable the other lysyl residue to be a ubiquitin acceptor ifthey are present in the same domain (Banerjee et al., 1993).
Recognition may be via dispersed structural features
The membrane-anchored yeast HMG-CoA reductase iso-zyme, Hmg2, has been shown (Jingami et al., 1987;Hampton, et al., 1996) to undergo a regulated degradation.This regulated degradation of Hmg2 requires informationdistributed over its entire N-terminal transmembranedomain of 523 residues (Gardner and Hampton, 1999).The authors have argued that its recognition is based on‘dispersed structural features rather than primary sequencemotifs’. The Kulka group has identified signals regulatingthe degradation of �-galactosidase and Ura3p fusionproteins via the same subsidiary pathway of the ubiquitin–proteasome system involving the E2s, Ubc6 and Ubc7,which are responsible for Hmg2 ubiquitination. Thispathway has been implicated in the degradation ofmisfolded and regulated proteins in the endoplasmicreticulum lumen and membrane. Gilon et al. (2000) recentlyshowed that a test sequence of 16 residues that forms aUbc6–Ubc7-specific target carries a patch of bulky hydro-phobic residues adjacent to a lysyl residue that isubiquitinated. In their experiments, certain substitutions ofbulky hydrophobic or positively charged residues withalanine created novel degradation signals, facilitating thedegradation of fusion proteins through an unidentifiedubiquitin–proteasome pathway not involving Ubc6 andUbc7.
Different structured regions of a protein may betargeted under different conditions
Cytochrome P450 2E1 (CYP2E1) levels are elevated inresponse to certain substrates (e.g. ethanol), and it has beenpostulated that substrate binding shields the CYP2E1protein from turnover by the ubiquitin–proteasome-depen-dent machinery (Roberts et al., 1995). Banerjee et al. (2000)
have used a reconstituted ubiquitination assay and identifieda determinant (residues 316–340) for ubiquitination con-taining two lysine residues in the free CYP2E1 protein.Modeling of CYP2E1 shows that this ubiquitinationdeterminant has partial amphipathic helical structure,containing hydrophobic residues in the helix that tend tobe buried in the protein. Preliminary experiments suggestthat an additional region of the protein is probably involvedin ER-associated CYP2E1 ubiquitination (Banerjee, unpub-lished observation) and also raise the possibility that twodifferent ubiquitination sub-pathways act on CYP2E1 incells.
In this note, apart from raising awareness of this complexrecognition phenomenon, we aim to provoke interest inpursuing the many open questions concerning recognitionmechanisms for ubiquitination, including the following:
(i) Given that ubiquitin-mediated protein degradation hasdiverse functions ranging from regulating levels ofspecific proteins to disposing of misfolded proteins, isthere a different type of recognition determinant foreach specific ubiquitination function which is catalyzedby a different E2 and accompanying ubiquitinationsub-pathway machinery?
(ii) How sensitive is ubiquitination to the conformation anddynamics of the substrate protein? Is, for example, aprotein that has undergone conformational changes inthe presence of ligand binding recognized by the sameubiquitination sub-pathway machinery as in the ab-sence of ligand binding, or is it recognized by adifferent ubiquitination sub-pathway machinery, ordoes it escape ubiquitination? Does substrate turnoverby a target enzyme (such as cytochrome P450) render itunrecognizable by its cognate ubiquitination machin-ery?
(iii) Do the turnover rates and cellular amounts of ubiqui-tination substrates depend on the action (and effi-ciency) of different competing/complementingubiquitination sub-pathway machineries that may varyin ‘activity’ for each substrate in a temporal and spatialmanner?
More research in this area of cellular regulation is neededto provide a clear understanding of the mechanisms bywhich protein substrates are recognized for ubiquitination incells. Once these recognition targets are identified andcharacterized, finding small molecules that can block thesetargets may not be unrealistic. Such compounds will have avast potential for use in analytical and therapeutic applica-tions as deregulated protein ubiquitination has been shownto contribute towards pathological changes in cells.
Acknowledgements
The authors’ research on protein ubiquitination is supported by NationalInstitutes of Health (USPHS) grant GM59467. We thank Peter Winn forcritical reading of the manuscript.
4 A. BANERJEE AND R. C. WADE
Copyright � 2002 John Wiley & Sons, Ltd. J. Mol. Recognit. 2002; 15: 3–5
REFERENCES
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ELUSIVE RECOGNITION DETERMINANTS 5
Copyright � 2002 John Wiley & Sons, Ltd. J. Mol. Recognit. 2002; 15: 3–5
