Gankyrin: a new oncoproteinand regulator of pRb and p53Simon Dawson1, Hiroaki Higashitsuji2, Anthony J. Wilkinson3, Jun Fujita2

and R. John Mayer1

1School of Biomedical Sciences, University of Nottingham Medical School, Queen’s Medical Centre, Nottingham, UK, NG7 2UH2Department of Clinical Molecular Biology, Graduate School of Medicine, Kyoto University, Shogoin Kawahararacho, Sakyo-ku,

Kyoto 606-8507, Japan3Structural Biology Laboratory, Department of Chemistry, University of York, York, UK, YO10 5YW

Gankyrin is a new oncoprotein with potent cell cycle and

apoptotic properties that is overexpressed early in

hepatocarcinogenesis and in hepatocellular carcinomas.

Gankyrin regulates the phosphorylation of the retino-

blastoma protein (pRb) by CDK4 and enhances the

ubiquitylation of p53 by the RING ubiquitin ligase

MDM2. Purified preparations of the 26S proteasome

contain gankyrin, which specifically interacts with the

S6b (Rpt3) ATPase of the 19S regulator. In conclusion,

gankyrin is a small versatile cell cycle regulator that

illustrates the essential interplay between the ubiquitin

proteasome system and gene expression in the cell.

Here, we discuss the activities of gankyrin and present a

model for its function in the regulation of pRb and p53.

Introduction

In multicellular organisms, cell growth must be tightlyregulated to prevent overproliferation, to enable therepair of damaged DNA and to eliminate damaged cellsby apoptosis. Crucial events in the cell cycle are regulatedby ‘check points’ that ensure each is completed before thenext occurs. Two of the most characterized checkpointcomplexes are the pRb–p16Ink4a–D cyclins and p53–ARF–MDM2. The retinoblastoma (pRb) tumour suppres-sor protein tethers transcription factors, including E2F. Atthe G1–S transition, hyperphosphorylation of pRbreleases E2F, which triggers the expression of DNAsynthesis genes. pRb is phosphorylated by the D cyclin-dependent kinases CDK4 and CDK6 and by cyclinE–CDK2. These activities are regulated by inhibitorsthat include p16Ink4a [1].

The p53 tumour suppressor is a transcription factorthat controls the expression of genes including the p21kinase inhibitor and apoptotic proteins. In turn, theactivity of p53 is controlled by many proteins includingthe RING ubiquitin ligase MDM2 and its inhibitor ARF,which are alternatively expressed from the pINK locus [2].Characteristically, cancer cells contain malfunctioningcheckpoint proteins and faults in the pathways involvingpRb–p16Ink4a–D cyclins and p53–ARF–MDM2 frequentlyoccur during human tumorigenesis [3,4]. Cancer occursbecause cells with faulty checkpoint proteins continue to

Corresponding author: Mayer, R.J. (john.mayer@nottingham.ac.uk).Available online 3 April 2006

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go through the cell cycle, leading to genomic instabilityand damage.

Molecules that alter cell cycle regulation and oncogen-esis are not discovered often. Gankyrin is one such newplayer that can control the activities of pRb and p53 [5,6].Gankyrin associates with CDK4 to control the phosphoryl-ation of pRb and with MDM2 to control the ubiquitylationof p53 (and possibly pRb).

Gankyrin discovery

Gankyrin is a small protein (25kDa with 226 amino acids)that contains seven ankyrin repeats [7] and is highlyconserved (Xenopus laevis–Homo sapiens: 78% identical,90% similar; Saccharomyces cerevisiae–H. sapiens: 32.5%identical, 45.7% similar). Gankyrin is a subunit of the 26Sproteasome [8] that specifically interacts with the S6bATPase (also know as Rpt3) of the 19S regulator [9]. The19S regulator contains six related ATPases of the AAAsuperfamily, probably in the form of a hexameric ring orcollar similar to prokaryotic counterparts [10], whichmediates the unfolding of proteins before their degra-dation in the 20S barrel-shaped proteolytic core of the 26Sparticle. In yeast, each of the six ATPases has non-redundant functions that control the activity of the 26Sproteasome [11]. In higher eukaryotic cells, interactionpartners of these ATPases include many cellular and viralproteins that modulate their ability to unfoldproteins [12].

Gankyrin was also discovered independently by con-structing subtracted cDNA libraries from non-cancerousliver and hepatocellular carcinoma (HCC). Importantly,gankyrin is overexpressed at the mRNA and protein levelsin most HCCs studied [5].

Gankyrin and pRb

The first clues to gankyrin function came from theidentification of an LxCxE motif in the C-terminal domain;this is characteristic of proteins that interact with pRb [5].Gankyrin might bind to pRb through this; however, thecrystal structure of gankyrin reveals that some residues inthis motif are buried in the a helix of the fifth ankyrinrepeat, which, in our opinion, precludes a similar bindingmechanism for gankyrin [7]. It is unlikely that gankyrinundergoes a conformation change to bind to pRb.

Opinion TRENDS in Cell Biology Vol.16 No.5 May 2006

. doi:10.1016/j.tcb.2006.03.001

Opinion TRENDS in Cell Biology Vol.16 No.5 May 2006230

Gankyrin binding to pRb, presumably through the S6bATPase of the 26S proteasome, increases the rate of pRbdegradation [5]. However, pRb might bind directly to the20S core (to the a7 subunit) of the 26S proteasome(promoted by MDM2) and mediate its own degradation[13]. In addition, gankyrin binding to pRb results in itshyperphosphorylation, release of the E2F transcriptionfactors and activation of DNA synthesis genes. A searchfor the interaction partners of transfected tagged gan-kyrin in cells showed an association with CDK4, which ispartly responsible for hyperphosphorylation of pRb [9]. Itis not known if gankyrin binds to monomeric CDK4 or theCDK4–cyclin D complex. Gankyrin does not bind to CDK6.The pINKs (inhibitors of cyclin dependent kinases) bind toand inhibit cell cycle kinases including CDK4, andgankyrin competes with p16INK4A for binding to CDK4.Gankyrin binding results in the activation of CDK4 andphosphorylation of pRb [14]. Several proteins, includinggankyrin, reversibly associate with the 26S particle in thepresence and absence of ATP [15]. We propose that such anATP-dependent mechanism, mediated by proteasomalATPase activities, could enable gankyrin to be present ina number of small complexes in the cell (e.g. with CDK4),in addition to being a regulatory subunit of the 19Sregulatory particle of the 26S proteasome [9]. Elucidationof the location and nature of these complexes will lead to agreater understanding of the roles of gankyrin in the cell.

Gankyrin and p53

When overexpressed, gankyrin has anti-apoptotic activityin tumour cells that have been exposed to DNA damagingagents and, conversely, the downregulation of gankyrininduces apoptosis in cells with wild-type p53 [6]. Increasedexpression of gankyrin inhibits apoptosis by causing thedegradation of p53 and, subsequently, the transcription ofp53-dependent genes. Gankyrin binds to MDM2 in vitroand in vivo, which increases p53–MDM3 association,thereby increasing the ubiquitylation and subsequentproteasomal degradation of p53. Gankyrin, MDM2 andp53 can be immunoprecipated from cell extracts thatcontain 26S proteasomes. The downregulation of gankyrinin cells by RNA interference (RNAi) reduces the amount ofMDM2 and p53 associated with the 26S proteasome [6].

Gankyrin is a component of the 26S proteasome [8,9],suggesting a biochemical pathway by which gankyrin can

1

SWIB/Mdm2domain

(p53-binding)ARF-bin

Acid

NES

NLS

Figure 1. Functional domains of the MDM2 ubiquitin protein ligase. The SWIB domain is a

binding region (aa 26–108). The acidic domain is a region rich in aspartic and glutamic a

binds to MDM2 in a region N-terminal to the RING-finger domain (aa 411–438). Abbrev

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not only control the rate of ubiquitylation of p53 throughMDM2, but might also be an adaptor protein that deliversubiquitylated p53 to the 26S proteasome for degradation[6]. This type of substrate delivery to proteasomalATPases might be common to many key regulatoryproteins [16]. For example, the Von Hippel Lindau tumoursuppressor protein, a component of a cullin-based SCFubiquitin ligase, similarly binds to proteasomal ATPaseS6a and delivers ubiquitylated hypoxia-inducible tran-scriptional factor (HIF) to the 26S proteasome fordegradation [17]. There are other routes of substratedelivery, including those through adaptor proteins thatcan bind to both ubiquitylated proteins and the protea-some [16]. Alternatively, individual ubiquitin proteinligases can bind directly to the 26S proteasome (e.g.Parkin [18]).

In addition to binding CDK4 and pRb, gankyrin bindsto the C terminus of MAGE A-4, one of the melanomaantigens [19]. The functions of this protein are unknownbut it is the only member of the large MAGE family to bindto gankyrin and inhibits its anti-apoptotic activity.Gankyrin function might be similarly counterbalancedby several proteins in the cell [19].

Gankyrin and MDM2

The mechanism by which gankyrin increases the bindingof p53 to MDM2 and facilitates the ubiquitylation of p53 iscurrently unknown. The activities of MDM2 are inhibitedby the ARF protein, which is an alternative transcript ofthe pINK gene locus [2]. Competition between gankyrinand the ARF protein for MDM2 binding has beendiscounted as part of the mechanism [6]. The difficultiesassociated with expression of soluble MDM2 preclude thedetermination of the crystal structure of the entireprotein, although the structure of the N terminus ofMDM2 has been solved [20]. The structure of gankyrin hasbeen solved in several laboratories and contains sevenankyrin repeats [7,21–23].

The solution of the co-crystal structure of gankyrinwith the gankyrin-binding region of MDM2 is necessary toexplain how gankyrin can increase the catalytic activity ofMDM2. Gankyrin binds to a region of MDM2 (aa 411–438)just N-terminal to the RING domain (Figure 1). This putsthe gankyrin-binding region of MDM2 in a centralposition, as the N terminus of MDM2 binds p53 and the

TRENDS in Cell Biology

491

RanBP2-typefinger

RING-typefinger

ding

ic region NLS

Gankyrin-bindingdomain

conserved region in the BAF60b proteins of the SWI/SNF family and spans the p53-

cid residues. RanBP2-type and RING-finger domains coordinate zinc ions. Gankyrin

iations: NES, nuclear export signal; NLS, nuclear localization signal.

Opinion TRENDS in Cell Biology Vol.16 No.5 May 2006 231

C terminus contains a RING site that enables associationwith an ubiquitin conjugating enzyme.

A model of gankyrin–MDM2 interaction

Mechanistic insight into the way that gankyrin controlsthe activity of MDM2 can be gained from the crystalstructure of another RING ubiquitin protein ligase, c-Cbl[24]. This enzyme ubiquitylates the C-terminal tails ofreceptor tyrosine kinases and is involved in receptordownregulation and the termination of growth factorsignalling. In the central region of c-Cbl is a linker region(aa 342–380) that starts with an a helix followed by aregion of low complexity and little structure. Mutations inthis region are associated with tumour formation,implying that it influences the ubiquitin ligase activityof the enzyme, perhaps by enabling the conformationalchanges that are required for the ligase-dependenttransfer of ubiquitin from an ubiquitin conjugatingenzyme to the receptor tyrosine kinase substrate. Inter-estingly, although there is no sequence homology in theunstructured regions of c-Cbl and MDM2, the MDM2gankyrin-binding domain is also part of a low complexitysequence (aa 360–438), which contains many polarresidues and is expected to have little overall secondarystructure. This region, on gankyrin binding, might changethe conformation of MDM2 and facilitate ubiquitintransfer to p53. If so, this is a new type of protein–proteininteraction for an ankyrin-repeat protein. Such a mech-anism might be necessary for the MDM2-dependentmonoubiquitylation of the multiple lysines in theC-terminal region of p53 and/or further polyubiquitylationof these residues [6]. MDM2 can attach ubiquitin and theubiquitin-like protein NEDD8 to p53 [25], therefore ifgankyrin alters the structure of MDM2, this could enablethe efficient ubiquitylation of p53 by MDM2 rather thanneddylation. p53 can also be sumoylated in this region,extending the complexity of posttranslational modifi-cations [26], but whether gankyrin is involved is notyet known.

Competitive conjugation of these ubiquiton molecules[16] has important consequences for p53 activity [25,26].The conjugatable lysine residues at the C terminus of p53are normally targeted by MDM2 but no alteration in theproperties and stability of p53 is observed if these aremutated to arginines in ES cells and transgenic mice[27,28]. This indicates that p53 can be ubiquitylated byMDM2 elsewhere [27]. Gankyrin also controls MDM2autoubiquitylation. DNA damage-activated autoubiquity-lation of MDM2 results in its destruction by the 26Sproteasome and is necessary for the elevation in p53 levelsrequired in DNA damage responses [29]. Gankyrin doesnot bind to the other p53 ubiquitylating enzymes, Cop1,Pirh2, p300 or the HECT ubiquitin ligase E6 AP [6];however, it is not yet known if gankyrin interacts withother RING enzymes, such as those involved in regulatingnuclear chromatin-related transcriptional events.

There are w500 genes encoding RING ubiquitinprotein ligases in the human genome, which controlmany cellular functions [16]. The roles of gankyrin andthe ubiquitin proteasome system in tumour suppressorregulation are therefore only part of an array of events

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(including nuclear events) that are controlled by theubiquitin system. It is now clear that the covalentconjugation of different ubiquitons to many proteins is atthe centre of nuclear events such as DNA synthesis andrepair, for example, in regulating the activities of theproliferating cell nuclear antigen [16]. Additionally,hormone-dependent gene expression (e.g. through oestro-gen receptors) [30], transcriptional regulation through theproteasome-related ATPases that are independent of 20S[31] and general transcriptional regulation [32] involvethe ubiquitin proteasome system. Some proteins, includ-ing p53, can be degraded by the proteasome independentof ubiquitylation [33]. It has been suggested thatassociation of ubiquitylated MDM2 with p53 is sufficientfor degradation of the ubiquitylated MDM2–p53 complex[27]. Further work is necessary to prove conclusively thatgankyrin facilitates p53 delivery to the 26S proteasomethrough MDM2 and, therefore, indirectly connects p53 tothe 26S proteasome. Finally, in the context of theregulation of both p53 and pRb by gankyrin (Figure 2),MDM2 also ubiquitylates pRb for degradation [34].

Gankyrin and DNA repair

In S. cerevisiae, gankyrin (known as Nas6p) is part of amultisubunit complex containing the Sem1p (or DSS1mammalian orthologue) protein [35]. Sem1p is also asubunit of the 26S proteasome [36]. The proteasome has arole in the repair of double strand DNA breaks (DSB) inyeast, and Sem1p deletions show synthetic growth defectswhen they are combined with mutations of otherproteasome subunits. Proteasomes are also present atDSBs [37].

DSS1 interacts with the DNA-binding domain of theBRCA2 protein [38], mutations in which contribute to bothovarian and breast cancer. With BRCA2, DSS1 regulatesthe activity of the RAD51 recombinase and the formationof RAD51-positive DNA repair foci in response to DNAdamage, in particular to DSBs [39]. DSS1 is required forDNA repair in mammalian cells [40] and in the geneticallymalleable fungus Ustilago maydis [41].

The BRCA2 protein forms part of a multi-subunitubiquitin protein ligase known as BRCC, which alsocontains BRCA1 and the RING-finger protein BARD1[42]. After DNA damage, BRCC localizes to RAD51 fociwhere p53 ubiquitylation occurs on the same C-terminalresidues of p53 that are targeted by MDM2. It would beinteresting to investigate whether gankyrin facilitates theubiquitylation of p53 through BRCC in these foci.

Gankyrin and liver cancer

Early observations that gankyrin overexpression occurs inmost HCCs [5] was confirmed and extended [43]. Anincrease in gankyrin expression in hepatocytes is one ofthe earliest molecular events in a chemical model of livercarcinogenesis [44]. Currently, it is unclear why gankyrinis overexpressed in HCC and not in other tumours.However, gankyrin is expressed in all eukaryotic cells.Its importance is illustrated by the fact that adenovirus-delivered gankyrin RNAi suppresses gankyrin expressionin HCC, resulting in reduced cell growth, enhanced pRbdephosphorylation and caspase8- and/or caspase9-

TRENDS in Cell Biology

Rb

Gankyrin

Rbp

E2F

DNA synthesis

p

CDK4

CDK4

p16INK4a

p16INK4a

RbRb

p53–dependentapoptosis

UbUb

UbUb

p53 p53

p53 p53

UbUb

UbUb

Mdm2

??

(a) (b)

(c) (d)

Gankyrin

Mdm2

Mdm2

Gankyrin

26SProteasome

Figure 2. Current understanding of the activities of gankyrin in cell cycle regulation and apoptosis. In the absence of gankyrin (a) pRb is not hyperphosphorylated and (b) p53

is inefficiently ubiquitylated by MDM2 and poorly degraded. In the presence of gankyrin (c) pRb is hyperphosphorylated and degraded, whereas E2F transcription factors are

released to trigger expression of DNA synthesis genes and (d) p53 is extensively ubiquitylated and degraded to inhibit p53 dependent apoptosis. Abbreviations: P, phosphate;

Ub, ubiquitin.

Opinion TRENDS in Cell Biology Vol.16 No.5 May 2006232

mediated apoptosis [45]. The oncogene encoding gankyrinis one of several genes that are upregulated in response toanti-apoptotic stimuli in the developing liver of Xenopuslaevis. There is a careful balance between cell division andapoptosis in developing hepatocytes [46]. Hepatocytes canregenerate following injury or resection in the adult liver.During regeneration, disturbances in the balance betweencell division and apoptosis by gankyrin overexpression,causing the inactivation of pRb and elimination of p53,could provide a mechanism of hepatocarcinogenesis.

Conclusion

Gankyrin is a new cell cycle regulator and oncoprotein thatcontrols the activities of two key tumour suppressors pRband p53. Gankyrin is a small ankyrin-repeat protein that isfound in different complexes involved in cell cycle controland functions in the efficient delivery of tumour suppressors(and other proteins) to the 26S proteasome for degradation.Future work will need to identify the full repertoire ofinteraction partners of gankyrin, particularly in the contextof the DNA repair and transcriptional regulation.

Acknowledgements

This work was sponsored in the UK by the EU Framework IV, the BBSRCand the University of Nottingham, and in Japan by grants from theMinistry of Education, Culture, Sports, Science and Technology of Japan,

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the Japan Society for the Promotion of Science and the Smoking ResearchFoundation of Japan.

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