(362), pe50. [DOI: 10.1126/stke.3622006pe50] 2006Sci. STKEMirko H. H. Schmidt and Ivan Dikic (21 November 2006)

Ubiquitin and NEDD8: Brothers in Arms`

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The ubiquitously expressed small molecule ubiquitin (Ub) be-came famous for its ability to mediate the destruction of target-ed proteins to which it is covalently attached. Ub is activated byan E1 enzyme in an initial adenosine triphosphate–dependentstep; it is then conjugated to an E2 enzyme through a thioesterbond and is finally attached to a target protein by means of anE3 ligase. Although only a few E1 and E2 enzymes are known,there are numerous E3 ligases, making ubiquitylation a highlyspecific process (1). More recently, it has become evident thatUb is involved in processes other than protein degradation, suchas cell signaling, membrane transport, and DNA repair (2). Thisis because Ub comes in different fla-vors. It can be attached to target pro-teins as a monomer or in the form ofdifferent polymeric structures. All ofthese arrangements of ubiquitin arerecognized by various specialized Ub-binding domains (UBDs) (3). Further-more, the way in which the individualmoieties of the polymer are linked af-fects the outcome of the Ub signal anddetermines whether a Ub-tagged targetprotein is destroyed or participates inother cellular processes (3). Finally,deubiquitinases (DUBs) that selective-ly cleave Ub moieties allow remodel-ing of Ub modifications into differenttypes; for example, a Lys48-linkedpolyubiquitin chain could be changedinto an alternatively branched Lys63-linked form (4). These actions create ahighly dynamic and flexible networkof signals that allow a cell to react todiverse environmental cues.

Adding to the complexity of theubiquitin system, there are at least adozen ubiquitin-like (Ubl) proteins(which include Ub itself) (5). Ub andUbls do not necessarily show greatsimilarity in their primary sequence,but all of them possess essentially thesame three-dimensional structure andall use a C-terminal glycine as the siteof substrate attachment. Despite thesesimilarities, the different Ubls are be-lieved to share neither E1, E2, and E3 enzymes nor cellularfunctions (5). Specificity is largely determined through Ub- orUbl-binding domains; indeed, every member of the Ubl family

of proteins has been thought to recognize only its own particu-lar set of interaction domains. However, a recent article byYarden and colleagues indicates that Ub and the Ubl Neuralprecursor cell–Expressed Developmentally Down-regulated 8(NEDD8) can act in a redundant fashion and share E3 ligasesand UBDs, properties that further increase the complexity ofUb and Ubl signaling (6).

NEDD8, known as Rub1 in yeast, is about 60% identical toUb (7, 8). Changes in NEDD8 abundance affect the signalingof various molecules, including auxin (9), interferon regulatoryfactor 3 (IRF3) (10), murine double minute 2 (MDM2) (11),

and p21 (12), but it isbest studied in thecontext of cullin-RINGubiquitin ligases. HereNEDD8 is covalentlyattached to cullins bythe E2 enzyme Ubc12and enhances theubiquitin-ligase activi-ty of the E3 ligasecomplex in vitro, po-tentially by facilitatingthe recruitment ofubiquitin-loaded E2s(13). In their currentwork, Oved et al. ex-pand the functionalspectrum of NEDD8to include one of thebest-studied Ub-de-pendent processes,namely trafficking ofmembrane proteins(2, 14).

The ligand-depen-dent endocytosis anddown-regulation of theepidermal growth fac-tor receptor (EGFR)are promoted by ubiq-uitylation mediated bythe E3 ligase Cbl (15).Cbl is recruited to theEGFR upon stimula-

tion with EGF and mediates the transfer of multiple Ubmolecules to the EGFR (2). This initiates the internalization anddown-regulation of the activated receptor complex, because theattached Ub molecules are recognized by Ub receptors in theendocytic sorting machinery (14), leading to termination of theEGF-induced signal and limiting EGF signaling spatially andtemporally. Oved et al. now show that, like Ub, NEDD8 is at-tached to the EGFR after ligand stimulation in a Cbl-dependentmanner (Fig. 1). EGFR neddylation resembles EGFR ubiquity-

Ubiquitin and NEDD8: Brothers in ArmsMirko H. H. Schmidt and Ivan Dikic*

(Published 21 November 2006)

P E R S P E C T I V E

Institute of Biochemistry II, Johann Wolfgang Goethe-UniversitySchool of Medicine, Theodor-Stern-Kai 7, D-60590 Frankfurt, Germany.

*Corresponding author. Telephone, 49-69-6301-83647; fax, 49-69-6301-5577; e-mail, Ivan.Dikic@biochem2.de

Eps15

Hrs

Ubc12

EGF EGF

Ub

Ub

Ub

PPPPP

N8

N8

N8

PPPPP

Eps15

Hrs

UbcH7

Cbl

STAMSTAM

EGFR

+

Fig. 1. Ub and NEDD8 (N8) are attached to the EGFR upon lig-and stimulation in a Cbl-dependent and cooperative manner bymeans of distinct E2 enzymes. The association of Cbl withUbcH7 leads to the conjugation of Ub moieties, whereas Ubc12promotes conjugation of NEDD8. Both Ubls induce the recruit-ment of Eps15, Hrs, and STAM. These proteins of the endocyticmachinery are needed for receptor down-regulation. This work in-dicates that Ub and NEDD8 function in a redundant manner incontrolling receptor endocytic pathways.

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lation in that the E3 ligase and the amino groups targeted in thereceptor are identical. The similarity of NEDD8 and Ub and thesimilarity of the RING domains of Cbl and the cullin-RINGubiquitin ligases might contribute to the partially redundantaction of both Ubls (16). Recruitment of the endocytic ma-chinery for receptor down-regulation also happens in a moreor less similar manner, because NEDD8 binds the same ubiq-uitin-interaction motifs (UIMs) in EGFR substrate 15(Eps15), hepatocyte growth factor–regulated tyrosine kinasesubstrate (Hrs), and the signal-transducing adapter molecule(STAM) as Ub (6). Finally, NEDD8 and Ub cooperate indown-regulation of the EGFR at low concentrations ofNEDD8, indicating the functional synergism of bothmolecules at physiological concentrations.

This work sheds new light on the interchangeability of Uband Ubls and their associated binding domains. It calls intoquestion the assumed specificity of Ubl-UBD binding. Appar-ently, NEDD8 and Ub can both be coupled to the EGFR andthen cause comparable actions. This indicates that bothmolecules can, at least in part, functionally substitute for eachother. Studies using the yeast two-hybrid system, in which morethan 30% of tested ubiquitin-binding proteins bind to NEDD8,suggest that UBDs other than UIM can interact with NEDD8.Moreover, a NEDD8 interaction partner termed NEDD8 ulti-mate buster–1 long (NUB1L) consists of three ubiquitin-associ-ated (UBA) domains (which are known to bind ubiquitin),through which it associates with NEDD8. These UBAs also bindto the small Ubl molecule FAT10, resulting in the increaseddegradation of FAT10 as well as FAT10-modified cargo (17).

Clearly, Ubl molecules are capable of associating with do-mains that have been assigned to other Ub-like modifiers. Thepurpose of this redundant binding of Ubls to various domainsremains unclear. Certain degradation modules such as theCOP9 signalosome, an evolutionarily conserved multiproteincomplex, which plays a role in the positive regulation of E3ligases, could benefit from fairly unspecific binding to variousUbls. This would enable the COP9 complex to associate withcargo proteins modified with different Ubls without carryingeach and every Ubl-binding domain necessary to recognize thevarious Ubls. One could think of lack of specificity in this con-text as an optimization process that allows COP9 to degradeUbl-modified cargo in a broad manner. On the other hand, thereare processes that require very specific Ubl-binding domaininteractions. For example, Ubls such as the SUMOs (smallUb-related modifiers) associate with specific interacting motifs thatare unable to bind to ubiquitin or other Ubls (18, 19). The lattertype of specific signaling is very important under physiologicalconditions and is often deregulated in pathologies such ascancer (20). It remains unclear to what extent Ub and NEDD8share redundant functions in vivo. However, this special case ofneddylation of the EGFR shows that Ub and Ubls can share

both E3 ligases and binding domains, indicating that Ub signal-ing is far more complex than we believed.

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16. N. Zheng, B. A. Schulman, L. Song, J. J. Miller, P. D. Jeffrey, P. Wang, C.Chu, D. M. Koepp, S. J. Elledge, M. Pagano, R. C. Conaway, J. W.Conaway, J. W. Harper, N. P. Pavletich, Structure of the Cul1-Rbx1-Skp1-FboxSkp2 SCF ubiquitin ligase complex. Nature 416, 703–709 (2002).

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Citation: M. H. H. Schmidt, I. Dikic, Ubiquitin and NEDD8: Brothers inarms. Sci. STKE 2006, pe50 (2006).

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