Degrons in cancer · is that they are transferable, and the attachment of such sequence elements by...

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1Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy ofSciences, 2 Magyar Tudósok krt, Budapest H-1117, Hungary. 2Structural and Computa-tional Biology Unit, European Molecular Biology Laboratory, Meyerhofstraße 1, 69117Heidelberg, Germany. 3Bioinformatics Platform, Berlin Institute for Medical Systems Bio-logy,Max-Delbrück Center forMolecularMedicine, Robert-Rössle-Strasse 10, 13125 Berlin,Germany. 4MTA-ELTE Lendület Bioinformatics Research Group, Department of Bio-chemistry, Eötvös Loránd University, Pázmány Péter stny 1/c, Budapest H-1117, Hungary.*These authors contributed equally to this work.†Corresponding author. Email: [email protected] (Z.D.); [email protected] (T.J.G.)

Mészáros et al., Sci. Signal. 10, eaak9982 (2017) 14 March 2017

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Degrons in cancerBálint Mészáros,1* Manjeet Kumar,2* Toby J. Gibson,2† Bora Uyar,3 Zsuzsanna Dosztányi4†

Degrons are the elements that are used by E3 ubiquitin ligases to target proteins for degradation. Most degrons areshort linear motifs embedded within the sequences of modular proteins. As regulatory sites for protein abundance,they are important for many different cellular processes, such as progression through the cell cycle and monitoringcellular hypoxia. Degrons enable the elimination of proteins that are no longer required, preventing their possibledysfunction. Although the human genome encodes ~600 E3 ubiquitin ligases, only a fraction of these enzymes havewell-defined target degrons. Thus, for most cellular proteins, the destruction mechanisms are poorly understood.This is important for many diseases, especially for cancer, a disease that involves the enhanced expression of onco-genes and the persistence of encoded oncoproteins coupled with reduced abundance of tumor suppressors. Loss-of-functionmutations occur in the degrons of several oncoproteins, such as the transcription factorsMYC andNRF2,and in various mitogenic receptors, such as NOTCH1 and several receptor tyrosine kinases. Mutations eliminatingthe function of the b-catenin degron are found inmany cancers and are considered one of themost abundantmuta-tions driving carcinogenesis. In this Review, we describe the current knowledge of degrons in cancer and suggestthat increased research on the “dark degrome” (unknown degron-E3 relationships) would enhance progress incancer research.

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Cancer as a Disease of Aberrant Protein DegradationCancer is well known to be a disease of gene mutation. More than halfof cancers have point mutations in the TP53 gene, compromising therole of the encoded protein p53 (also known asTP53) as the guardian ofthe genome (1–3). However, cancer is also a disease of aberrant expres-sion of genes. For example, gene duplication, aneuploidy (an imbalancein the number of chromosomes), or remodeling of chromatin state cancause either increases in expression of oncogenes encoding tumor-promoting proteins (oncoproteins) or decreases in expression of genesencoding tumor suppressors (4–7). Gene mutations and gene expres-sion changes are often tightly connected. For example, a mutation thatinactivates a tumor suppressor that functions as a negative regulator ofgene expression can lead to increased expression of an oncogene. Thus,as a prerequisite to understand any given cancer, both the set of muta-tions and the gene expression changes need to be fully cataloged.

In this Review, we focus on the importance of degrons in onco-genesis. As the destruction signals in cellular proteins, degrons serveas a nexus between genemutation and protein abundance in cancer. Ifdegron function is impeded by any form ofmutation, the result is thata protein will have a longer half-life, enhancing and prolonging itsfunction in the cell. Nevertheless, with the exception of the frequentlymutated degron in themultifunctional protein b-catenin (also known asCTNNB1) (8, 9), mutations that affect degron function have a lowprofile in the cancer literature. To demonstrate the importance of de-gronmutations in cancer, we provide an overviewof themanyways thatdegrons, their mutations, and mutations in the E3 ligase systems thatgovern their recognition and degradation of the degron-containing pro-teins influence cellular transformation and oncogenesis.

Details of the Ubiquitin-Proteasome SystemHow the ubiquitin-proteasome degradation pathway worksIntracellular protein degradation in eukaryotes is mainly achieved bythe ubiquitin-proteasome system (UPS) (10). The UPS plays an essen-tial role inmaintaining cellular homeostasis by participating in proteinquality control, for instance, by recognizing and rapidly degrading in-correctly folded or assembled proteins. In addition to the eliminationof aberrant proteins, this system is responsible for the dynamic proteinturnover involved in cell regulation. The spatially and temporally regu-lated degradation of proteins is critical for diverse cellular processes, in-cluding cell cycle progression, signaling, differentiation, and growth (10).

The UPS has a sophisticated network of enzymes that targets pro-teins for degradation. The core degradation machine of this system isthe 26S proteasome, which consists of twomain structural components:the 20S core particle, which functions as a gate throughwhich substratesenter and are degraded, and the 19S regulatory particle, which controlsthe opening of this gate to selectively let substrate proteins with theproper degradation signals into the core particle (11, 12).

Although various posttranslational modifications, such as poly–adenosine 5′-diphosphate-ribosylation (PARsylation) (13) and poly-sumoylation (14), can target substrates to the 26S proteasome, thecanonical signal for 26S-mediated proteasomal degradation are theLys48 (K48)–linked polyubiquitin chains, which are generated by at-tachment of ubiquitin to a lysine residue of the substrate protein and thenrepetitive attachment of ubiquitin to Lys48 of the last-attached ubiquitin.A chain of at least four ubiquitinmoieties targets the substrate protein tothe 26S proteasome (15, 16).

The machinery of ubiquitinationThe covalent transfer of ubiquitin, a 76–amino acid proteinwith amassof 8 kDa, to substrate proteins is mediated by three types of enzymes:the E1 ubiquitin-activating enzyme, the E2 ubiquitin-conjugating en-zyme, and the E3 ubiquitin ligase enzyme (Fig. 1) (17). The humangenome contains 2 genes encoding E1 enzymes, 41 genes encodingE2 enzymes, and more than 600 genes encoding E3 enzymes (18),which reflects the importance of the variation of E3 enzymes in thecomplex spatiotemporal regulation of posttranslational protein stabil-ity in the cells. Ubiquitin is activated by the E1 enzyme in an adenosine

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5′-triphosphate–dependentmanner. The activated ubiquitin is, in turn,transferred to an E2 conjugating enzyme. The interaction of the E2 en-zyme with a substrate-specific E3 enzyme leads to the formation of anisopeptide bond between the C-terminal glycine residue of ubiquitinand a lysine residue of the substrate protein (19). The attached ubiqui-tin proteins can be K48-ubiquitinated to form polyubiquitin chains


attached to the substrate protein, thustargeting the substrate protein for 26Sproteasome–mediated degradation (16).

Proteins and protein complexes thathave E3 ligase activity are divided intotwomain families on the basis of the coreprotein domain that facilitates the trans-fer of ubiquitin to the substrates. The firstfamily of E3 ligases contain the HECT(homologous to E6-AP C terminus) do-main (20), and the second family of E3ligases contain the RING (really inter-esting new gene) domain or variants ofit (19). For HECT E3 ligases, ubiquitinis sequentially transferred from the E2to theHECTdomain and then to the sub-strate, whereas for the RING E3 ligases,the ubiquitin is directly transferred to thesubstrate (Fig. 1) (19, 20). HECTE3 ligasescomprise ~5%of the knownE3 ligases; therest are RING E3 ligases.

Some E3 ligases function as part of acomplex and others function individually.The additional subunits that may be partof the complex include adaptors, scaf-folds, and substrate receptors (Fig. 2). Theclass of RINGE3 ligase complexes with themost members and diversity of subunits isthe CRL complexes (21), which are furtherclassified according to the scaffold proteinCullin in the complex (22–26). Most ofthese complexes are named for the adap-tor subunit, Cullin scaffold subunit, andthe substrate receptor subunit (Fig. 2).Dif-ferent substrate receptor subunits enabledistinct substrate recognition. The ECVand ECS complexes are different from theothers in that the ECV and ECS complexeshave two adaptor subunits: Elongin B andElongin C. The ECV complex also onlyforms with a single receptor subunit,von Hippel-Lindau (VHL), whereas theothers canhave various receptor subunits.CRL3 is different in that it lacks an adap-tor subunit and only contains the E3 li-gase, the Cullin scaffold, and one of fivesubstrate receptors. Another E3 ligasecomplex with RING-type E3 ligase activ-ity is the APC/C, which controls cell cycleprogression (27).APC2 is the scaffoldwiththe Cullin domain, and APC11 is a RINGbox 1 (RBX1)–like RING finger proteinthat is the catalytic E3 subunit. Although

the APC/C organization resembles that of CRL complexes, it is a largercomplex thanotherCullin-containingE3 ligase complexeswith at least 11subunits including the E3, scaffold, adaptor, and receptor subunits (27).Most RING E3 ligases contain the conventional RING domain; however,some RING E3 ligases contain a variant of the RING domain, such astheU-box (28, 29), the B-box (30), or the RBR (RING-between-RING)

Ub + ATP

Ubiquitin activation

Ub-tagged substrate degradation

26S proteasome

HECT-type E3 ligases Individual RING-typeE3 ligases

E1

Ub

UbUbUb

AMP + PPi

ATP ADP + Pi

Ubiquitin transfer to E2 Ubiquitin attachment to substrate

Details for specific E3 ligases

Ub

E2

E2

E2

E1

E1

UbUbUbUb

E2

E3

E3

E3

Substrate

Substrate

UbUbUbUb

Substrate

UbUb

Ub

Ub

Substrate

UbUb

E2 SubstrateSubstrate

Substrate

CDC20/CDH1

Adaptor

Cullin-RING E3ligase complexes

RING-between-RINGE3 ligases

E3

Ub Ub

E2 Receptor

Adaptor

APC/C complex

Ub Ub

AdaptorAPC11

+

+

E2

1 2

1 2

HECTE3

UbUbUb

E2 Substrate

CULLIN

RBR E3

APC2

Fig. 1. The steps to 26S proteasome-mediated degradation. (Top) The overall process by which ubiquitin (Ub) istransferred to substrate proteins. (Middle) Mechanismof transfer for the different families of E3 ligases and E3 ligase complexes.HECTdomain–containing E3 ligases forman intermediate bondwith theubiquitin, whereas RINGdomain–containing E3 ligases,in general, provide aplatform for the direct transfer of ubiquitin to the substrate,with the exceptionof RBRE3 ligases, whichmayform intermediate bonds like the HECT-type E3 ligases (359). Some RING-type E3 ligases can function individually; some ligasesfunction as part of protein complexes, such as Cullin-RING E3 ligase (CRL) complexes or anaphase-promoting complex/cyclosome (APC/C). Apart from the RING domain–bearing subunit, E3 ligase complexes may include additional subunitssuch as adaptor proteins, scaffold proteins, substrate receptor proteins, and accessory proteins. (Bottom) Degradation ofpolyubiquitinated substrates by the 26S proteasome in an energy-dependent reaction. The ubiquitin moieties attached tothe substrate are recycled. AMP, adenosine 5′-monophosphate; PPi, inorganic pyrophosphate; Pi, inorganic phosphate.

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domain,which consists of anN-terminalRING-likedomain, aC-terminalRING-like domain, and an IBR (in-between RING fingers) domain inthe middle of the two RING-like domains (31).

Alternative consequences of ubiquitinationThe fate of ubiquitinated proteins does not have to be degradation bythe 26S proteasome (32). The activity of deubiquitinating enzymes


(DUBs) that remove ubiquitins from thesubstrates enables this posttranslationalmodification to be reversible. Even in theabsence of DUB activity, the type of ubi-quitin signal dictates the fate of the ubiqui-tinated protein. Substrate proteins can bemodified by a single ubiquitin (monoubi-quitination), atmultiple sites by single ubi-quitins (multimonoubiquitination), or bypolyubiquitin chains. Polyubiquitin chainscanbe grownby successive additionof ubi-quitin to one of the seven lysine residues ofubiquitin (K6, K11, K27, K29, K33, K48,and K63) or to the N-terminal methionine(M1). Depending on the combination ofwhich of these residues are modified ateach round of ubiquitin addition duringchain formation, the signal (“ubiquitincode”) on the substrate protein can be in-terpreted differently in the cell (33, 34).WhereasK48- andK11-linkedpolyubiqui-tin chains usually serve as signals for pro-teasomal degradation (16, 35), M1- andK63-linked chains may signal other pro-cesses. Monoubiquitination, in contrast toK48-linked polyubiquitination, targetstransmembrane proteins at the plasmamembrane for internalization and deliveryto lysosomes, rather than to the 26S pro-teasome, for degradation. For instance,monoubiquitination of Notch1 by theRING domain–containing individual E3ligase casitas B-lineage lymphoma (CBL)targets Notch1 to the lysosome for deg-radation (36). Ubiquitination may alsoserve completely different functionsother than as signals for protein degra-dation (32, 37–39). For instance, mono-ubiquitination may serve as a signal forintracellular protein transport in thesecretory and endocytic pathways (40).Moreover, K63-linked polyubiquitina-tionof proteins contributes toDNArepair(41), regulation of protein translation(42), and regulation of cell signalingthrough proteasome-independent activa-tion of protein kinases, such as IkB kinase(43).Other sites of polyubiquitin chain for-mation have also been associated with var-ious nondegradation-related cellularprocesses such as DNA repair (K6, K27,and K33), nuclear factor kB (NF-kB)

signaling (M1), and post-Golgi trafficking of proteins (K33) (44).

Definition of degrons and the structural basis ofdegron recognitionFine-tuning the abundance of intracellular proteins usually involvesE3 ligases that specifically recognize localized sequence elements,called degrons, in their substrates. An important property of degrons

E3 ligase types

RING domain–containingE3 ligases

Cullin-RING E3 ligase(CRL) complexes

APC/C

RING domainvariants

U box B box RBR

HECT domain–containingE3 ligases

~95%

HERC family

ARF-BP1

E6AP

CRL1 (SCF)

PPIL2CHIP

CBLCOP1

MDM2RNF4PIRH2 TRIM

subfamily(TRIM13)

ParkinHOIL1HOIP

~5%

CRL7 CRL3ECV/ECS CRL4

RING or RING-likedomain–containingE3 ligase

Cullin or Cullin-likedomain–containingscaffold protein

Substrateadaptor subunit

Substratereceptor subunit

IndividualE3 ligases

NEDD4 family

CDC20/CDH1

CDC20

FZR1 (CDH1)

Adaptors

APC11

APC2

R

CULLIN1

R

CULLIN7

R

CULLIN3

R

CULLIN2/CULLIN5

R

CULLIN4A/CULLIN4B

SKP1

F-box

SKP1

F-box

DDB1

DCAF

BTB

Elongin C

VHL/SOCS box

Elongin B

SKP2

β-TrCP1

FBXL2

FBXO31

FBXO4

FBXW7

FBXW8 VHL

SOCS [1–7]

KEAP1

SPOP

CRBN

DET1

Fig. 2. Classification of E3 ligase proteins and complexes. Representative examples of the main families of E3ligases are shown. A subset of the possible substrate receptor subunits for the Cullin-RING E3 ligase complexes isshown beneath each complex.

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is that they are transferable, and the attachment of such sequenceelements by means of genetic engineering confers instability on oth-erwise long-lived proteins (45). The degron itself is usually a shortlinear motif (46, 47) characterized by a specific sequence pattern.The key amino acid residues that are critical for the interaction showstrong evolutionary conservation. The largest collection of linear mo-tifs is collected in the Eukaryotic LinearMotif (ELM) database (http://elm.eu.org), which currently contains 15 different classes of degronswith 93 experimentally verified instances in 83 unique protein se-quences in human (48).We extended the list with additional examplescollected from the literature (table S1; http://dosztanyi.web.elte.hu/CANCER/DEGRON/TP.html).

A well-characterized example of an E3 ligase–degron pair is the de-gron in p53 and the E3 ligaseMDM2 (murine doubleminute 2), whichis a RINGdomain–containing individual E3 ligase (49). In the absenceof DNA damage or other stress signals, MDM2 targets the constantlyproduced p53 for degradation. The structure formed between MDM2andp53 (Fig. 3A) shows that a short segment on theN-terminal regionof p53, corresponding to the degron motif, forms an a-helical stretchthat binds to the SWIBdomain ofMDM2 (50). The three hydrophobicamino acids of p53 (Phe19, Trp23, and Leu26) form critical contacts withMDM2 and occupy a relatively deep hydrophobic cleft on the surface of


MDM2 (Fig. 3A). Other substrates ofMDM2 and the structurally similarMDM4 exhibit a similarmethod of degron binding and share a commonmotif (Table 1). This sequence motif highlights three key hydrophobicamino acids (a Phe, a Trp, and a Val, Ile, or Leu) that must be spacedsuch that they are located on the same side of a helix and incorporatesthe incompatibility of proline residues as part of this recognition motif.

In many cases, the interaction with an E3 ligase requires post-translational modification of the degron motif. For example, a phos-phodegronmotif is a degron that contains a phosphorylation site, andphosphorylation of this site enables the phosphorylated degron (phos-phodegron) to interact with the E3 ligase. The cell cycle inhibitor p27KIP1

(also knownasCDKN1B) contains a phosphodegron (51). Substrate rec-ognition of this protein requires SKP2, a receptor subunit of the SCFfamily of E3 ligases, and the accessory protein CKS1 (CDK regulatorysubunit 1). The degron is characterized by its correspondingmotif (Table1) centered around a phosphorylated threonine that binds into a positivelychargedpocket onCKS1withhigh specificity (Fig. 3B) (52). Several othermembers of the SCF family, such as SCFb-TrCP1 and SCFFBXW7, recognizedoubly phosphorylated motifs through a WD40 b-propeller structure(Fig. 3C) (53). b-TrCP recognizes a degronmotif in its targets with twophosphorylated residues (54), and FBXW7 (F-box and WD repeat do-main containing 7) targets contain a similar yet distinct motif with

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either two phosphorylated residues ora glutamic acid substituting for the sec-ond phosphorylated residue (55).

The COP1 E3 ligase is an individualRING domain E3 ligase and also uses theconserved surface of its WD40 domainto interact with its degron, which is usuallyunmodified (Table 1), such as in the solvedstructure of COP1 with the TRB1 (Tribbleshomolog 1) degron (Fig. 3D). The CBL E3ligase targets degron motifs containing aphosphotyrosine residue (56). Substraterecognition by the ECV complex, whichcontains the receptor subunit VHL, crit-ically depends on another posttranslationalmodification, prolyl hydroxylation (57).The substrates of VHL include hypoxia-inducible factor 1a (HIF-1a), which regu-lates gene expression in response to oxygenavailability (58). Whereas HIF-1a is themost important cancer-linked target ofVHL, other targets (such as hsRPB7, b2-adrenergic receptor, or SPRY2) also dependon hydroxylation to exhibit a functionaldegron. Other posttranslational modifica-tions, such as methylation, can promotethe interaction with E3 ligases to mediatedegradation, and methylation-dependentdegradation is especially important forproteins involved in DNA repair (59, 60).

The complexity ofdegron regulationVarious mechanisms control degron-dependent degradation. One key regu-latory element is the binding strengthof the interaction between the E3 ligase

A B

C D

Fig. 3. E3 ligase–degron complexes. (A) p53-MDM2, the p53 degron peptide enters a deep hydrophobic pocket onMDM2. The three key hydrophobic residues fromp53 peptide are shown [Protein Data Bank (PDB): 1YCR]. (B) Structureof SKP1-SKP2-CKS1 in complex with the p27KIP1 phosphodegron peptide. SKP2 is rendered in gray surface, whereasCKS1 is represented in dark gray surface, with red (oxygen) and blue (nitrogen) polar functional groups. The smallpeptide from the p27KIP1 is rendered in ribbon representation with purple color: Its phosphothreonine interacts withpositively charged CKS1 surface (blue region), which provides the phosphospecificity. SKP1 is not shown for clarity(PDB: 2AST). (C) Doubly phosphorylated b-catenin degron motif (see Table 1) in complex with b-TrCP1 and SKP1. Bothof these molecules are shown in gray surface representation, whereas the b-catenin fragment is rendered as purpleribbon (PDB: 1P22). (D) COP1 E3 ligasewith TRB1 degron. TRB1 binds on the conserved surface of COP1 in an extendedmanner. COP1 with WD40 repeats (b-propellers) is rendered as ribbon, whereas TRB1 peptide is rendered in stickrepresentation and colored purple. (PDB: 5IGQ). Interchain H bonds are represented in magenta. Motif-definingpositions have been rendered as yellow sticks for all the cases.

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and its substrate. The small interface formed between the proteinsegments that contain the degron motif and their partner E3 ligaseusually enables only a low affinity for the interaction. However, thisinterface is not the only interaction that occurs between some E3 li-


gases and their substrates. For example, ERG [erythroblasttransformation–specific (ETS)–related gene] is recognized and ubi-quitinated by the CRL3 E3 ligase with the receptor subunit Speckle-type POZ protein (SPOP). The binding region of ERG for SPOP

Table 1. Degron motifs and cancer-associated dysfunction.

Genename

Proteinname

E3 ligase or subunit-recognizing degron

Instance
Startingposition
Motif*
Source† Associated mechanism in cancer
TP53
p53 MDM2 FSDLWKLL 19 F [^P]{3}W[^P]{2,3}[VIL]
DEG_MDM2_1
Increased degradationby increased E3ligase activity
CDKN1B
p27KIP1 SKP2-CKS1 SVEQTPKK 183 ..[DE].(T)P.K DEG_SCF_SKP2-CKS1_1
Increased degradationby increased E3ligase activity

CTNNB1
b-catenin b-TrCP1 DSGIHS 32 D(S)G.{2,3}([ST]) DEG_SCF_TRCP1_1 Missense mutationsin degron
MYC
MYC FBXW7 LLPTPPLS 55 [ LIVMP].{0,2}(T)P..([ST])
DEG_SCF_FBW7_1
Translocation, mutation of theposttranslational modification siteneeded for E3 ligase binding,increased gene expression,
and stabilization by MCV-mediatedinhibition of FBWX7

ERG
ERG SPOP ASSSS 42 [ AVP].[ST][ST][ST] (61) Gene deletion by chromosomaltranslocation
HIF1A
HIF-1a VHL L APAAGDTIISLDF 400 [IL]A(P).{6,8}[FLIVM].[FLIVM]
DEG_ODPH_VHL_1
Stabilization by E3 ligaseinactivation
NOTCH1
NOTCH1 FBXW7 PFLTPSPE 2508 [ LIVMP].{0,2}(T)P..E
DEG_SCF_FBW7_2
Truncating mutations in C-terminalPEST region
TP63
p63 ITCH PPPY 540 PP.Y (342) Reduced geneexpression
NFE2L2
NRF2 KEAP1 DEETGE 77 [ DNS].[DES][TNS]GE
DEG_Kelch_Keap1_1
Missense mutations in both
degrons, NRF2 deletion induces

NFE2L2
NRF2 KEAP1 QDIDLGV 26 QD.DLGV DEG_Kelch_Keap1_2
ETV1
ETV1 COP1 DEQFVPDY 67 [STDE]{1,3}.{0,2}[TSDE].{2,3}
VP[STDE]G{0,1}[FLIMVYPA]

DEG_COP1
Translocation deletingthe N-terminal degron
CSF1R
CSF-1R CBL LLQPNNYQFC‡ 963 [DN].(Y)[ST]..P (157) Mutations abolishing posttranslationalmodification site needed for E3
ligase binding

MET
c-Met(HGFR)
CBL
DYR 1002 D(Y)R (157) Mutations in posttranslationalmodification site needed for E3 ligasebinding/exon skipping/translocation
deleting the degron

SH2B
APS CBL RAVENQYSFY 623 RA[VI].NQ(Y)[ST] (157) —
AURKB
Aurorakinase B
CDH1
QKENS 3 .KEN. D EG_APCC_KENBOX_2 —
CCNA1
Cyclin A1 CDC20/CDH1 FDIYMD 135 [ILVMF].[ILMVP][FHY].[DE]
LIG_APCC_ABBA_1
—
CCNB1
Cyclin B1 CDC20/CDH1 PRTALGDIG 41 .R..L..[LIVM]. DEG_APCC_DBOX_1 —
PTTG1
Securin CDC20 DKENG 8 .KEN. D EG_APCC_KENBOX_2 —
*Definition of regular expressions describing linear motif sequence conservation: [LIV], [] refers to group of amino acids, in this case, Leu, Ile, and Val are allallowed; [^P], anything but Pro is allowed; a period (.) denotes any kind of residue; (T), modified residue (that is, phosphorylated Thr); .{0.2}, variable lengthposition, in this case, 0, 1, and 2 positions of any kind of amino acid. †Known motifs are represented as standard regular expressions and were taken fromthe ELM database (48) or from the references cited. ‡Does not meet standard motif definition.

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extends N-terminal of the phosphodegron motif, which would likelyconfer stronger binding (61, 62).

Several E3 ligases recognize their substrates through a multisitesubstrate recognitionmechanism. An example ofmultisite recognitionis presented by the interaction between the substrate NRF2 (nuclearfactor erythroid 2–related factor 2) and KEAP1 (Kelch-like erythroidcell–derived protein with CNC homology–associated protein 2), a dif-ferent receptor subunit of the CRL3 E3 ligase (63). NRF2 contains twodegrons in close proximity, which act in synergy, mediating binding toa KEAP1 dimer. Although both degrons bind to Kelch domains onrespective subunits of the partner KEAP1 dimer, their affinities are dif-ferent. TheN-terminal DLG degronmotif has a 200-fold lower affinityfor KEAP1 than the high-affinity C-terminal ETGE degronmotif (64).The finely tuned affinity of these two sites is key to the stabilization andactivation of NRF2 in response to electrophilic stress. Cyclin E alsocontains two degrons, which are recognized by the SCF family E3 li-gase containing the FBXW7 receptor subunit. FBXW7 can exist bothas a monomer and as a dimer in normal cells. Like the pair of NRF2degrons, the ones in cyclin E also have very different affinities, with theN-terminal one forming a much weaker interaction. The two degronsin cyclin E are involved in cooperative binding that is coupled to thedimerization of FBXW7 (65). As withNRF2, the multisite degrons en-able tight control for cyclin E turnover (66).

Cooperative interactions likely mediate E3 ligase–mediated degra-dation of members of the Gli family of transcription factors. Gli2 andGli3 are critical players in the transcriptional regulation by the Sonichedgehog pathway (67, 68) and have multiple degron motifs. The de-gron motifs in these proteins are phosphodegrons that are rich in ser-ine and threonine residues that, when phosphorylated, are recognizedby the SPOP-containing CRL3 E3 ligase. Mutation of a subset of thesemotifs generates resistance to SPOP-mediated protein degradation inGli2, which suggests that their binding with SPOP happens in a coop-erative fashion and uses multiple sites for interaction (69). In contrastto Gli2 and Gli3, Gli1 has fewer Ser- and Thr-rich patches, has no ca-nonical SPOP-recognized degron, and is poorly targeted for degrada-tion by SPOP (69). Instead, Gli1 contains two degrons that arerecognized by b-TrCP, a receptor subunit of the E3 ligase CRL1 (alsoknown as SCF). Gli1 lacking both of these degrons is resistant tob-TrCP–dependent degradation (70). b-TrCP–mediated degradationis not the only E3 ligase–dependent degradation mechanism for Gli1.Gli1 has two PPxYmotifs and a C-terminal pSPmotif (71), whichme-diate the ubiquitination by HECT-type E3 ligase ITCH in a NUMB(adaptor protein for ITCH)–associated manner, and this mechanismcontrols Gli1 stability in the nucleus (72).

Regulation of degron recognition often involvesmolecular switchesthat can turn on or turn off interactions in response to environmentaland cellular cues (73). Posttranslationalmodifications, including phos-phorylation, represent the most common mechanism for either pro-moting or inhibiting substrate recognition by E3 ligases. Substratephosphorylation can control E3 ligase–mediated degradation. For ex-ample, phosphorylation of Thr18 of p53 blocks its interactions withMDM2, resulting in stabilization of p53 (Fig. 4) (74). Other forms ofposttranslational modifications, including acetylation, sumoylation, orglycosylation (59), can also regulate substrate recognition byE3 ligases.

Combinations of posttranslational modifications enable fine-grainedcontrol of protein abundance in response to various signals. For example,the formation of phosphodegrons often involves “priming” phospho-rylation events that are necessary before an adjacent residue in the de-gron can be phosphorylated, and these priming events are often


carried out by kinases that are different from the ones that targetthe residue in the phosphodegron. Such a scenario occurs for theb-TrCP–mediated degradation of b-catenin, for which casein kinaseI (CK1) is the priming kinase and glycogen synthase kinase 3b(GSK-3b) is the secondary kinase, with phosphorylation of both sitesrequired for the recognition of the phosphodegron (75). In contrast,the binding of the individual E3 ligase CBL to the phosphorylatedtyrosine in the degron of epidermal growth factor receptor (EGFR)is blocked by the phosphorylation of neighboring serine residues (76).The HECT-type E3 ubiquitin ligase ITCH (the homolog of mouseItchy) recognizes tandem PPxY motifs. For the substrate thioredoxin-interacting protein (TXNIP), phosphorylation of the tyrosine residuein the motif abolishes binding to ITCH, instead promoting the inter-action of TXNIP with a p-Tyr–binding SH2 domain–containing pro-tein (77). The FBXW7-recognized degron of JUN (Fig. 4) is primed atSer243 (possibly by DYRK2) for the GSK-3b–activating phosphoryl-ation of Thr239 (78). Conversely, the intrinsically active ITCH degronin JUN (Fig. 4) can be inhibited by ABL phosphorylation of Tyr170 inthe PPxY motif (79). For polo-like kinase 4 (PLK4), the biological ac-tivity of this enzyme is coupled to its owndestruction. PLK4 controls thenumber of centrioles in the cell. After completion of PLK4-mediatedcentriole biogenesis (80), these kinases trans-autophosphorylate them-selves in a region located in the vicinity of the kinase domain. This cre-ates a phosphodegron that is recognized by b-TrCP, leading to thedegradation of PLK4 (81).

Subcellular localization and alternative splicing provide additionalmechanisms to regulate protein recognition by E3 ligases and control pro-tein stability. For p27KIP1, degradation is controlled by phosphorylation-dependent changes in subcellular localization. Unphosphorylatedp27KIP1 resides in the nucleus and is bound to cyclin E–CDK2 orcyclin D–CDK4 complexes, halting cell cycle progression. Phospho-rylation of Ser10 triggers the nuclear export of p27KIP1 to the cytoplasmwhere it is recognized and ubiquitinated by cytoplasmic CRL E3 li-gases with the receptor subunit SKP2 and its accessory proteinCKS1 (Fig. 4) (82). As exemplified by the transcription factors p53and HIF-2a (83, 84), alternative splicing can also remove degronsand produce isoforms that have similar domain structure and func-tionality but differ in stability. Therefore, the relative expression ofthese different isoforms can also influence the half-lives of the encodedproteins. Chromosomal deletion or fusion events can also influencestability of the encoded proteins, as exemplified by ETV1, whichcan either undergo a truncation event that removes the degron inthe N-terminal region or undergo a chromosomal translocation eventleading to the replacement of the N-terminal part of the protein withtransmembrane protease, serine 2 (TMPRSS2) (Fig. 4). Either of theseevents increases the stability of the resulting protein by elimination ofthe degron recognized by the individual E3 ligase COP1.

Molecular switches can also regulate the E3 ligase by altering itsassembly, activity, or subcellular localization. E3 ligases can also becontrolled by UPS-mediated degradation, for example, by autoubiquiti-nation, as observed forCBL (85). Another layer of regulatory complexityexists in which one E3 ligase targets another E3 ligase for destruction.TRUSS (tumor necrosis factor receptor–associated ubiquitous scaffold-ing and signaling protein), a subunit of the CRL4 E3 ligase complex, isone such example. TRUSS contains a phosphodegron that is phos-phorylated by GSK-3b, creating a binding site for SKP2 and targetingtheCRL1E3 ligase to tagTRUSS for proteasomal degradation. Thus, theabundance of TRUSS inversely correlates with the abundance of SKP2(86). Similarly, SKP2 is a substrate for APC/C (87).

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A key structural feature that contributes to the successful operationof the UPS involves intrinsic protein disorder. The function of manyproteins and protein regions relies on highly flexible conformationalstates instead of a single well-defined conformation. Although someproteins, like p27KIP1, are completely disordered, others have a mod-ular architecture that involves a combination of ordered globular do-mains and intrinsically disordered regions. Most known degronmotifs, like linear motifs in general, are located within intrinsically dis-ordered regions andundergo disorder-to-order transition upon bindingto their partner proteins (88, 89). Disorder can also be important for theregulation of the degron motifs by ensuring that they are accessible for


posttranslational modifications. The po-sition of the degron motif(s) varies withsome degrons located near the N-terminalend, near the C-terminal end, or in themiddle of the sequence (Fig. 4). However,within the human proteome, degrons tendto be less common in the middle of a pro-tein sequence (Fig. 5).

Many individual proteins are regulatedby multiple E3 ligases that are activatedunder different conditions. A well-studiedexample is JUN, which contains degronsfor at least three different E3 ligases (Fig.4). Among these, the binding of COP1does not depend on phosphorylations.However, the interaction with the CRL1receptor subunit FBXW7 requires doublephosphorylation of JUN on Thr239 andSer243. The activity of ITCH, a HECTdomain–containing E3 ligase, dependson phosphorylation by the kinase JUNN-terminal kinase (JNK) (90). This createsa negative feedback loop: JNK both acti-vates and enables degradation of JUN(91). In contrast, ABL-mediated phospho-rylation of the JUN degron recognized byITCH protects JUN from interacting withITCH and thus stabilizes JUN (79). De-spite knowing that multiple E3 ligasesregulate some proteins, including MYC,p53, andNOTCH1, formost cases, the de-gron and E3 ligase binding site are un-known. Without knowledge of the degron,it is difficult to establish direct or indi-rect mechanisms of E3 ligase–dependentdegradation.

Given the large number of E3 ligasesand the combinatorial possibilities to usethem, E3 ligase–degron pairs can poten-tially provide exquisite specificity for theUPS. However, substrates and the cor-responding degron motifs have been de-scribed only for a few. The direct targetsof most of the nearly 600 E3 ligases areunknown. Even for well-characterizedE3 ligases, additional unknown partnersmay exist. Moreover, there could be ad-ditional signals that tag proteins for deg-

radation, such as the N-degron, which is an N-terminal signal thatreduces protein half-life (92). Efficient degradation may also dependon additional sequence cues in the target proteins, including the presenceof disordered segments. The degradation signal may have three parts(93): (i) the primary degron that is recognized by the E3 ligase, (ii) sec-ondary sites of single or multiple lysine residues that can be efficientlyubiquitinated, and (iii) a structurally disordered segment that initiatessubstrate unfolding at the 26S. Althoughmuch of the “degrome” remainsto be explored, understanding the details of the controlled degradation ofindividual proteins has provided insights into the regulation of cellularprocesses, transcription factors, and signaling pathways.

MDM2

NES

TAD 4X

pT NLS USP7dock

pS

1 393

p53 p53 DBD

SKP2-CKS1Cyclin

box

iCyc-CDK

1

p27KIP1

pY

pT

pS

COP1

ITCH FBXW7

ABLp-Site7

GSK3p-Site

CDKp-Site

bZIP

1 331

Jun

COP1

TMPRSS2-ETV1 fusion

1 477

ETV1 ETS DBD

pT

198

Fig. 4. Selected functional elements in four regulatory proteins with modular architectures and well-studieddegrons. Only the DNA binding domains (DBD; in black) of p53 and ETV1 are natively folded; the remaining parts ofthese proteins are intrinsically unstructured, with larger functional regions indicated in light blue. The sites of de-grons are indicated with red arrows and labeled with the cognate E3 ligase. The degrons recognized by FBXW7 andSKP2-CKS1 must be phosphorylated. Phosphorylation of or near the degrons recognized by ITCH and MDM2 ab-rogates recognition. Other classes of linear motif are shown as light brown rectangles, and phosphorylation sites areshown as circles. TAD, transactivation domain; 4X, tetramerization domain; NLS, nuclear localization signal; NES,nuclear export signal; USP7 dock, USP7 binding motif; bZIP, basic leucine zipper (coiled coil) dimerization andDNA binding domain; ETS DBD, DNA binding domain found in ETS-like transcription factors; TMPRSS2-ETV1 fusion,site of cancer gene fusion event that has deleted the ETV1 N-terminal degron; iCyc-CDK, inhibitory module thatbinds across the cyclin-CDK dimer; Cyclin box, docking motif that mediates binding to cyclins.

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Processes and Pathways Regulated by DegronsThe cell cycleDegrons can be found in a diverse collection of cell signaling and reg-ulatory pathways. Defects in degron-mediated degradation by theUPSthat are particularly important in cancer include the dysfunctional de-grons that impair regulation of the cell cycle. The cell cycle consists offour main phases: G1 phase (growth), S phase (DNA replication), G2

phase (more growth and preparation for mitosis), and mitosis (alsoknown as M phase, during which segregation of chromosomes and cy-tokinesis occur). The cycle is promoted by the activity of cyclins andCDKs and is controlled at many checkpoints by cell cycle regulatoryproteins, such as CDK inhibitors (94). The accurate progression ofthe cell depends on a fine-tuned and tightly controlled complex networkof protein interactions, for which the exact amount of each protein isoften critical. Therefore, one of the most important mechanisms for theaccurate progression of the cell cycle is the ubiquitin-mediated degra-dation of cell cycle regulatory proteins (27, 95, 96).

Understanding the substrate-specific activity of E3 ligases in theregulation of the cell cycle involves characterization of the degronsthat serve as targets for specific E3 ligases. Although exceptions exist(97), most of the well-characterized degrons for the cell cycle are re-cognized by either the APC/C or the SCF complexes. The substratereceptor subunit of the APC/C complex is either CDC20 or CDH1(Fig. 2). The APC/C complex can bind to three types of degrons impor-tant for the cell cycle: the D-box, the KEN box, and the ABBA motif(Table 1). In contrast, the substrate receptor subunit of the SCF complexcan be one of more than 60 different F-box proteins (18). However, sofar, only three of these have dedicated degrons that function in the cellcycle: FBXW7, SKP2, and b-TrCP (95). Although there is some overlapbetween the sets of cell cycle regulatory proteins targeted for degrada-tion byAPC/C and SCF complexes, the degrons recognized byAPC/Cor SCF differ in terms of the cell cycle phases during which they areused to regulate the stability of substrate proteins (Fig. 6). Most APC/


C-targeted degrons are critical for the nor-mal mitosis, especially the latter parts ofchromosomal segregation and cytokinesisand early G1 phases, whereas most SCF-targeted degrons are critical for G1-S pro-gression, S phase, and early parts ofmitosis(27, 96). During the mitosis, APC/C reg-ulates the stability of PLK1 (98), Geminin(99), Cyclin B (100), Securin (101), Bub1(102), andCyclin A (103). G1 phase targetsof APC/C are SKP2 (87), mE2-C (104),CDC6 (105), CDC20 (106), Nek2A (107),Aurora A (108), and AXIN-2 (109).

With three different receptor subunitsthat are important for targeting cell cycleregulatoryproteins, the SCFcomplex func-tions in all phases of the cell cycle (Fig. 6)and controls the stability of both proteinsthat promote the cell cycle and proteinsthat inhibit the cell cycle. The F-box pro-teins FBXW7 and SKP2 have antagonisticeffects on cellular proliferation (27). FBXW7mainly acts as a tumor suppressor becauseit targets growth-promoting factors, suchasMYC (110) and JUN (78), for degrada-tion at late G1 or the G1-S transition. The

result of SKP2-mediated targeting of substrates is generally the promo-tion of the cell cycle. SKP2-recognized degrons occur in tumor sup-pressors, such as CDK inhibitors p21CIP1 (111), p27KIP1 (112), andp57KIP2 (113). Other SKP2-recognized degrons in proteins relevant tothe cell cycle are found in cyclinD (114), ORC1 (115), CDT1 (116), cyclinE (112), E2F1 (117), and p130 (118). Degrons recognized by b-TrCPare found in a diverse set of oncoproteins and tumor suppressors.Most of the characterized b-TrCP degrons function in mitotic onsetand mediate the degradation of BORA (119), FBXO31 (120), WEE1A(121), and CDC25B (122). Other cell cycle–relevant b-TrCP degronsoccur in proteins distributed throughout the cell cycle: b-catenin (123),CDC25A (124), PLK4 (125), CREB-2 (also known as ATF4) (126), andTFAP4 (127).

The final E3 ligase with a regulatory role in the cell cycle is CRL3.To the best of our knowledge, the only known cell cycle–relevant sub-strate of CRL3 is p60Katanin, which is recognized by the KLHL42(Kelch-like protein 42) that forms the CRL3 complex with CULLIN3and binds p60Katanin. During mitosis, the Kelch domains of KLHL42bind p60Katanin, and degradation of p60Katanin is required for normalprogression through mitosis in mammalian cells (97). Even the pre-sumably incomplete picture of degrons involved in regulation of thecell cycle reveals that degrons are indispensable functionalmodules forthe normal progression of the cell cycle (Fig. 6).

p53 pathwayThe p53 pathway is regarded as the major tumor-suppressor pathwayin multicellular organisms (128). This pathway enables the appropriateresponse to a range of stress factors, especially those that cause DNAdamage, such as ionizing and ultraviolet (UV) radiation and genotoxicagents. Furthermore, the p53 pathway is also involved in the cell’s reac-tion to nutrient deprivation (129, 130), hypoxia (129), and thermalshock (131, 132). The outcome from the activation of the p53 pathwaycan be either cell cycle arrest, which provides the necessary time frame

Degron position1: N terminus; 100: C terminus

Dens

ity

10050250 75

0.000

0.003

0.006

0.009

Positional density of degrons in protein sequences

Fig. 5. Positional density of degrons in protein sequences. The plot shows the relative density of degron loca-tions in protein sequences. All protein sequences that contain at least one degron (166 proteins from table S1 andhttp://dosztanyi.web.elte.hu/CANCER/DEGRON/TP.html) were split into 100 equal bins. The bins were numbered from1 to 100 in the direction of N terminus to C terminus of the protein sequence. The number of degrons overlappingeach bin was counted, and a density diagram was generated using ggplot2 geom_density function (360).

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for DNA repair or resolution or adaptation to other stresses, or the in-duction of apoptosis, in case the cellular stress proves to be too severe forthe cell to survive (133–135).

Although this pathway integrates a wide range of signals andcontrols a similarly wide spectrum of reactions, at the molecular level,regulation is focused primarily on the protein p53, encoded by thegeneTP53. This contrastswith the spatially and temporally distributednature of cell cycle control. The activity of the pathway depends on theconcentration of p53. In healthy cells, p53 is constantly produced, butit is present in only low amounts because of constant degradation(136). The main ubiquitin ligase controlling p53 abundance isMDM2, which recognizes a short degron in the N-terminal part ofp53 (between residues 17 and 29) (Fig. 4). Not only does MDM2 ubi-quitinate p53, targeting it for degradation, it also promotes the exportof p53 from the nucleus to the cytoplasm (50). Upon the activation ofstress signal–conducting pathways, several kinases can phosphorylatep53 on its N-terminal region, which overlaps with the degron. Thesekinases relay signals through arms of the mitogen-activated proteinkinase pathway that respond to oxidative stress, osmotic stress, or heatshock, among others. In addition, several other kinases, such as ATMor CHK1, phosphorylate p53 in response to activation of the genomeintegrity checkpoint (128).N-terminal phosphorylationdisruptsMDM2


binding, leading to p53 accumulation inthe nucleus. As a result, p53 activates thetranscription of a wide range of genes,including those encoding proteins con-nected to the inhibition of cell cycle (forexample, p21KIP1), the induction of ap-optosis (for example, the cell surface deathreceptor FAS), and other antiproliferativeprocesses (for example, the dual-specificityprotein phosphatase PTENor extracellularmatrix protein thrombospondin-1) (128).

AlthoughMDM2 is themain E3 ligasefor p53 that mediates its destruction (49),there are several other UPS componentsthat fine-tune p53 abundance under var-ious cellular conditions. The individualRING domain–containing E3 ligasesCOP1, PIRH2 (p53-induced protein witha RING-H2 domain), and synoviolin; theRING-type E3 ligase MKRN1; the HECTdomain–containing E3 ligase ARF-BP1(HECT); the U box–type E3 ligase CHIP;and the F-box–containing E3 ligase com-plexes with JFK are all reported to targetp53 for proteasomal degradation, albeitthroughmechanisms that may be distinctfrom those used byMDM2 (137). For ex-ample, as opposed to MDM2 and COP1,which cannot recognize p53 phosphoryl-ated at the N-terminal region, PIRH2 re-cognizes N-terminally phosphorylatedp53, and thus, PIRH2 is the primary E3ligase for p53degradationunder conditionsof irreparableDNAdamage (138). Further-more, some E3 ligases bind other sites inp53, suggesting that there are other de-gronswithin the central portion of the pro-

tein, such as residues 149 to 155, which are recognized by the F-boxreceptor subunit JFK (139).

Degradation processes in the p53 pathway add additional layers ofcomplexity because several p53-targeting E3 ligases, includingMDM2and COP1, also have autoubiquitination activity (140, 141). Further-more, genes encoding the E3 ligases COP1, MDM2, PIRH2, and JFK,all of which target p53, are induced by the transcriptional activity ofp53 (138, 139, 142–144). In addition, p53 ubiquitination by these E3ligases is reversible by DUBs. The primary DUB targeting ubiquiti-nated p53 is ubiquitin-specific–processing protease 7 (USP7), whichstabilizes p53 even in the presence of high amounts of MDM2. How-ever, USP7 also deubiquitinates and stabilizes MDM2 (145, 146). Theinherent complexity in p53 degradation is also related to the existenceof multiple isoforms (nine isoforms exist in UniProt database for p53;accession no. P04637), some of which lack the MDM2 binding motifin the N-terminal region and thus exhibit prolonged half-lives (147).The subcellular localization of p53 is also regulated by nuclear local-ization and export signals, which also influence the degradation of p53by controlling its accessibility to different E3 ligases (Fig. 4). Thesemultilayered and redundant degradation processes ensure the precisecontrol of p53 that is necessary for efficient tumor suppression andmaintenance of genome integrity.

Mitosis Interphase S

G2Prophase

Metaphase

Anaphase

Telophase G1

MitosisBUB1 (mitotic exit)

PLK1 (anaphase)

Securin (anaphase)

Geminin (metaphase-anaphase)

Cyclin B (metaphase)

Cyclin A (prometaphase)

p60Katanin

BORA

FBXO31 (prophase)

WEE1A (mitotic onset)

CDC25B (mitotic onset)

G1 phaseSKP2

mE2-C

AXIN2

Aurora A

Nek2A

CDC6

CDC20

β-Catenin

Degron types in cell cycleAPC/C-D-box

APC/C-KEN box

APC/C-ABBA motif

SCF (FBXW7)

SCF (SKP2)

SCF (β-TrCP)

CRL3

G1-S transitionp57KIP2

MYC

JUN

S phasep21CIP1

p27KIP1

Cyclin D

Cyclin E

PLK4

ORC1

CDT1

E2F1

CDC25A

G2 phaseCyclin E

TFAP4

CREB-2

p27KIP1

p130

E2F1

Fig. 6. Cell cycle phase diagram showing where degrons function in the cycle. Degron-containing proteinnames are categorized adjacent to the cell cycle phase during which the degradation of the protein enables pro-gression of the cell cycle.

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Wnt/b-catenin pathwayCanonicalWnt signaling regulates various key biological processes duringembryogenesis, including those that specify cell fate and contribute toorganogenesis (148). A key downstream component of this pathway isb-catenin, which controls gene transcription in response to the glyco-proteins of the Wnt family of ligands. In the absence of Wnt, cytosolicb-catenin is degraded by a multisubunit destruction complex. Thisdestruction complex consists of AXIN, adenomatous polyposis coli(APC), the Ser/Thr kinases GSK-3b andCK1, and the protein phospha-tase PP2A. The destruction complex marks b-catenin for degradationin a phosphorylation-dependent manner (149). At the destructioncomplex, CK1 phosphorylates Ser45 of b-catenin, which enables thephosphorylation of Thr41 by GSK-3b. Once Ser45 and Thr41 are phos-phorylated, GSK-3b phosphorylates Ser33 and Ser37. These last twosites mark the boundaries of the degron, which, in its phosphorylatedstate, is recognized by the SCFb-TrCP E3 ligase. SCFb-TrCP attaches K48-linked polyubiquitin chains to the Lys19 and Lys49 residues of b-catenin(54), targeting it for proteasomal degradation.

Once the Wnt signaling pathway is activated, the destruction com-plex translocates to the cytoplasmic side of the transmembrane Frizzledreceptors, leading to a series of posttranslational modifications on theadaptor proteins Dishevelled and AXIN. Among these modifications,dephosphorylation of AXIN reduces its binding affinity for b-catenin(150). Moreover, Tankyrase targets AXIN for PARsylation, which iscritical for the ubiquitination of AXIN. Once PARsylated, AXIN is re-cognized by E3 ligase RNF146, thereby targeting AXIN for degradationin the proteasomal machinery (151). Consequently, b-catenin accumu-lates in the cytoplasm, translocates into the nucleus, and recruits cofactorsof the TCF/LEF family to regulate transcription of its target genes (152).Many of the activated genes encode proteins connected to cell growth,proliferation, or survival, such asMYC, cyclinD, and JUN (153). There-fore, SCFb-TrCP-dependent degradation of b-catenin is a critical factormaintaining the “off” state of the canonicalWnt pathway, andRNF146-dependent degradation of AXIN is critical for turning on this pathway.

Receptor tyrosine kinase pathwaysProtein ubiquitination plays an important role in regulating the activitiesof receptor tyrosine kinases (RTKs). Protein tyrosine kinases, bothRTKs andnon-RTKs, are primarymediators of cell-to-cell communica-tion in multicellular organisms, playing essential roles in most develop-mental and homeostatic processes, including the regulation of cell growth,proliferation, motility, survival, and differentiation (154). EGFR, colony-stimulating factor-1R (CSF-1R), andMet [the hepatocyte growth factor(HGF), also known as scatter factor (SF) receptor] are members of theRTK family that are particularly important for cancer and exhibit E3ligase–dependent regulation (155). As single-pass transmembrane pro-teins, the RTK transmembrane helix connects the extracellular ligandbinding domain to the cytoplasmic region, which contains a conservedtyrosine kinase domain and additional regulatory sequences. RTKs areactivated by the binding of growth factors or other polypeptide ligands,which triggers receptor dimerization, oligomerization, or conformationalchange and activation of the kinase domain. The activated RTK phos-phorylates several tyrosine residues on the oligomerized receptors, whichform docking sites for cytoplasmic proteins that relay the signal. Simul-taneously, RTK activation sets in motion various mechanisms, such asreceptor-mediated endocytosis and either receptor degradation or re-cycling, that will ultimately terminate signaling. One of the major me-chanisms of RTK down-regulation involves the E3 ligase family calledCBL (156).


The CBL family is evolutionarily conserved and contains three mem-bers in mammals: CBL, CBL-B, and CBL-C (56, 156). These proteins arecharacterized by a commonN terminus that includes a phosphotyrosinebinding (TKB) module connected by a linker helix to a RING fingerdomain. The TKB module contains an embedded SH2 domain thatselectively recruits substrates having phosphorylated tyrosine–containingmotifs (Table 1) (157). The RINGdomain binds to E2 proteins andmed-iates the transfer of ubiquitin to the substrates. In addition to the conservedN terminus, CBL andCBL-B also have additionalC-terminalmodules thatcan mediate multiple protein-protein interactions, as well as an ubiquitin-associated domain, whichmediates homodimerization of these twomem-bers of the CBL family. This C-terminal region confers adaptor-likefunctions onCBLandCBL-B. It is ensured at two levels that only activatedRTKs are targeted byCBL. First, substrate recognitionbyCBL requires thephosphorylation of tyrosine residues on the receptors, which is carried outby the activated receptors themselves. Second, the ligase function of CBLonly becomes active as a result of the phosphorylation of a conservedtyrosine (Tyr371 in CBL), which is located within the linker helix regionconnecting the TKB module and the RING domain (158, 159).

The recruitment of CBL proteins to specific phosphotyrosine residuesof the activated RTKs leads to ubiquitination of RTKs at the plasmamem-brane and induces their internalization through the endocytic pathway(160, 161). Through several sorting steps, RTKs can be degraded or re-cycled back to the cell surface depending on the type and concentrationof the ligands. In contrast to themore commonmechanisms that targetproteins for proteasomal degradation by tagging them with K48-linkedubiquitination, RTKs are associated with a distinct pattern of modifica-tions, featuringmultiplemonoubiquitination events andK63-linked poly-ubiquitination and are targeted to the lysosome fordegradation (162–164).CBL also has intracellular substrates, such as the kinases ZAP70 and SRCfamily kinases, which follow the more canonical degradation pathwaythrough the proteasome (165). To fine-tune receptor signaling, there areseveralmechanisms that regulate CBL-dependent ubiquitination. Theseinclude the autoubiquitination of CBLs, the interaction with adaptormolecules that recruit CBL to targets, and proteins that inhibit CBL function. For example, EGFRdown-regulation byCBL involves the adaptorprotein GRB2 (166), and a similar mechanism may function in CBL-mediated down-regulation of KIT (167). Sprouty 2 (SPRY2) is a targetand an inhibitor of CBL. Activated RTKs induce transcriptional up-regulation and the phosphorylation of SPRY2, thereby creating a bindingsite for CBL, which targets it for degradation. However, the interactionbetween SPRY2 and CBL can also attenuate the ubiquitination andendocytosis of some RTKs (168).

Modulation of Protein Degradation in CancerDegradation processes can be altered in and contribute to cancer (Fig. 7).In some cancers, the alteration is in the substrate: Specific degron motifsare affected by genetic alterations. The accumulation of these mutationsacross various types of cancer is consistent with thesemutations having adriver role in oncogenesis (169). Altered E3 ligase activity can also con-tribute to cancer. Furthermore, these basicmechanisms are intertwined inincreasingly complex regulatory subsystems, presenting multiple mech-anisms by which altered function of the UPS affects cancer progression.

Impaired degron functionality by mutation, gene fusion,or truncationOneof themost studieddegrons that ismodifiedby cancermutations is thedegron in b-catenin (8, 9). TheN-terminal phosphodegron in b-catenin

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is disrupted by mutations in many forms of cancer, including hepato-cellular carcinoma, colorectal cancer, ovarian and breast cancers, lungcancer, and glioblastoma. Most often, the degron mutations are single-nucleotide variations that manifest as missense mutations and, less fre-quently, occurring in-frame deletions, which are concentrated at thedegron region and the adjacent GSK-3b and CK1 phosphorylationsites. The COSMIC database—a collection of mutations sequenced fromtumor samples (170)—lists more than 5000missensemutations for b-ca-tenin, 70% of which affect one of the four phosphorylation sites (Ser33, Ser37,Thr41, and Ser45) that are directly or indirectly responsible for SCFb-TrCP-mediated degradation. In addition, most of the identified deletionsaffect the degron region aswell, showing that, formutations fallingwithinthe CTNNB1 gene, the increased b-catenin activity that leads to cancer isprimarily achieved through the inhibition of degradation.

A similarmechanism is responsible for the oncogenicity of the nuclearSET-binding protein (SETBP1), which is another target of cancer muta-tions. Although the exact biological role of SETBP1 is poorly understood,its involvement in myeloid malignancies is well documented (171).SETBP1 contains a phosphodegron that is recognized by SCFb-TrCP

(172). The degron motif contains almost half of known SETBP1 cancermutations despite representing only 0.4% of the protein sequence. Al-though the sameubiquitin ligase regulates SETBP1 andb-catenin, SETBP1mutations primarily occur in various forms of leukemia and myelo-


proliferative diseases (172), a more restricted range of cancers than thosethat contain b-catenin mutations.

The transcription factor NRF2 lies at the core of the primary pathwayfor cellular defense against oxidative stress (173). NRF2 binds to the anti-oxidant response elements (AREs) in the genome and stimulates theexpression of target genes encoding cytoprotective enzymes, such asGSTA2 (glutathione S-transferase A2) and NQO1 [NADPH (reducedformofNADP+) quinone oxidoreductase 1]. In the absence of oxidativestress, NRF2 is recognized and ubiquitinated by theCRL3 E3 ligase withthe BTB receptor subunit named KEAP1. KEAP1 sequesters NRF2 inthe cytoplasmand ubiquitinatesNRF2, targeting it for proteasomal deg-radation. NRF2 has two degrons, which bind KEAP1 dimers synergisti-cally but with different affinities. Despite this asymmetry in the bindingstrengths, ubiquitination and subsequent degradation of NRF2 requirebinding of KEAP1 to both degrons. Thus, missense point mutations ineither of the two degrons result in excess NRF2 activity. Not surprisingly,increased NRF2 abundance is present in a wide range of cancers, mostoften of the lung, breast, head and neck, ovaries, and the endometrium(174, 175). Themutations found inNRF2 are spread through the residuesin the degrons without forming apparent mutational “hotspots.” Thispattern contrasts with phosphodegrons, in which the phosphorylatedresidues are predominantly mutated, and is consistent with KEAP1 re-cognition of NRF2 not involving posttranslational modifications.

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ERG belongs to the ETS transcriptionfactor family and functions as a transcrip-tional regulator, affecting differentiation,embryonic development, and cell death(176). In normal cells, this protein is tar-geted for degradation by CRL3 with theSPOP receptor subunit, which recognizestheN-terminal degronmotif of ERG. Sim-ilar to b-catenin and NRF2, disruption ofERG degradation is connected to cancer,in particular, prostate cancer (177) andhumanmyeloid leukemia (178). This deg-radationprocess is impaired in a substantialfraction (~65%) of prostate cancer cases.However, in contrast to the previous exam-ples, the ERG degron is not affected bypoint mutations. Instead, gene fusionevents,most commonlywith theTMPRSS2protein, disrupt or eliminate the degron.Transcripts for the TMPRSS2:ERG fusionprotein are associated with androgen-independent prostate cancer (177, 179),and this fusion protein is resistant to pro-tein degradation by CRL3. The twomostcommonprostate cancer–associated fusionscontain the first exon of TMPRSS2 andeither the first four or five exons of ERG[TMPRSS2 exon 1–ERG exon 4 (T1-E4)andTMPRSS2exon1–ERGexon5(T1-E5)].In T1-E4 and T1-E5, the first 39 and 99residues of ERG are lost, respectively. Al-though there aremultiple putative degronsin ERG, the N-terminal degron (residues42 to 46)has critical residues for the SPOP-ERG interaction. In the case of the T1-E4fusion protein, the core degron is present,

E3

E3

E3 ligase or E3 ligase complex

Target Example of procancer event

Oncoprotein

Tumorsuppressor

Mutations in β-catenin degron impairbinding to SCFβ-TrCP complex

Mutations in SPOP receptor subunitimpair binding to ERG

Epigenetic reduction in TRIM33 E3ligase abundance impairs SMAD4degradation

Increased abundance of MDM2increases degradation of p53

E3

CULLIN1

Skp1

β-TrCP1

E3

CULLIN3

Skp1

SPOP

TRIM33

MDM2

MDM2

MDM2

MDM2

β-Catenin

β-Catenin

β-Catenin

ERG

ERG

ERG

SMAD4

SMAD4

SMAD4

p53

Fig. 7. Cancer-promoting mechanisms of impaired protein degradation. Cellular abundance of proteins can bederegulated because of genetic mutations that lead to loss of binding to the E3 ligase or substrate receptor of an E3ligase complex. Partial or complete loss of the degron-containing region of a protein or mutations in the degronrecognition surface of an E3 ligase (or complex) may impair binding to the target substrate. If the target substrateis an oncoprotein, accumulation of such a protein can have a tumorigenic effect. Alternatively, cellular abundance ofproteins can be deregulated because of anomalies in the expression patterns of the E3 ligases or E3 ligase complexesthat tag the substrate proteins for 26S proteasomal degradation. In the absence of mutations in either the E3 ligase orthe substrate degron, down-regulation of an E3 ligase (or complex) that targets an oncoprotein or the up-regulation ofan E3 ligase that targets a tumor suppressor could have a similar cancer-promoting impact on the cell.

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but the N-terminal flanking region is not. Because the N-terminalflanking region substantially adds to the binding strength, the fusionevent leads to an unstable interaction with SPOP and hence impairedubiquitination of the fusion protein. In the T1-E5 fusion protein, thecritical degron is completely lost owing to the absence of the first 99 re-sidues, which makes ERG degradation-resistant. The diminished orabolished degradation eventually leads to the accumulation of thesefusion proteins, which drives prostate cancer metastasis (61, 62).

TMPRSS2 also forms genetic fusions through chromosomal trans-location with the gene encoding ETV1. ETV1, ETV4, and ETV5 repre-sent the PEA3 subfamily of the E26 transformation-specific transcriptionfactors (180, 181). TheN-terminal regionsof thePEA3 subfamilymemberscontain degronmotifs recognized by the E3 ligase COP1 (Fig. 4). In ad-dition to the TMPRSS2 fusion, other fusions arising through chromo-somal translocations of ETV1 (and, less commonly, ETV4 and ETV5)often produce N-terminal–truncated mutants in human prostatecancers. These degron-lacking ETV proteins evade COP1-mediateddegradation (182).

The degradation of NOTCH1 is also often misregulated in cancer,including head and neck squamous cell carcinoma, breast cancer, CLL(chronic lymphocytic leukemia), and lymphoma (183–186),with trun-cating mutations as the most commonly occurring genetic alterations.NOTCH1 regulates cell fate decisions and differentiation. NOTCH1 isa single-pass transmembrane receptor that is activated by ligands pres-ent on the surface of adjacent cells. Ligand binding triggers NOTCH1cleavage, releasing the Notch intracellular domain (NICD), whichtranslocates to the nucleus and promotes transcriptional programsthat stimulate cell division and prevent differentiation. NOTCH1has been implicated in various cancer types. Multiple ubiquitin ligases,including ITCH, CBL-B, and CBL-C (187), may target NOTCH1.However, the CRL1/SCF E3 ligase with the receptor subunit FBXW7plays a crucial role in regulating the transcriptional activity of theNICD.FBXW7 recognizes the natively unstructured C-terminal region, whichis rich in proline, aspartic acid, serine, and threonine residues (a so-called PEST region). PEST regions are associated with short molecularhalf-life (188). The NICDPEST region harbors a phosphodegronmo-tif anchored by Thr2512, the recognition of which depends on phos-phorylation by CDK8 (189). A major mutational hotspot zone inNOTCH1 is in the PEST region, and most of these cancer mutationsare frameshift or nonsense truncatingmutations that eliminate the de-gron recognized by FBXW7 and increase NICD stability.

Degradation impaired through decreased E3 ligase activityAs the other side of the coin, degradation can also be impaired throughvarious mechanisms that decrease the functional activity of E3 ligases.Although truncating mutations in NOTCH1 frequently eliminate thedegron recognized by FBXW7, in T cell acute lymphoblastic leukemia(T-ALL), mutations in FBXW7 are also common. However, cancerswith increased NOTCH1 abundance have either the NOTCH1 PESTmutations or the FBXW7mutations (190–192). This mutual exclusivityis similar to that seen for other substrates and E3 ligases or receptor sub-units, such as the substrate ERG and the receptor subunit SPOP. Im-paired E3 ligase activity of CBL and TRIM33 has also been reported invarious cancers.

Like ERG translocation events, genetic alterations of the SPOP-encoding gene are also connected to prostate cancer (193).Most of SPOPmutations are concentrated in the MATH domain (for example, Y87Cand F133Vmutations), which is crucial for substrate recognition (194).Recognition of the ERG degron is facilitated by a shallow groove on the


MATH domain where the ERG degron motif interacts through anextended conformation. Mutations in this evolutionarily conservedand structurally important region lead to the loss of E3 ligase functiontoward SPOP-dependent targets (194, 195). As with translocations thateliminate the function of the ERG degron, mutations in SPOP thatcompromise substrate binding result in increased ERG in prostate cancercells. There is a mutual exclusivity in the TMPRSS2-ERG fusions (194)and SPOPmutations, consistent with both genetic alterations being ableto promote the cancer-associated increase in ERG abundance at an earlystage of tumorigenesis (62, 194).

As a phosphotyrosine-directed E3 ligase that controls the degrada-tion of a range of target proteins connected to transducing growth factorsignals, many of which are oncoproteins, CBL is also linked to variousforms of cancer. CBL mutations are frequently observed in acute mye-loid leukemia (AML) and othermyeloproliferative diseases, as well as innon–small cell lung cancer (196, 197). Surprisingly, the mutation hot-spots largely spare the TKB domain, which is responsible for substraterecognition. Instead, mutations accumulate in the RING domain andthe linker helix region, therefore interfering with E2 binding. The highestfrequencyofmissensemutationsoccurs atTyr371within the linker region,which has a critical role in ligase activity by properly positioning andorienting the TKB-bound substrate relative to the catalytic RINGdomain(158, 159). CBL mutants that bind RTKs but not the E2 may protect theRTKs against degradation, even in the presence of a second functionalCBL allele by preventing access to the substrate.

Tripartite motif containing 13 (TRIM13) is a B-box ubiquitin ligasethat is involved in the endoplasmic reticulum (ER)–associated degrada-tion, a process that degrades secretory and membrane proteins synthe-sized, folded, and assembled in the ER (198). TRIM13 also has cytosolicsubstrates, and these are the substrates relevant to cancer. The oncogenicrelevance of TRIM13 comes from its ability to target MDM2 (the mainE3 ligase for p53) and AKT1 (a kinase that activates MDM2) for degra-dation, thus increasing the abundance of p53 and promoting apoptosis.Physical mapping of the regions in chromosome 13 that are frequentlydeleted in B cell CLL (B-CLL) has pinpointed TRIM13 to be a core tu-mor suppressor cancer gene in B-CLL development (199). In contrast toE3 ligases SPOP and CBL, TRIM13 functionality is not typically im-paired by mutation; instead, the whole gene is lost.

TRIM33 (also known as transcription intermediary factor 1 g) playsa key role in erythropoiesis (production of red blood cells) by promot-ing the differentiation of the red cell precursors (200). TRIM33inactivation has been shown to lead to chromosomal defects, and re-ducedTRIM33 expression correlated with an increased rate of genomicrearrangements (201). Accordingly, TRIM33 down-regulation isconnected to various forms of cancer, most notably chronic myelomo-nocytic leukemia (202) and hepatocellular carcinoma (203). In ~35% ofleukemic cells, TRIM33 expression was undetectable as a consequenceof various epigenetic factors, includingCpGhypermethylation and spe-cific histone modifications in the gene promoter (202). At the proteinlevel, the effect of epigenetic gene repression is functionally equivalentto that of gene deletion, similarly to TRIM13. However, expressionof TRIM33 can be at least partially restored by demethylating agents,representing—in contrast to previously described mechanisms—areversible modification of a compromised degradation process.

Increased degradation through up-regulatedE3 ligase activityIn the alterations of degrons andUPS components described so far, theeffects of these modulations are stabilization and increased abundance

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of the degron-containing proteins. Thus, impaired ubiquitination pro-motes tumorigenesis, which means that the target protein has an on-cogenic role and the E3 ligase has a tumor suppressor role. However,target proteins that are tumor suppressors can also be involved incancer-promoting alterations of the UPS (Fig. 7). In this case, thenet effect of the modulations of the degradation process is a reductionin the abundance of the target protein,meaning that the activity of cer-tain E3 ligases is increased. At the level of degrons, enhanced degrada-tion cannot be achieved by a simplemodification of an already existingdegron. Although, in theory, a de novo cancer-promoting degroncould be formed by a simple mutation or a fusion event, no suchscenario has been documented. However, a gene fusion event couldintroduce additional lysines that could be ubiquitinated, leading toenhanced degradation, an event that could contribute to cancer (204).

The dominant oncogenic mechanism that leads to the increaseddegradation of tumor suppressor proteins involves an increase in theabundance of their cognate E3 ligase. A well-known example isMDM2, the primary E3 ligase for p53, which is increased in activitythrough a range of different molecular mechanisms in various typesof cancer. First, transcription of MDM2 can be increased by severalmechanisms. A single-nucleotide polymorphism in the promoter ofMDM2 increases the binding affinity of the transcription activatorSP1, resulting in an increase in themRNAandof the translated protein(205). This increase in MDM2 attenuates activity of the p53 pathwayboth in vivo and in vitro and is associated with accelerated develop-ment of three different human cancers (205, 206). Gene amplificationofMDM2 also increases the activity of this E3 ligase and is common inosteosarcomas, esophageal carcinomas, and soft tissue tumors (207).Incidentally, gene amplification in MDM4, encoding a paralog ofMDM2 that also targets p53 for degradation, is also common in reti-noblastoma (Rb) (4).

MDM2 abundance and activity can also be increased without directmutations or changes to the MDM2 gene. Increased transcription ofMDM2 is due to increased growth factor signaling by transforminggrowth factor–b1 (TGF-b1), which activates the heterodimeric tran-scription factor complex SMAD3/4. Many late-stage carcinomas ex-hibit excess SMAD3/4 activation, which is associated with highMDM2 abundance (208). Increased MDM2, but without an increasein its transcript, contributes to the development or the severity of sev-eral types of cancer (209). The abundance of p53 is indirectly enhancedby p14ARF, which sequesters MDM2 in the nucleolus. Expression ofthe gene encoding p14ARF is stimulated by the RB/E2F pathway, apathway that is frequently down-regulated in cancer. Without thisnegative regulation, MDM2 activity increases and p53 abundance de-creases. In general, cancers with increased MDM2 activity havenormal p53, indicating that MDM2 up-regulation is sufficient for tu-morigenesis in the absence of genetic aberration of TP53 (210).

Similar observations linking cancer to overexpression at the tran-script level exist for other E3 ligases that function within the p53pathway, including COP1, PIRH2 (also known as RCHY1), ARF-BP1, and several others (211), as well as for E3 ligases that target othertumor suppressors for degradation (212). The CDK inhibitor p27KIP1

represents an interesting example because at least three E3 ligasescontrol its degradation. The SCFSKP2 E3 ligase complex, the RINGdomain–containing individual PIRH2, and the dimeric RING domain–containing E3 ligase complex KPC1 (KIP1 ubiquitination-promotingcomplex 1) act sequentially, depending on the phase of the cell cycleand cellular location. The E3 ligase with the SKP2 receptor subunitand the accessory subunit CKS1 recognizes the phosphodegron in the


C terminus of p27KIP1 (Fig. 4) and targets p27KIP1 for degradation dur-ing the S andG2 phases in the nucleus. Although p27

KIP1 is seldommu-tated in cancer, the abundance of p27KIP1 is frequently reduced becauseof an increased abundance of SKP2 or CKS1. Increased abundance ofPIRH2, which is mainly active from late G1 to S phase, may also triggerenhanced degradation of p27KIP1 in malignant cancers (213). In con-trast, KPC1, which targets p27KIP1 exported from the nucleus by thenuclear exporter Chromosomal Maintenance 1 protein in G0-G1

transition phases, has not been implicated in cancer. The importanceof proper regulation of p27KIP1 in cancer is evidenced by decreasedabundance of p27KIP1 serving as a prognostic factor predictive of a poorprognosis in many malignancies (214). There are many E3 ligases thatare overexpressed, leading to increased transcript and protein abun-dance in various cancers (table S2; http://dosztanyi.web.elte.hu/CANCER/DEGRON/E3_TRS.html). However, formany of these E3 li-gases, the targets and the exactmechanisms by which they contribute tocancer have not been explored.

Disruption of one degron: b-catenin/b-TrCPAlthough we have presented relatively simple examples of cancer-associated changes in degrons and E3 ligase activities, the affectedproteins participate in complex and intertwined networks within cells.Consequently, the effects of the degradation—and in many cases thedegradation process itself—of certain proteins are interconnected andcannot be separated fromone another.We present a set of increasinglycomplex scenarios showing how cancer and protein degradation arelinked in the context of regulatory subnetworks.

The properties of the canonical Wnt/b-catenin pathway enablecancer to arise from simple disruption of a single degron: (i) b-catenintarget genes include oncogenes (215); (ii) b-catenin is constitutivelyproduced and degraded in the absence of Wnt signaling through amechanismdependent on a single degron (216); (iii) b-catenin appearsto be the main or even sole transducer of the Wnt signal into the nu-cleus. Thus, the pathway lacks redundancies or feedback loops thatcould overcomedegron-disruptingmutations.Degradationof b-cateninrequires not only a degronmotif but also a functional ubiquitin ligase—in this case, SCFwith the receptor subunit b-TrCP. Theoretically, cancercould depend on impaired b-catenin degradation due to disruptedb-TrCP function. However, mutations of b-TrCP are relatively rareevents. We predict that the reason for this asymmetry between degronand ligase modulation in cancer is that b-TrCP has a wide range of tar-gets, some with oncogenic and some with tumor suppressor functions.Thus, the effect of b-TrCP inactivationmay not be tumorigenesis. How-ever, some of its oncogenic targets exhibit degron-disruptingmutationssimilar to those observed for b-catenin.

Disruption of synergistic degrons and the correspondingE3 ligase: NRF2/KEAP1Although theNRF2/KEAP1 system is similar to the b-catenin/b-TrCPsystem [constitutive production and degradation of the substrate withsignal-mediated stabilization of the substrate and its translocation tothe nucleus (217)], a key difference is that the CRL3 E3 ligase with theKEAP1 receptor subunit has few substrates (218). Like b-catenin, thedegron mutations of NRF2 promote resistance to degradation, result-ing in constitutively active NRF2. Because the primary role of KEAP1is to limit NRF2 abundance, KEAP1 mutations that disrupt the inter-action with NRF2 or epigenetic changes that suppress the expressionof the gene are common in cancers (219, 220). The mutational patternof KEAP1 is broadly distributed with many missense mutations

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scattered along the substrate-binding Kelch domain. Whereas mono-allelic mutations in NRF2 are enough to increase NRF2 abundance,usually both alleles ofKEAP1 aremutated in cancer (220, 221) becauseeven one functioning copy of the KEAP1 gene can produce the func-tional E3 ligase. Mutational binding studies showed that KEAP1 mu-tations that either attenuate or enhance the interaction with NRF2both decreased NRF2 degradation (222), showing that the interactionis optimized in terms of affinity and thus mutations that alter the af-finity in either direction could contribute to tumorigenesis.

Disruption of multiple E3 ligase components for one target:Cyclin D1/SCF E3 ligasesIn contrast to the degradation of b-catenin or NRF2, the machineryinvolved in regulating cyclin D1 abundance is complex and inter-twined, involving multiple E3 ligases that act at different cellular loca-tions and different phases of the cell cycle. Cyclin D1 is a promoter ofthe G1-S transition in cell cycle and contains a phosphodegron recog-nized by SCF E3 ligase with the FBXO4 or the CRL7 E3 ligase with theFBXW8 receptor subunits (223–225). Cyclin D1 functions in the G1

phase when bound to CDK4. In response to mitogenic signals, the cy-clin D1–CDK4 complex phosphorylates and inhibits RB, releasingtranscription factors of the E2F family and thus enabling gene expres-sion that aids progression through the G1 phase. In its active state dur-ing G1 and early S phases, cyclin D1 is in the nucleus. However, at lateS phase, cyclin D1 is phosphorylated at the C-terminal Thr286 residue,translocates to the cytoplasm, and is targeted for degradation by CRLcomplexes SCFFBXO4 and CRL7FBXW8 (223, 224). Under normal cir-cumstances, cyclin D1 has a short half-life of less than 30 min (114),which enables the reduction in abundance that occurs at the end of theS phase and is a prerequisite for cell cycle progression. After nuclearexport, cytoplasmic cyclin D1 is bound by one of three CRL ubiquitinligase complexes with different receptor subunits: FBXO4 (226),FBXW8 (224), and FBXO31 (227). The ubiquitination by SCFFBXO4

and CRL7FBXW8 is required for normal cell cycle control, with SCFFBXO4

representing the main pathway. SCFFBXO4-mediated cyclin D1 degrada-tion requires the cofactor aB crystallin (223). Degradation of cyclin D1throughCRL7FBXW8-mediated ubiquitination ismore restricted; FBXW8and cyclinD1are not usually present together inmost cells. Instead, thisE3 ligase complex is activated in response to genotoxic stress and is notrequired for normal cell cycle control (224). In addition to SCF E3ligase–mediated degradation, cyclin D1 is also degraded through aphosphorylation-independent process in which an N-terminal degronis recognized byAPC/C. Similar to CRL7FBXW8-mediated ubiquitination,the APC/C-mediated degradation pathway is activated in response toionizing radiation and other conditions that cause genotoxic stress (228).

Increased abundance of cyclin D1 occurs in breast and esophagealcancers, but direct gene amplification is responsible for a few of theseevents, suggesting that disruption of cyclin D1 degradation is theprimary mechanism underlying this protein’s contribution to tumor-igenesis (229). The complex nature of the regulation of cyclin D1 deg-radation offers multiple target sites for tumorigenic alterations thatcould promote cyclin D1 stability. Direct degron mutations are rela-tively rare, although degron mutations are connected to endometrialcancer (230, 231). Instead, the primary mechanism for E3 ligase–degron binding disruption is mutations in the SCF and CRL7 targetrecognition components. For FBXO4, most mutations affect the di-merization site (232), diminishing the ubiquitin ligase activity, whichdepends on dimerization for activity. Reduced abundance of aB crys-tallin, which is associated with breast cancer (229), impairs the inter-


action between cyclin D1 and FBXO4, thereby stabilizing cyclin D1and enhancing the progression of cells into S phase.

CyclinD1and cyclinD3are closely related,withnearly identical tissueexpression and cellular localization patterns. Furthermore, both exerttheir main function by inhibiting RB, are linked to mitogen-stimulatedactivation of the cell cycle, and are controlled through SCF- and CRL7-mediated degradation. However, the receptor subunit that recognizes cy-clin D3 is primarily FBXL2 (F-box and leucine-rich repeat protein 2)(233). Despite the similarities between the structure, function, and controlof both cyclins, their mutation patterns associated with cancer showstrikingdifferences. Insteadofmutations that primarily affect the receptorsubunit, as occurs in cancers with increased cyclin D1, for cancers asso-ciatedwith increased cyclinD3, theC-terminal degron is oftenmutated atseveral sites. In contrast to cyclin D1, the degron in cyclin D3 does notrequire phosphorylation to be functional, and its recognition by FBXL2can be disrupted equally well by mutations at many of the degron resi-dues. Furthermore, the cancers associated with these differentmutationalevents are different: Increased cyclin D1, due to mutations or changes inabundance of substrate recognition subunits, is associated with breast(234) and esophageal cancer (235), whereas increased cyclin D3, due todegron mutations, is associated with Burkitt lymphoma (236). Thisshows how the molecular mechanisms that contribute to tumori-genesis are connected to the precise molecular context of the proteinwith altered abundance. Seemingly marginal differences, such as dif-ferences in substrate recognition components, can shift the balancebetween the prevalence of tumorigenic mutations affecting degronsor mutations affecting E3 ligase subunits and accessory factors.

Disruption of one E3 ligase with multiple functionallyrelated targets: RTKs/CBLIn contrast with E3 ligases with one or a few targets (such as KEAP1 orthe SCF and CRL7 target recognition components for cyclin D1), thethree members of the CBL E3 ligase family (consisting of CBL, CBL-B,andCBL-C) have several targets with oncogenic potential. Consequently,deregulation of CBL-mediated ubiquitination provides a potential mech-anism that can lead to cancer through altered abundance ofmultiple dif-ferent oncogenic targets (237). We describe four examples of particularimportance in stomach, lung, blood cell, andbrain cancers, amongothers.

Multiple mechanisms of genetic alteration that disrupt CBL recog-nition of the receptor MET have been observed in several differenttypes of cancer. Some gastric carcinomas are associated with muta-tions around Tyr1003 in the juxtamembrane region of MET, whichis the site of CBL binding (238). Disruption of the CBL binding siteleads to prolonged half-life and increased transforming activity. Exon14 skipping in the MET gene commonly occurs in lung cancers andalso disrupts CBL recognition, thereby stabilizing MET (239, 240).Another oncogenic form of MET that lacks the CBL binding site isTPR (translocated promoter region)–MET,which is generated througha chromosomal translocation that results in combining the dimeri-zation domain of TPR (a scaffolding element of the nuclear porecomplex) with the kinase domain of MET. The resulting fusion pro-tein, which is detected in some gastric cancers, is constitutively activeand lacks the binding site for CBL and thus is not targeted for deg-radation (238, 241, 242).

The juxtamembrane region of the receptor KIT (mast/stem cellgrowth factor receptor) also contains a binding site to which CBL isrecruited, either directly or through adaptor proteins. This region isfrequently mutated or deleted most frequently inmelanomas and gas-trointestinal stromal tumors and enhances the transforming activity of

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KIT (237). Mutation in this region impairs not only degradation butalso an autoinhibitory function of the receptor (243).

The oncogenic potential of CSF-1R results from the loss ormutationof the CBL binding site (244). The CBL binding site is a phosphodegronat the C-terminal region containing Tyr969, which is autophospho-rylated upon activation of the receptor. BecauseCSF-1Rplays an essentialrole in the regulation of survival, proliferation, and differentiation ofmac-rophage andmonocyte precursors, it is not surprising thatmutationsof Tyr969 are frequently observed in human myelodysplasia andAML (245).

Some glioblastomas havemutations that ablate regions in EGFR thatare involved in CBL binding. Although mutation of the degron recog-nized by CBL is rare, several types of genetic alterations can interferewith CBL-dependent down-regulation of EGFR. Truncated versionsof EGFR lacking the binding site for TKB in the EGFR C-terminalregion (246) and deletion of exons 2 to 7, which encode parts of theextracellular domain of EGFR, are observed in some glioblastomas.In both cases, the mutant protein does not effectively trigger CBL-dependent down-regulation. For the receptor lacking part of theextracellular domain, although the receptor has less autophospho-rylation, which is the mechanism that compromises CBL recognition,the receptor is still tumorigenic despite its decreased activity (246).CBL-mediated destruction of EGFR can also be impaired by increasedabundance of other members of the EGFR family. When humanepidermal growth factor receptor 2 (HER2) is in excess, as occurs fre-quently in many cancers, including breast, ovary, prostate, and braintumors, the increased HER2 shifts the balance between EGFR homo-dimers and HER2:EGFR heterodimers toward heterodimers, whichare not recognized as efficiently byCBL as EGFRhomodimers, leadingto increased EGFR signaling (237).

Disruption of a single E3 ligase with functionally diversetargets: SCFFBXW7

Although SCF with the FBXW7 receptor subunit is the primary E3ligase of NOTCH1, this E3 ligase has many other targets. WhereasNOTCH1 and FBXW7 alterations represent two sides of the same tu-morigenic story, dysfunction of FBXW7 has far-reaching effect. Othercancer-associated proteins are targeted for degradation by SCFFBXW7,including the transcription factors MYC and KLF5 (Kruppel-likefactor 5). Chromosomal translocation ofMYC to one of the immuno-globulin or T cell receptor loci may be one of the primary events inmalignant transformation in Burkitt lymphomas (247). However, thisevent is often accompanied by additional hotspot mutations, includ-ing mutations at Thr58 in the phosphodegron of the MYC part of thefusion protein (248, 249). Phosphorylation of this residue is requiredfor FBXW7-dependent ubiquitination and degradation of MYC andthe fusion protein (250). Thus, mutation of this residue stabilizes theprotein and contributes to tumorigenesis, a similar effect of MYCamplification and overexpression that is frequently observed in othercancer types (251). KLF5 is another transcription factor that is regu-lated by GSK-3b–mediated phosphorylation of a phosphodegron andFBXW7-mediated degradation. Once again, mutations of the criticalresidues of the phosphodegron motif were observed in colon cancersamples, as well as other carcinomas, and these mutations increasedthe KLF5 half-life and transcriptional activity (252, 253).

Large-scale cancer genome studies have revealed that FBXW7is one of the most commonly mutated cancer genes. In addition toT-ALL, FBXW7 mutations were observed in diverse human cancertypes, including colorectal adenocarcinoma, uterine carcinosarcoma,


uterine endometrial carcinoma, and bladder carcinoma (190). The on-cogenic potential of FBXW7 substrates and frequent allelic loss in hu-man cancers, which is supported by mouse models, indicate thatFBXW7 can function as a tumor suppressor in many human cancers(190, 254). However, the mutational spectra also revealed “hotspots”in FBXW7 mutations. A large number of these variations causedamino acid substitutions in three arginine residues that form criticalcontacts with the phosphodegron motif and are required for high-affinity interactions with the substrates (65, 190, 255). Most tumorswith these mutations retain a normal second FBXW7 allele. This isuncommon for most tumor suppressors, for which both alleles typi-cally need to be inactivated or produce dysfunctional protein. Thepresence of one mutant and one normal FBXW7 allele may suggesta dominant-negative mechanism of action of the mutant protein ormay indicate that some residual FBXW7 function is necessary for cellsurvival. Other mutational hotspots outside the region involved indegron recognition are isoform-specific and likely influence specificsubcellular localization (256). FBXW7 function can also be impairedby oncogenic microRNAs or by various mechanisms that reduceFBXW7 expression, for example, through promoter hypermethyla-tion (190). Regulation of FBXW7 function can also be perturbed incancer by changes that affect its degradation or phosphorylation-dependent heterodimerization of various FBXW7 isoforms. Overall,FBXW7 has a complex cancer-associated pattern of genetic altera-tions affecting the FBXW7 gene and those encoding other partnersor regulators. This pattern depends on the tissue where the canceroriginated and the mutational mechanisms. This way tumorigenesisoccurs through various molecular events that depend on the exactalterations of FBXW7, presumably through differential effects onspecific substrates (190).

Disruption of UPS function by pathogenic interferenceIn all examples discussed so far, the mechanistic background of tu-morigenic processes has been the accumulation of mutational or ep-igenetic changes to the cellular genome that affect the degradationprocess. However, these alterations in UPS function and protein sta-bility are often modulated in a way that confers advantage on cancerformation or growth in the absence of genetic or epigenetic alterations.These modulations involve interactions between elements of the UPSand either pathogenic proteins or pathogen-induced alteration of UPSfunction. Although a connection between bacterial infection andaltered UPS function has not yet been reported, many viruses asso-ciated with cancer interfere with the UPS. The relevance of this tocancer is evidenced by viruses being collectively responsible for~16% of worldwide cancer incidence (257).

To date, seven human oncoviruses have been identified: Epstein-Barr virus (EBV), hepatitis B virus (HBV), human T-lymphotropicvirus 1 (HTLV I), human papillomaviruses (HPV16 and HPV18),hepatitis C virus (HCV), Kaposi sarcoma-associated herpesvirus(KSHV), and Merkel cell polyomavirus (MCV) (258). All seven ofthem use various strategies of interfering with certain degradationevents of the host cell (258, 259). Similar to other viruses, oncovirusesmodulate the host UPS (258) during viral entry and early after viralentry into the cell (260), as well as during other steps of viral life cycle(261, 262). Although the molecular details of the host-pathogen in-teractions vary among the various viruses, the main human path-ways important for cancer that are virally targeted are the p53pathway (to suppress apoptosis by down-regulating p53), the Rb/E2F pathway (to induce entry into the cell cycle by activating E2F

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proteins), NF-kB pathway (to modulate the immune response), andthe Wnt pathway (to enhance proliferation and tissue regenerationthrough b-catenin activation). Viruses modulate the host cellsignaling at these points to overcome cellular senescence and to forcethe cell to express and activate genes required for progressionthrough the cell cycle so that the virus can exploit the host DNA rep-lication machinery.

Although the main target host pathways are similar for differentviruses, the oncoviruses use divergentmolecular mechanisms to inter-fere with these common pathways. Both HPV and EBV interfere withp53 activity by promoting its degradation, but the detailed mecha-nisms differ. The HPV protein E6 interacts with the HECT-type hostE3 ligase E6AP, thereby promoting p53 degradation (263, 264). EBVtargets a different element of the host UPS to achieve the same effect.USP7 is a deubiquitinase that stabilizes p53 by removing ubiquitinfrom p53, counteracting the MDM2-mediated degradation. TheEBVnuclear antigenEBNA1bindsUSP7, inhibiting its deubiquitinaseactivity and preventing it frombinding to p53 (265), therefore enhanc-ing p53 degradation.

Viruses typically modulate the RB/E2F pathway by disrupting theinteraction betweenRB and the transcription factors of the E2F family;RB maintains E2Fs in an inactive state. Many viruses produce a pro-tein that binds RB, leaving the E2Fs free to drive transcription of genesneeded during the cell cycle. The HPV E7 protein binds not only RBbut also the CULLIN2 E3 ligase, promoting RB degradation (266).

Like cancer-causing geneticmutations that disrupt the b-catenin de-gron, viral oncogenesis also targets Wnt signaling through b-catenin.Increased b-catenin stability is achieved by HBV through the viral Xprotein (Hbx), which activates the kinase Src, which, in turn, suppressesGSK-3b activity (267) and thus prevents b-catenin phosphorylation andsubsequent E3 ligase recognition and targeting for degradation. KSHValso targets GSK-3b through the viral latency-associated nuclear anti-gen, which sequesters GSK-3b in the nucleus (268). HCV also targetsb-catenin activity by activating the kinase AKT, which phosphorylatesb-catenin on Ser552 and enhances transcriptional activity. Furthermore,patientswith liver cancer and underlyingHCV infection exhibit a higherrate of mutation of b-catenin. Activation of AKT by the HCV non-structural proteinNS5A increased the transcriptional activity ofb-cateninand also stabilized the protein through unknown mechanisms (269).

The main oncogenic effect of b-catenin stabilization is the up-regulation of the expression of b-catenin target genes,most notably thegene encoding MYC. MCV enhances MYC abundance through ab-catenin–independent mechanism by impairing its degradation.MCV has two main oncoproteins: the large T antigen (LT) and thesmall T antigen (sT). LT targets the RB/E2F interactions to promotethe cell cycle (270). However, this effect is counteracted by the host bytargeting LT for degradation by the SCFFBXW7E3 ligase. Viral sT coun-teracts this protective mechanism by inhibiting SCFFBXW7, therebypreventing LT degradation. Because MYC is also targeted for degra-dation by SCFFBXW7, sT also triggers a pronounced increase in MYCstability (271).

In addition to pathways common tomost oncoviruses, some virusesalter other aspects of UPS function, producing additional oncogenicconsequences. HPV E5 protein blocks the interaction between EGFRand CBL, suppressing EGFR ubiquitination (272). The increased EGFRactivity contributes to uncontrolled cell division. Apart from interferingwith the host UPS machinery, some viruses also encode proteins withubiquitin ligase or deubiquitinase function. These include the K3 andK5 E3 ligases of KSHV, which interfere with antigen presentation


(273), and the BPLF1 deubiquitinase of EBV, which enhances the pro-duction of infectious particles (274). Viruses may also alter APC/Cfunction through as yet undefined mechanisms with unknown physio-logical consequences (275).

Multiple degron mutations are found in viral and cellular proteinsin EBV-induced cancers. Under sustained exposure to pathogen orexposure to environmental carcinogens, EBV-infected cells can be-come cancerous. EBV is a causative agent for two cancers: Burkitt lym-phoma in endemic malaria zones and nasopharyngeal carcinoma(NPC), mainly in southern parts of China (276). In addition, EBVis often a cofactor in other leukemias, such as Hodgkin’s lymphoma.MYC is frequently translocated in Burkitt lymphoma, and the pointmutations resulting in T58N and S62P substitutions were identified inthe sequences of translocatedMYC of culturedBurkitt lymphoma cells(277). These mutated residues are key determinants of the phospho-degron recognized by FBXW7 and contribute to the stability of aber-rant MYC in the lymphoma cells (278). The EBV latent membraneprotein 1 (LMP1) also has phosphodegron mutations that are impor-tant for driving cancer. In this case, a pair of phosphodegrons recog-nized by b-TrCP act as pseudosubstrates because LMP1 lacks Lysresidues that can be ubiquitinated, resulting in sequestration of E3 li-gases with the b-TrCP receptor subunit. Both degrons are frequentlymutated in NPC, enabling SCFb-TrCP to target IkB for degradation,which then stimulates NF-kB proliferative signaling (279). Thus,cancer-driving mutations in degrons are common features of EBV-induced cancers.

Tumorigenesis: Somewhere Between Neutralityand LethalityThe cancer-related changes in protein degradation are between neutralaberrations (a change with little or no phenotypic effect) and lethal aber-ration (a change that causes death). The effect of the enhancing or im-pairing the degradation of a gene product depends on the gene’s functionand the function of the genes downstream of it. Neither ubiquitin itselfnor either of the two known human E1 ubiquitin–activating enzymes aremutated in cancer. This is not surprising because those mutations wouldimpair the whole UPS and are likely lethal aberrations. For similar rea-sons, with the exception of UBE2Q1 (E2 ubiquitin–conjugating enzymeQ1), E2 enzymes are generally not implicated in cancer either becausetheir inactivation would affect hundreds or even thousands of proteins.Differential expression of UBE2Q1 has been linked to cancer, with somecolorectal cancer cells showing overexpression in both the transcript andthe protein (280). This is likely possible because UBE2Q1 presumablyfunctions with only two E3 ligases, STUB1 and RNF5 (as assessed by in-teraction data in the STRING database). In contrast to the limited linksbetween cancer and E2 enzymes, many E3 ligases and their targets areencoded by cancer-driving genes.

Analyzing the link between cancer mutations and various degrada-tion processes uncovers a wide range of possible effects for tumorigenicalterations. In general, germlinemutations have a lower impact becausethesemutations are present in all cells and the organismmust be able toadapt to the mutations to develop and survive. Because somatic muta-tions occur in a limited number of cells, they can cause a greater rewiringordisruptionof signalingmechanisms.Anexample of germlinemodula-tion of the degradation of a cancer-driving gene is the gene encodingVHL protein (VHL). VHL is the receptor subunit of an E3 ligase com-plex that contains Elongin B, Elongin C, and CULLIN2 (named ECV)(Fig. 2). ECV targets a relatively small number of proteins for degradation,

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most notably the hypoxia-inducible factor HIF-1a. Patients sufferingfrom the VHL syndrome carry inherited mutations on one allele ofVHL and thus have a predisposition for developing benign or canceroustumors, includinghemangioblastoma, clear-cell renal carcinoma (ccRCC),pancreatic tumors, and pheochromocytoma (PCC) (281). However,monoallelicVHL inactivation has amild effect, and both benign tumor-igenesis and cancerous tumorigenesis require the inactivation of theother allele or a secondary mutation in another gene. Biallelic VHL in-activation impairs HIF-1a degradation, enabling HIF-1a to induce an-giogenesis,which confers adistinct advantage for tumorgrowth.However,cancer does not always result from biallelic VHL inactivation andincreased HIF-1a activity. Often, additional mutations of other genesare required (282), showing that cancer development connected toVHL inactivation is a multistep process involving the additive effect ofmutations that each carry a small advantage toward tumorigenesis (58).

In contrast to germline mutations, somatic mutations only exerttheir effects in the cells forming the tumor and can havemore aggressivephenotypic results. This is the domain of most known cancer genes.Similar to cancer-associated germline mutations, cooperativity amonggenetic and epigenetic aberrations is a hallmark of cancer-related mod-ulations across genes harboring somatic mutations. However, somati-cally targeted genes reveal the balance between tumorigenesis andlethality. One such example is EGFR, which is activated by variousgrowth factors (most notably EGF and TGF-a) that induce EGFR di-merization, activation, and downstream signaling that promotes cellproliferation. Accordingly, increased EGFR abundance and activityare linked to various forms of cancer, including squamous cell carcino-ma of the lungs, glioblastoma, and head and neck carcinoma. Becausethe result of increased abundance due to gene amplification or impaireddestruction is similar, EGFR degron mutations linked to cancer wouldcome as no surprise; however, they are not common. EGFR is targetedby two isoforms of the E3 ligase CBL through distinct mechanisms(283). Although activatingmutations in the intracellular kinase domainof EGFR are very common in cancer, the degrons are almost nevermu-tated, suggesting that the effect of gene amplification and the inhibitionof degradation are different for the cell. EGFR is recycled through in-ternalization, and the fate of activated EGFR is decided at the early en-dosomal stage:Ubiquitination byCBL targets the receptor for lysosomaldegradation; otherwise, EGFR recycles to the plasmamembrane. EGFRactivity resulting from gene amplification is at least partially counter-acted by internalization and subsequent degradation. There is no apparentbalancing mechanism for impaired degradation. The lack of degronmutations suggest that EGFR overactivation balances on the verge ofcancer and lethality, with gene amplification and kinase-activating mu-tations falling on the viable side and degronmutations on the lethal side.

Oncogenes and tumor suppressors in the UPSGenes implicated in cancer are usually classified either as (proto)oncogenes or as tumor suppressors. Oncogenes are genes with apotential to promote tumorigenesis such that their (over)activationconfers advantages for cancer formation, growth, metastasis, or all three.Tumor suppressors are genes that protect the cell from tumorigenesis,typically by counteracting the functions and activity of oncogenes. Thus,oncogenes are typically overexpressed or their encoded products areoveractivated in cancer, whereas either tumor suppressors are lessexpressed or the encoded products are inactivated. Accordingly, pro-teins harboring degron mutations have disrupted degradation and aregenerally expected to be oncogenic, and their corresponding E3 ligasesare presumably tumor suppressors. Whereas there are some relatively


clear-cut examples of oncogenes and tumor suppressors among E3ligases and their targets, in certain cases, single proteins cannot beassigned to either category. Although the hundreds of known sub-strate recognitionmodules of E3 ligases are highly specific,most of themrecognize more than one target protein. The biological effect resultingfrom the decrease or loss of function of these ligases emerges throughthe functions of its substrates. VHL can be categorized clearly as a tumorsuppressor because it has a very limited number of targets, most ofwhich perform functions that confer advantages to tumorigenesis.Many other ligases have targets that are either oncogenic or tumor sup-pressors. The two-hit model for tumor suppressors is inadequate, andmany other mechanistic models have been proposed (284, 285) thatmove away fromconsidering separate allelic aberrations as distinct stepstoward a continuum model of tumor suppression. Although thesemodels cover a considerably larger spectrumof gene perturbations, theyare unable to correctly describe genes with dual oncogenic and tumorsuppressor functions. For many elements of the UPS, the classical rolesand categories used for cancer genes need to be reassessed.

Defying classification as an oncogene or tumor suppressorFor typical oncogenes, such as EGFR, enhanced activity promotescancer, but reduced activity does not cause cancer (286) and, to a cer-tain extent, does not threaten viability, making EGFR a typical on-cogene. However, this asymmetry in the physiological response toabnormal amounts of a protein is not universal. For many proteins,especially E3 ligases and their targets that control the expression ofa large number of genes (usually through transactivation), tippingthe delicate scale of normal abundance in either direction can havedetrimental consequences, although the change may not result in can-cer. For example, by inhibiting several cyclin/CDK systems, cyclin-dependent kinase inhibitor 1C (p57KIP2) inhibits many hallmarks ofcancer, including cell invasion, metastasis, tumor differentiation, andangiogenesis. This classifies p57KIP2 as a tumor suppressor (287). Ac-cordingly, the p57KIP2 degron targeted by the SCFSKP2 E3 ligase (113)is generally not mutated in cancer; rather, expression of the gene en-coding p57Kip2 is down-regulated through epigenetic changes, suchas DNA methylation and repressive histone marks at the promoter(288). However, mutations in the degron and the corresponding in-crease in p57KIP2 result in other diseases, collectively called IMAGesyndrome, which is characterized by intrauterine growth restriction,metaphyseal dysplasia, congenital adrenal hypoplasia, and genitalanomalies (289). Thus, deviation from the normal p57KIP2 abundancein either direction promotes different disease states.

In certain other components of the UPS, however, any deviation inabundance or function can promote tumorigenesis. Exhibiting eitheroncogenic or tumor suppressor effects in response to up- or down-regulation, respectively, is characteristic of many UPS componentsat the E3 level. Examples include COP1 and E3 ligase complexes withSPOP receptor subunits. For example, although the E3 ligase COP1mainly targets oncogenic substrates, such as JUN and members ofthe ETV family, COP1 also targets p53 in an MDM2-independentmanner (290). Consequently, both the increase and the decrease ofCOP1 abundance are observed in cancer. The possibility of a clear cat-egorization of COP1 is hindered not only by its mixed targets but alsoby the complexity of its mechanisms of action as well. Oncogenic tar-gets are recognized by COP1 as part of the CULLIN4-DDB1-RBX1-DET1 E3 complex (291). COP1 mediates p53 ubiquitination as anindividualE3 ligase and also acts as anMDM2cofactor, thereby enhanc-ing p53 degradation (292). To add an additional layer of complexity,

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COP1 also promotes its own degradation in response to UV radiation(293). The different substrates (oncogene or tumor suppressor) are alsoreflected in whether a cancer exhibits an increase (breast, ovarian, liver,and pancreatic cancers) or a decrease (prostate cancers, AML, andmel-anoma) in COP1 (294, 295).

The CRL3E3 ligase containing SPOP acts as a tumor suppressor bypromoting the degradation of several oncogenes, including ERG andCDC20 (296), an activator of the APC/C E3 ligase. Consequently, mu-tations that inactivate SPOP function are present in 15% of prostatecancers (193). Increased SPOP is associated with other forms ofcancer, most notably ccRCC. The mechanism by which an increasein SPOP contributes to tumorigenesis is different from themechanismby which SPOP inactivation leads to cancer (297). The gene encodingSPOP is stimulated by HIF-1a. Thus, hypoxia or mutations that in-activate ECV, the E3 ligase that targets HIF-1a (mutations in the re-ceptor subunit VHL are common in ccRCC), increase the abundanceof SPOP. By targeting several tumor suppressors, such as the phospha-tase PTEN, ERK phosphatase, and transcription factors such asDAXX and GLI2, the increase in SPOP impairs apoptosis and pro-motes cell proliferation (297). Thus, E3 ligases with both oncogenicand tumor suppressor targets can promote cancer development whenthe abundance of the E3 ligase deviates from normal, independentlyfrom the direction of the deviation.

The ambiguous nature of many E3 ligases manifests in their actingas oncogenes or tumor suppressors in response to different alterationsin their abundance. However, in other cases, the discriminating featurebetween oncogenic and tumor suppressor behavior for an E3 ligase isnot the direction of shift in protein abundance but the cellular context.Genes encoding proteins related to certain biological processes canswitch between the two states based on factors other than abundance,such as stage of the cancer or the cell type fromwhich the cancer arises.

A prime example of such a system is NRF2 and its correspondingE3 ligase, which contains the receptor subunit KEAP1. Under normalconditions, NRF2 is kept inactive and targeted for degradation byKEAP1. Exposure or the accumulation of highly oxidative molecules,such as reactive oxygen species (ROS), deactivates KEAP1, inhibitingNRF2 degradation (298). NRF2 induces gene expression throughAREs or electrophile response elements, which encode proteins thatcounteract cellular oxidative stress. NRF2-induced gene products notonly detoxify endogenous oxidative agents but also prevent oxidativecarcinogens from interacting with vital biomolecules, such as DNA,RNA, and proteins, and prevent the accumulation of reactive speciesthat result from cellularmetabolism. Accordingly,NRF2-null mice areprone to tumor formation, showing that, in normal and premalignantcells, NRF2 activity has an antitumorigenic effect. The concept ofmany chemopreventive agents is enhancement of this oxidative stresspathway (299, 300). On the other hand, NRF2 and KEAP1 mutationsthat keep NRF2 constitutively active are often found in various formsof cancer (175, 301). The reason behind this seemingly paradoxical be-havior is that increased resistance to ROS offers a survival advantagefor tumor cells and confers resistance to some forms of chemotherapy,decreasing the efficacy of chemotherapy in NRF2–up-regulatedcancers (302). A protein’s oncogenic or tumor suppressor status notonly can change with the progression of malignancy but also can de-pend on cell or tissue type. This dependence is evident withNOTCH1, which is up-regulated through impaired degradation inCLL (185). As opposed to this oncogenic role, in certain tissues(and, correspondingly, certain cancer types), NOTCH1 acts as a tu-mor suppressor by promoting cell cycle arrest through stimulation


of expression of the cell cycle inhibitor p21 (303). Although less fre-quent than the occurrence of degron-eliminating truncation muta-tions in CLL, tumor suppressor roles of NOTCH1 have beenreported in basal cell carcinoma (304, 305), lung squamous cell carci-noma (306), and small cell lung cancer (307).

This type of context dependence is not only characteristic ofNOTCH1,but the transcription factor Krüppel-like factor 4 (KLF4) also exhibitscell type–dependent oncogenic or tumor suppressor roles. KLF4 is a keyregulator of the cell cycle, apoptosis, differentiation, and stem cell re-newal (308). KLF4 is one of few transcription factors that orchestratethe reprogramming of somatic cells into induced pluripotent stem cells(309). KLF4 plays both tumor suppressor and tumor-promoting rolesin cancer as a result of its wide range of targets and its function in severalkey pathways important for maintaining control over cell proliferativepotential. Tumor suppressor functions of KLF4 relate to its ability toinduce expression of genes encoding the CDK inhibitors p21WAF1 andp27KIP1 and to reduce expression of the genes encoding cyclin D1, cyclinB, and FOXM1 (310–312), negatively regulating proliferation in gastriccancer, bladder cancer, lymphoma, and lung carcinomas, among others(313–316). Tumor-promoting activity arises from the ability of KLF4 torepress the expression of the gene encoding p53 (317) and, consequently,reduce expression of p53 target genes encoding proapoptotic proteins,such as BAX (318). The oncogenic activity of KLF4 has been reported inbreast cancer and squamous cell carcinoma (317, 319). BecauseKLF4 andits regulatory factors are distributed unevenly across various cell types, thedominant function of KLF4 is tissue-specific. Consequently, aberrantalterations in KLF4 abundance can either lead to tumorigenesis or tumorsuppression, depending on the tissue and the direction in the shift inabundance (320). One of the main mechanisms through which KLF4abundance is altered in cancer is through the modulation of KLF4 deg-radation by one of its degrons. KLF4 is targeted by at least three differentE3 components of theUPS:VHL,APCCDH1, and SCFb-TrCP.Degradationby VHL plays a role in colorectal cancer: Mutations that increase VHLabundance promotes KLF4 degradation, indicating that KLF4 functionsas a tumor suppressor role in this context (321). This highly specificscenario linked to one cancer type results from not only the tumor sup-pressor role of KLF4 but also a switch by VHL from its classical tumorsuppressor role to an oncogenic role. This oncogenic role of VHL is op-posite its classical tumor suppressor function observed in most othercancers associatedwith alteredVHL function, such as renal cell carcinoma(RCC) (322).

The oncogenic role of VHL in certain forms of colorectal cancer isan exceptional deviation from its otherwise universal tumor suppres-sor function. However, there are other less obvious context-dependentfeatures of VHL mutations. Cancerous manifestations of the VHLsyndrome often present as multiple tumors in various tissues, includ-ing RCC, retinal hemangioblastoma, pancreatic neuroendocrine tu-mors, and PCC. On the basis of the presence or absence of thesetumor types, VHL diseases are clustered into phenotypic categories(323). The VHL syndrome subtypes reflecting tissues of tumor occur-rence correlate with certain genotypic features. For example, mostpatients with truncating VHL mutations or exon deletions do not de-velop PCCs (and are classified as type 1), likely because complete VHLinactivation is lethal for PCC cells. In contrast, germline mutations inVHL generally lead to PCC development. These characteristic muta-tion patterns correlate not only with cancer and tissue type but alsowith the molecular details that result from the impaired degradation,most notably the balance between the impairment of HIF-dependentand HIF-independent processes.

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In general, both oncogenes and tumor suppressors in the UPS canexhibit context dependence at various levels. However, there is an-other group of proteins linked to cancer development that does notfit into either category. These proteins, which are encoded by genescollectively termed nononcogene addiction (NOA) genes (324), arenot tumorigenic themselves but are necessary for sustaining the tu-morigenic state. The function of proteins encoded by NOA genes isusually tissue type– and cancer type–specific. A NOA gene encodinga component of theUPS isRNF4, a RING-type E3 ubiquitin ligase. Inits canonical function, RNF4 targets polysumoylated proteins (in-cluding PML, PEA3, CENPI, EPAS1, and PARP1) for degradation,controlling chromosome alignment and spindle assembly (325).This function is not directly related to cancer development, andRNF4 is usually not altered (neither genetically nor epigenetically)in cancer. However, RNF4 can also recognize phosphodegrons inproteins such as b-catenin and MYC and polyubiquitinates thesesubstrates (326). RNF4-mediated polyubiquitination does not targetthe proteins for degradation; instead, the substrates are stabilized. Incontrast to the regular K48-linked ubiquitination, RNF4 catalyzesthe formation of K11- and K33-linked polyubiquitin chains, whichare not compatible with the UPS degradation pathway (327).Through this mechanism, RNF4 stabilizes a number of oncogenesby translating a transient signal (phosphorylation) into long-termstability, enhancing the tumor phenotype, and in certain tissues, thiseffect is essential for cancer cell survival (326). Thus, RNF4 repre-sents a distinct class of UPS components that contribute to tumori-genesis solely through the change in their context (in this case, thetargeted proteins) without involving a mutation, epigenetic altera-tion, or change in abundance. This also shows that even the mostbasic elements of the UPS, such as degrons or polyubiquitination,can have context-dependent meanings and functions in cancer, inthis case, changing from the canonical “degradation” role to a detri-mental “stabilization” role.

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Implications for Drug DesignBecause the UPS regulates the stability and functions of several cancer-associated proteins, this system provides attractive opportunities fortherapeutic interventions. The potential success of this approach inthe treatment of cancer was first highlighted by the proteasome inhib-itor, bortezomib, which has developed into an important treatment formultiple myeloma and mantle cell lymphoma (328). The proteasomefeatures three dominant proteolytic activities, the trypsin-like, thechymotrypsin-like, and the peptidyl-glutamyl-peptide–cleaving (acidic)activity. Bortezomib inhibits the chymotrypsin-like activity by re-versibly binding to the b5 subunit of the 20S proteasome (Fig. 8A). Sur-prisingly, inhibition of the proteasomal activity showed selectivecytotoxicity to certain myeloma and lymphoma cells. This is largelyattributed to the increased proteotoxic stress that characterizes tumorcells because of their higher proliferation and rapid growth rates. Manypatients can benefit from treatmentwith bortezomib, but the side effectscan be dose- or treatment-limiting (329). Inhibiting the proteasome ishitting the most central and least specific target in the protein degrada-tion machinery; thus, it is perhaps not surprising that treatment is notfeasible for most other tumor types, notably solid tumors (329).

Specific inhibitors have also been developed to inhibit theUPS at thelevel of E1 enzymes. These include PYR-41 and PYZD-4409, the firstcell-permeable compounds that are currently in preclinical develop-ment stage. Their therapeutic potential originates from their ability to


inhibit the catalytic activity of UBE1, the main ubiquitin-activating en-zyme in humans (330). However, similar to proteasome inhibitors, thesedrugs affect many substrates, which could limit their therapeutic value.Neddylation is a largely similar process to ubiquitination, withNEDD8-activating enzyme (NAE) playing a largely equivalent role to E1 activatingenzymes (331). The small molecule, MLN4924, a potent and selectiveinhibitor of NAE, acts as an indirect inhibitor of Cullin-RING ligases byblocking neddylation that is essential for their activation.MLN4924wasshown to have antitumor activities and is currently undergoing phase 2clinical trials (332).

Because of their abundance, diversity, and potential for selectivity,the E3 ligase–substrate pairs offer the most targeted approach to ther-apeutically altering protein abundance at the levels of single or sets ofprotein substrates. However, current drug design strategies target re-latively small, deep pockets, such as the active site of enzymes. This is apractical approach to drug design of enzymatically active proteinsbecause the active site can be readily targeted by small molecules. Incontrast, the action of E3 ligases primarily involves protein-proteininteractions that, for many years, have been regarded as practicallyundruggable (333, 334). Results that indicate that protein-proteininteractions can be effectively targeted and have shown promise fordirect regulation of protein abundance by targeting the activity of sub-strate recognition of E3 ligases have started to emerge.

A prime example of drugs that directly target an E3 ligase–substrateinteraction involves MDM2 and p53. The detailed knowledge of thisinterface (Fig. 3A), together with high-throughput small-moleculescreening, led to the discovery of nutlins, a group of small moleculesthat can block theMDM2binding site bymimicking the peptidemotifof p53 (Fig. 8B) (335, 336). Nutlins stabilize p53, leading to the acti-vation of cell cycle arrest and apoptosis pathways. Although nutlinsand relatedmolecules do not fit into the binding pocket as well as naturalligands, they still were effective competitive inhibitors for p53 (335).Their competitive binding arises because interactions of native degronmotifs involve a disorder-to-order transition that entails a large entropiccost, resulting in a relatively weak overall binding (337). This concept isexploited in the design of stapled peptides, in which synthetic a-helicalpeptides corresponding to the native bindingmotif are stabilized throughcovalent linkage (338). Stapled helices not only ensure that the structureand biological activity aremaintained but also enable the engineering ofleadmolecules with optimized pharmacokinetic properties. In addition,stapled helices also bindMDM4, an additional E3 ligase that targets p53,which could be important for treating a broader range of cancers thanthose currently treated with nutlins (339). These and several other newclasses of small inhibitors targeting the MDM2-p53 binding interface orinterferingwith this interaction in otherways are undergoing clinical trialsfor the treatment of various solid and hematological cancers (340, 341).

The other twomembers of the p53 family, p63 and p73, are targetedfor degradation by the E3 ligase ITCH (330). Because p63 and p73 sharesome of the functions of p53, blocking their degradation may be apromising therapeutic strategy, and cyclic peptides that interfere withthe binding of ITCH to these targets have been identified (342).

Smallmolecules or bicyclic peptides that inhibit the activity ofmem-bers of the HECT family of E3 ligases have also been identified ordeveloped. Rather than interfering with substrate recognition, theseinhibitors obstructed the E2 binding site specifically for individual HECTligases or worked as allosteric inhibitors with a broader specificity formultiplemembers of theHECT family (343). Another attractive E3 ligasecomponent that could be exploited for cancer therapy is SKP2.Among itssubstrates, p27KIP1 is themost relevant to the oncogenic function of SKP2

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(344). The structure of the complex (Fig. 3B) (52) formed between thedegron motif of p27KIP1, and the SCFSKP2 complex revealed severalpockets that could be targeted by small molecules. This structuralinsight led to the identification of compounds that block various inter-faces formed between SKP2 and other components or accessory pro-teins of the E3 ligase complex (SKP2 with SKP1 or SKP2 with CKS1)or with the substrate (SKP2/p27KIP1) (330, 345).

Inhibition of E3 ligase activity by directly targeting either the en-zyme itself or its substrate recognition is only potentially useful incancers, or other diseases, involving excessive substrate degradation.In many cases, however, the disease state originates from the oppositescenario: insufficient ubiquitination-dependent degradation to main-tain appropriate protein abundance either through impaired E3 ligasefunction or through increased production of the substrate. These casespresent more challenges than E3 ligase inhibition because they requirerestoring the function of a defective E3 ligase or retargeting E3 ligasestoward substrates that they do not normally recognize. An excitingnew approach to retarget E3 ligase activity toward novel substrates isbased on PROTACs (proteolysis targeting chimeras) (346). This


involves chimericmolecules that combinea degron-mimicking compound to recruitan E3 ligase coupled to another com-pound to recruit a specific target. Theseconstructs effectively tether the ligase tothe substrate, inducing its ubiquitinationand degradation. The viability of this con-cept was demonstrated by combining thedegron of IkBa (nuclear factor of k lightpolypeptide gene enhancer inB cells inhib-itor a) with ovacilin, a drug that covalentlybinds to methionine aminopeptidase 2(METAP2). The degron was recognizedby b-TrCP, which targeted METAP2 forproteasomal degradation. PROTAC tech-nology has already been successfully ap-plied to various E3 ligases in preclinicalexperiments (347, 348).

A remarkable story of retargeting anE3 ligase combined with drug repurposingis provided by thalidomide derivativesbinding to the protein cereblon (CRBN).CRBN is an unusual receptor subunit of aDCX (CUL4-DDB1-RBX1-CRBN) E3ligase complex. CRBN has a deep hydro-phobic pocket that is the ligand bindingsite for thalidomide (Fig. 8C), the terato-genic molecule that has been repurposedin lymphoma and leprosy treatments(349, 350). Binding of thalidomide andmore recently derivedCRBN-binding “im-munomodulatory” molecules, such aslenalidomide and pomalidomide, switchesthe ligand binding preference of CRBNfrom substrates such as MEIS2 to sub-strates such as the hematopoietic transcrip-tion factors Ikaros and Aiolos, therebyreducing cell division (351). Lenalidomide-bound CRBN also targets the E3 ligase toCK1a, which contributes to the clinical ef-

ficacy of this drug in 5q-deletion associated myelodysplastic syndrome[del(5q) MDS] (352). An additional immunomodulatory molecule,CC-885, was shown to have an antitumor activity by inducing theCRBN-dependent degradation of the translation termination factorGSTP1 (353). Structural analysis revealed degrons with a b-hairpin loopin each of GSPT1 and CK1a (Fig. 8D) (352, 353), and degradation ofthese proteins depends on the binding interface provided by lenalido-mide bound to CRBN.

The activity of the CRBN-containing E3 ligase is likely modulatedby at least one natural cellular molecule that thalidomide mimics.Uridine binds in the pocket and may be one natural modulator, al-though towhat targets uridine directsCRBNorhowuridine affectsCRBNfunction is not known (354). In plants, the concept of endogenoussmall-molecule regulation of substrate receptor subunits of E3 ligasesis well established. The plant hormone auxin induces the degradationof the Aux/IAA family of transcriptional repressors by binding to theF-box component TIR1 of a multisubunit E3 ubiquitin ligase (355).Currently, there are no solved structures for CRBN in complex with alinear motif degron, with or without any small-molecule modulators.

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Fig. 8. Complexes of docked drugs. (A) Proteasome:bortezomib (whole proteasome and outtake box of the boundbortezomib). The structured is rendered using PDB: 5LF3. Inset shows drug in binding pocket of one of the proteasomesubunit (H bonds colored magenta). Proteasome surface of a subunits is colored in different shades of blue, whereassurface corresponding to b subunits is colored gray. (B) MDM2–Nutlin-3A structure (nutlinmolecule is drawn in stick andMDM2 in surface representation). Binding of Nutlin-3A on MDM2 blocks degron interaction interface on it. (C) Thalid-omide (analog)–lenalidomide in complex with cereblon. Lenalidomide (represented in stick with cyan color) is bound inthe deep hydrophobic pocket on cereblon (surface representation in gray). (D) Lenalidomide in complex with cerebloncreates a novel binding surface for CK1a (ribbon colored green). The loop from CK1a can be seen interacting with thesurface on cereblon. Inset with zoomed-in drug (stick representation, colored cyan with mesh over it) shows the inter-actions on the cereblon where it is surrounded by tryptophans (stick representation in gray).

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Whether CRBN recognizes both b-hairpin loop degrons and linearmotif degrons is unknown but represents an example of the challengeof thinking only in “canonical” terms about substrate recognition byE3 ligases and the potential for the identification of additional types ofdegron structures in the proteome.

These examples ofCRBN-targetedmolecules highlight the possibilityof identifying or developing small molecules to restore proper degrada-tion by acting as a molecular “glue.” Similar approaches have identifiedsmall molecules that restore the function of mutant p53 by interactingwith its DNA binding domain and stabilizing native folding impaired bypoint mutations (356). FBXW7, which contains hotspot mutations in itssubstrate recognition domain in various cancers, is one of the E3 ligasesubstrate receptor subunits that may be a good candidate for this ap-proach. Specific compounds could stabilize the binding interface withthe substrate and reactivate FBXW7 mutants.

The proper functioning of ubiquitin-mediated degradation dependsnot only on the active E3 ligase but also on the active (recognized) formof the degron. Recognition of degron motifs can be regulated bymultiple mechanisms, involving posttranslational modifications or bycompeting interactions that can enhance or mask the degron motif.These regulatory mechanisms also offer potential strategies for drugdesign, as demonstrated for n-MYC. n-MYC is a neuronal memberof the family of MYC proteins and is specifically amplified in neuro-blastoma (357). The degradation of n-MYC involves a phosphodegronmotif that is recognized by FBXW7. However, in neuroblastoma cells,Aurora kinase (AURKA) binds to the n-MYC:SCFFBXW7 complex andprevents access of FBXW7 to the degron, thus blocking ubiquitinationand stabilizing n-MYC. n-MYC degradation can be restored by smallmolecules that induce a distortion in the conformation of Aurora A,preventing its interaction with n-MYC (358).

Besides the clinically approved bortezomib and thalidomide deriv-atives, there are several small molecules targeting various componentsof the UPS that are under investigation in preclinical studies or are inclinical trials. The most specific effects are expected from targetingthose E3 ligases that have the fewest substrates. However, because thesets of targeted proteins vary for different cell types, the effect of E3ligase–degron inhibitors is also expected to vary for different cancers.This is true for the E3 ligase modulator thalidomide, which has effica-cy for multiple myeloma but not for solid tumors (349). Inhibiting theE1-E2-E3 interactions is intermediate in complexity between specificdegron blocking and nonspecific inhibition of the proteasome itself.When choosing novel drug targets, the ability to predict the therapeu-tic effect of any novel inhibitor molecule is extremely limited. There-fore, it makes sense to produce inhibitors for any practical target in thecell and then test them against a range of cell types and disease modelsfor useful effects. The other key use of small-molecule inhibitors is asreagents in experimental research; even inhibitors that are too toxic orotherwise inappropriate for medical use can be very useful researchtools. These considerations suggest that all E3 ligase–degron interac-tions are valid targets for small-molecule inhibition.

Future DirectionsThe relative and absolute amounts of many proteins are critical factorsin the establishment and maintenance of cancerous cells. Degrons ononcogenes and tumor suppressors are the targets of E3 ligases that directthe proteins with the degrons for destruction through the UPS. If thedegron is not known, it is difficult to evaluate whether posttranslationalmodifications modulate the E3 ligase–degron interaction and whether


there is a direct relationship between an E3 ligase and a putative sub-strate protein. Cancer-associated mutations compromise degrons inseveral key oncogenes. By contrast, a lack of mutations in degron se-quences in a proteinwith a proposed role in cancer biologymay indicatethat the protein is a tumor suppressor.We recommend that scoring de-gron mutational status should be a routine part of cancer genomescreening workflows. The availability of defined E3 ligase–degroncomplexes enables the screening of compound libraries for inhibitorsof these interactions that may have antitumor activity. In summary,knowing the degrons enhances understanding of and therapeutic inter-ference with the destruction systems operating in different cancers. Yet,compared to the ~600 E3 ligases in the human proteome, only about25 classes of degron motifs have been defined. Have degrons beenneglected?We think so and we believe that putting more effort into de-gron discovery will benefit biomedical research, providing payoffs inimproved cancer treatments.

SUPPLEMENTARY MATERIALSwww.sciencesignaling.org/cgi/content/full/10/470/eaak9982/DC1Table S1. List of known degron motifs.Table S2. List of E3 target recognition subunits.Additional information can be found at http://dosztanyi.web.elte.hu/CANCER/DEGRON/.

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Acknowledgments: We thank the researchers who have helped build up the ELM resourceand provide insights into linear motif functions within the cell. B.M. and Z.D. thank A. Reményiand A. Zeke (Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academyof Sciences) for many helpful discussions. Funding: B.U. acknowledges funding received aspart of the RNA Bioinformatics Center of the German Network for Bioinformatics Infrastructure[031 A538C RBC (de.NBI)] from the German Federal Ministry of Education and Research. M.K.is thankful for the postdoctoral fellowship from theHumboldt Foundation. M.B. was funded by the


postdoctoral fellowship of the Hungarian Academy of Sciences and the OTKA grant (K115698).Z.D. acknowledges the support of the “Lendület” grant from the Hungarian Academy of Sciences(LP2014‐18) and OTKA grant (K108798). Competing interests: The authors declare that theyhave no competing interests.

Submitted 23 December 2016Accepted 27 February 2017Published 14 March 201710.1126/scisignal.aak9982

Citation: B. Mészáros, M. Kumar, T. J. Gibson, B. Uyar, Z. Dosztányi, Degrons in cancer. Sci.Signal. 10, eaak9982 (2017).

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