sumo-1 modification of pml

INTRODUCTION

The PML gene was first identified as a result of a reciprocaltranslocation event t(15;17) associated with acutepromyelocytic leukaemia, which also disrupted retinoic acidalpha (RARA;17). This produced the fusion proteins PML-RARA and RARA-PML (for a review see Grimwade et al.,1997). The PML protein in normal cells is a nuclearphosphoprotein, localising to discrete matrix-associatedsubnuclear domains, as part of a multi-protein complextermed ND10, Kr bodies, PML oncogenic domains (PODs)or PML nuclear bodies (for a recent review see Hodges et al.,

1998a). In the t(15;17) acute promyelocytic leukaemia (APL)diseased state, PML nuclear bodies become disrupted to formnumerous PML foci. However, treatment with all-transretinoic acid results in the reformation of normal PML bodiesand a release of the block in promyelocytic differentiation(Daniel et al., 1993; Dyck et al., 1994; Koken et al., 1994;Weis et al., 1994). PML nuclear bodies are also the target fora variety of virus-derived proteins and it is thought that theloss of organisation of these domains is an important phaseof the viral infection cycle (reviewed in Hodges et al., 1998a).It has also been shown that PML nuclear bodies are disruptedin the development of Spinocerebellar Ataxia type I (Skinner

381Journal of Cell Science 112, 381-393 (1999)Printed in Great Britain © The Company of Biologists Limited 1999JCS4651

PML is a nuclear phosphoprotein that was first identifiedas part of a translocated chromosomal fusion productassociated with acute promyelocytic leukaemia (APL).PML localises to distinct nuclear multi-protein complexestermed ND10, Kr bodies, PML nuclear bodies and PMLoncogenic domains (PODs), which are disrupted in APLand are the targets for immediate early viral proteins,although little is known about their function. In a yeasttwo-hybrid screen, we first identified a ubiquitin-likeprotein named PIC1 (now known as SUMO-1), whichinteracts and co-localises with PML in vivo. More recentstudies have now shown that SUMO-1 covalently modifiesa number of target proteins including PML, RanGAP1 andIκBα and is proposed to play a role in either targetingmodified proteins and/or inhibiting their degradation. Theprecise molecular role for the SUMO-1 modification ofPML is unclear, and the specific lysine residues within PMLthat are targeted for modification and the PML sub-domains necessary for mediating the modification in vivoare unknown. Here we show that SUMO-1 covalentlymodifies PML both in vivo and in vitro and that themodification is mediated either directly or indirectly by the

interaction of UBC9 with PML through the RING fingerdomain. Using site-specific mutagenesis, we have identifiedthe primary PML-SUMO-1 modification site as being partof the nuclear localisation signal (Lys487 or Lys490).However SUMO-1 modification is not essential for PMLnuclear localisation as only nuclear PML is modified. Thesequence of the modification site fits into a consensussequence for SUMO-1 modification and we have identifiedseveral other nuclear proteins which could also be targetsfor SUMO-1. We show that SUMO-1 modification appearsto be dependant on the correct subcellularcompartmentalisation of target proteins. We also find thatthe APL-associated fusion protein PML-RARA isefficiently modified in vitro, resulting in a specific andSUMO-1-dependent degradation of PML-RARA. Ourresults provide significant insights into the role of SUMO-1 modification of PML in both normal cells and the APLdisease state.

Key words: SUMO-1, PML, Ubiquitin-like, UBC9, Modification,Acute promyelocytic leukaemia

SUMMARY

SUMO-1 modification of the acute promyelocytic leukaemia protein PML:

implications for nuclear localisation

Estelle Duprez 1,‡, Andrew J. Saurin 1, Joana M. Desterro 2, Valerie Lallemand-Breitenbach 3, Kathy Howe 4,Michael N. Boddy 1,§, Ellen Solomon 4, Hugues de Thé 3, Ronald T. Hay 2 and Paul S. Freemont 1,*1Molecular Structure and Function Laboratory, Imperial Cancer Research Fund, 44 Lincolns Inn Fields, London WC2A 3PX, UK2School of Biomedical Sciences, University of St Andrews, St Andrews KY16 9AL, Scotland, UK3Centre Hayem, Hôpital Saint-Louis, 75010 Paris, France4Division of Medical and Molecular Genetics, Guys and St Thomas’s Medical and Dental School, Guys Hospital, London SE1 9RT,UK*Author for correspondence (e-mail: [email protected])‡Present address: Unite INSERM 496, Centre Hayem, Hôpital Saint-Louis, 75010 Paris, France§Present address: Departments of Molecular Biology and Cell Biology, Scripps Research Institute, La Jolla, CA 92037, USA

Accepted 26 November 1998; published on WWW 13 January 1999

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et al., 1997). Thus, it appears that PML nuclear bodies arefunctionally important multi-protein complexes which, whendisrupted or reorganised, have profound cellularconsequences.

The PML protein contains a number of well-characterisedzinc-binding domains that include a RING finger (Borden etal., 1995; for a review see Saurin et al., 1996) adjacent to twocysteine/histidine-rich motifs known as B-boxes (Borden et al.,1996; Reddy et al., 1992). These domains, together with apredicted α-helical coiled-coil domain, form a conserved motif(known as RBCC) that is found in several other functionallyunrelated proto-oncoproteins (Reddy et al., 1992; reviewed inSaurin et al., 1996). The PML RING finger is required for PMLnuclear body formation, as deletion of the RING and/or pointmutations of the zinc-binding ligands abrogate PML nuclearbody formation in vivo (Boddy et al., 1997; Borden et al.,1995; Kastner et al., 1992; Le et al., 1996). The B-box andcoiled-coil domains also appear important in nuclear bodyformation by mediating PML hetero-and homo-oligomerisation, respectively (Borden et al., 1996; Kastner etal., 1992; Perez et al., 1993). In some experimental systemsPML has been shown to act as a growth supressor (Liu et al.,1995; Mu et al., 1994), an activity which depends on theintegrity of PML nuclear bodies (Le et al., 1996). Recently, apossible mechanism for PML-associated growth suppressionhas been proposed, which involves an induction of cell deathrequiring the RBCC domain (Fagioli et al., 1998), suggestingthat PML overexpression is pro-apoptotic (Borden et al., 1998;Le et al., 1998; Quignon et al., 1998).

We first reported the identification of a novel ubiquitin-likeprotein that specifically interacts with PML, which we calledPIC1 for PML I nteracting Clone-1 (Boddy et al., 1996).Subsequently, PIC1 was cloned and named independently byseveral other groups and found to interact with a variety offunctionally distinct proteins including the death domains ofFas/Apo-1 (called Sentrin; Okura et al., 1996), RAD51/52(called UBL1; Shen et al., 1996) and RanGAP1 (calledGMP1/SUMO-1; Matunis et al., 1996; Mahajan et al., 1997).PIC1/SUMO-1 (Small Ubiquitin-like MOdifier) is part of agrowing family of ubiquitin-like proteins with significantsequence homology to ubiquitin (18% identity for SUMO-1;for a recent review see Hodges et al., 1998b). Similar toubiquitin, SUMO-1 has been shown to covalently modify anumber of target proteins (Kamitani et al., 1997), the best-characterised of which are RanGAP1 (Mahajan et al., 1997;Matunis et al., 1996) and IκBα (Desterro et al., 1998). Themodification of RanGAP1 does not lead to degradation (unlikeubiquitin) but targets RanGAP1 specifically to the nuclear porecomplex. Furthermore, conjugation is via the C terminus ofSUMO-1 (Gly 97; after removal of the four C-terminalresidues) and an ε-amino group of a specific Lys (K526) inRanGAP1 (Mahajan et al., 1998). IκBα is another SUMO-1-modified protein, and functions as an inhibitor of thetranscription factor NF-κB, which is targeted for ubiquitin-mediated degradation as part of the NF-κB signal-inducedactivation pathway (Desterro et al., 1998). However, SUMO-1-modified IκBα is resistant to signal-induced degradation,with the same lysine (Lys21) that is modified by ubiquitin inNF-κB activation, covalently modified by SUMO-1 (Desterroet al., 1998). Thus, SUMO-1 modification can directly inhibitubiquitin conjugation of the same protein, perhaps allowing

increased cellular regulation of specific protein concentrations(Desterro et al., 1998; Everett et al., 1998).

A number of reports have now firmly established that theE2-conjugating enzyme for SUMO-1 is UBC9 (Desterro et al.,1997; Gong et al., 1997; Johnson et al., 1997a; Saitoh et al.,1998). For RanGAP1, conjugation of SUMO-1 has an absoluterequirement for UBC9 (Saitoh et al., 1998). UBC9 wasoriginally identified as a gene essential for G2-early M-phasecell-cycle progression, the product of which was thought tofunction in ubiquitin-mediated degradation of specific proteins(Seufert et al., 1995), although this is now likely to involveSUMO-1. The E1 enzyme for SMT3P, the yeast homologue ofSUMO-1, has also been identified and exists as a heterodimerwith sequence similarity to ubiquitin E1 (Johnson et al.,1997b). Thus, the SUMO-1 modification system is similar tothe ubiquitin system, although unlike ubiquitin, modified targetproteins are not degraded (for a review see Hodges et al.,1998b).

Three reports have provided evidence for the covalentmodification of PML by SUMO-1 (Kamitani et al., 1998;Müller et al., 1998; Sternsdorf et al., 1997) and another PMLnuclear body component SP100 (Sternsdorf et al., 1997). Here,we extend our initial observations on the interaction betweenPML and SUMO-1/PIC1 and show that SUMO-1/PIC1covalently modifies PML both in vivo and in vitro.Furthermore, we show that SUMO-1-PML modification ismediated by the interaction of UBC9 with PML, through theRING finger domain. We also identify candidate lysineresidues in PML that are SUMO-1-modified and show thatonly nuclear PML is modified by SUMO-1. It has beensuggested that SUMO-1 modification of PML targets PML tothe nuclear matrix and PML nuclear bodies (Müller et al.,1998). We discuss our results in light of these recent reports.

MATERIALS AND METHODS

Mammalian expression vectorsHuman PML 69 (69 kDa isoform; PML3B: Accession numberM80185) and PML 97 (97 kDa isoform; PML1: Accession numberM79462) cDNAs were subcloned into a mammalian expression vectorcarrying the MLV enhancer (pMLV-plinkS2; Dalton and Treisman,1992) using the NcoI site. PML∆COOH was produced by restrictiondigest of PML69-MLV with XmaI, deleting PML from amino acid448. PML∆NH2 and PML∆RING were produced by partial PCRcloning to delete the first 46 or 98 amino acids, respectively, andcloned in pMLV-plinkS2 at NcoI and BamHI sites.

Site-directed mutagenesis of PML was performed using the PCR‘stitching’ method of Mullis et al. (1986) as described in Boddy et al.(1997). The following ‘internal’ mutant primers (forward and reverse)were used to introduce the specific mutations: for PML Cys57,60∆ala, 5′-CAG TTT CTG CGC GCC CAG CAA GCC CAGGCG-3′ and 5′-CGC CTG GGC TTG CTG GGC GCG CAG AAACTG-3′; for PML Lys 65,68∆ala, 5′-GGC GGA AGC CGC GTGCCC GGC GCT GCT GCC TTG-3′ and 5′-CAA GGC AGC AGCGCC GGG CAC GCG GCT TCC GCC-3′; for PML Lys 476,478∆arg,5′-CAA CGA CAG CCC AGA GGA GGA GGT GCA GC-3′ and 5′-GCT GCA CCT CCT CCT CTG GGC TGT CGT TG-3′; for PMLLys 487,490∆arg, 5′-CCA GGA GGG TCA TCA GGA TGG AGTCTG AG-3′ and 5′-CTC AGA CTC CAT CCT GAT GAC CCT CCTGG-3′; for PML Lys 515 ∆arg, 5′-CCA GCA CCT CCA GGA CAGTCT CAC CAC C-3′ and 5′-GGT GGT GAG ACT GCC CTG GAGGTG CTG G-3′.

E. Duprez and others

383SUMO-1 modification of PML

Human SUMO-1 and UBC9 cDNAs were subcloned into thepMLV-plink2T vector, resulting in an in-frame fusion at the aminoterminus of the human c-myc and T7 promoter epitope tags,respectively. PML-RARA was subcloned in pCDNA3 (Invitrogen).

Yeast two-hybrid assayThe wild-type and mutant human forms of PML, SUMO-1 and UBC9cDNAs were inserted either in-frame with the Gal4 DNA-bindingdomain in the yeast expression vector pAS2-1 (Clontech) or in-framewith the Gal4 activation domain in the yeast expression vector pACT2(Clontech). Constructs were transformed into the Saccharomycescerevisiaestrain Y190 (Clontech) and their ability to interact wasmeasured by β-galactosidase activity using a liquid assay forquantification. Single colonies on selective plates were picked andgrown overnight in selective liquid medium, before diluting andgrowth for a further 4 hours. Approximately 2×108 cells werecollected by centrifugation and resuspended in 600 µl of Z buffer (100mM phosphate buffer, pH 7.0, containing 10 mM KCl and 1 mMMgSO4). Cells were permeabilised with repeated freeze/thaw cyclesby dipping into liquid nitrogen for 10 seconds and rapidly warmingto 20°C. ONPG (o-nitrophenyl β-D-galactopyranoside; Sigma) wasadded as substrate, and the reaction was carried out at 30°C for 2hours. Colour development was stopped by the addition of 250 µl of1 M Na2CO3, and cell debris removed by a brief centrifugation. Theoptical density of the reaction was measured (OD420), and β-galactosidase activity was calculated using the formula described byClontech. All data presented are the average of at least fourindependent experiments.

In vitro SUMO-1 conjugation assayTranscription/translation of proteins was performed using 1 µg ofplasmid DNAs and the wheat germ-coupled transcription/translationsystem according to the instructions provided by the manufacturer(Promega). 35S-methionine was used in the reactions to generateradiolabelled proteins. In vitro-translated PML proteins labelled with35S-methionine (0.5 µl) were incubated with 3 µl of HeLa cell fraction(fraction II.4) containing SUMO-1 E1 activity (Desterro et al., 1997)in a 10 µl reaction including an ATP-regenerating system (50 mMTris, pH 7.6, 5 mM MgCl2, 2 mM ATP, 10 mM creatine phosphate,3.5 i.u./ml of creatine kinase and 0.6 i.u./ml of inorganicpyrophosphatase), SUMO-1 (200 µg/ml) and UBC9 (50 µg/ml).Reactions were incubated at 37°C for 2 hours. After terminating thereactions with SDS-sample buffer containing β-mercaptoethanol,reaction products were fractionated by SDS-PAGE (8.5%) and thedried gels analysed by phosphor imaging (Fujix BAS 1500, MacBASsoftware). SUMO-1 and UBC9 were expressed and purified aspreviously reported (Desterro et al., 1997).

Mammalian cell transient and stable transfectionsFor transient transfection, 2C4 (HT 1080; human fibrosarcoma) cellswere transfected using Superfect transfection reagent (Qiagen)according to the manufacturer’s instructions. Briefly, 15 µl Superfectwas mixed with 2 µg of DNA in DMEM medium and added toapproximately 1×106 cells. After 3 hours, the medium was removedand cells were cultured in fresh medium supplemented with 10% FCSovernight. Transfected cells were assayed for protein expression bywestern blotting and indirect immunofluorescent staining.

For stable transfections, chinese hamster ovary (CHO) cells weretransfected using transfect reagent (Promega). 3 µg of PML cDNAand 0.3 µg Dspthygro selection vector were mixed to 6 µl usingtransfection reagent (Promega) in OptiMEM medium (Gibco).Transfection mix was added to approx. 1×107 cells, previously serum-starved for 10 minutes, and the transfection completed using DMEMsupplemented with 10% FCS. After 48 hours, cells were selected inHygromycin (500 µg/ml; Gibco) for 2 weeks. Stable-transfected cellswere analysed by western blot analyses.

Western blottingCells were lysed and boiled for 5 minutes in SDS-sample buffer andprotein extracts (20 µg) were separated by SDS-PAGE (10%) andelectrophoretically transferred onto nitrocellulose membranes(Amersham). Membranes were blocked with 3% fat-free milk in PBS,then incubated with the polyclonal antiserum directed against PML(Boddy et al., 1996) in blocking solution for 1 hour at roomtemperature. Subsequently, membranes were incubated with an anti-rabbit peroxidase-conjugated secondary antibody in block solution(Dako). Each of the incubation steps were followed by 3 washes for10 minutes in PBS containing 0.1% Tween 20. Development wasperformed as described in the ECL protocol (Amersham).

Immunofluorescence staining and confocal analysesTransfected cells were fixed in methanol at −20°C for 10 minutes andpreblocked with block solution (PBS containing 0.1% Tween 20 and10% fetal calf serum). In single-labelling experiments, myc-taggedPML was detected with a 9E10 anti-myc monoclonal antibody (a giftfrom G. Evan). For double labelling, PML was detected with therabbit polyclonal antiserum directed against PML and SUMO-1 wasdetected with the 9E10 anti-myc monoclonal antibody. Primaryantibodies were diluted in block solution and incubated with thepermeabilised cells for 1 hour at room temperature. Secondaryantibodies used included a Texas Red-coupled anti-mouse antibody(Amersham) and an FITC-coupled anti-rabbit antibody (Dako) andwere incubated with cells for 30 minutes at room temperature. Allincubations were followed by three washes in PBS followed by a finalwash in PBS containing 0.05% Tween for 10 minutes. Preparationswere examined by confocal laser scanning microscopy using an MRC1000 inverted confocal microscope (Bio-Rad) or an inverted Diaphot300 microscope (Nikon). Images were collected using an oilimmersion lens (60×, NA I.4 plan Apochromat) and using excitationwavelengths of 488 nm (for FITC) and 543 nm (for Texas Red).

RESULTS

Identifying PML domains required for modificationby SUMO-1The E2-like conjugating enzyme, UBC9, has been shown to beessential for the SUMO-1 modification of RanGAP1 (Johnsonet al., 1997a; Saitoh et al., 1998) and IκBα (Desterro et al.,1997, 1998). As it is likely that UBC9 is the E2-likeconjugating enzyme for all SUMO-1 modifications, we testedthe ability of UBC9 to interact with the PML 69 kDa isoform(PML3B; Goddard et al., 1991), used previously to isolateSUMO-1 (Boddy et al., 1996). Using the yeast two-hybridsystem, we show that PML 69 kDa can interact with UBC9and SUMO-1 (see Fig. 1B), thus implicating UBC9 as acandidate protein for mediating the SUMO-1-PML interaction.

To better characterise the domains in PML required forinteractions with SUMO-1 and UBC9, we constructed grossdeletions of the PML 69 kDa isoform, which remove the N-terminal proline-rich region, the RING finger domain and theC-terminal region including the nuclear localisation signal(NLS; Fig. 1A). Using the yeast two-hybrid system, we co-transformed these PML deletions with either SUMO-1 orUBC9 and tested any interaction using the β-galactosidaseassay system (Fig. 1B). Removal of the N-terminal proline-richregion has no effect on the UBC9 interaction but, surprisingly,increases the ability of SUMO-1 to interact with PML (Fig.1B). However, deletion of both the proline-rich region and theRING finger domain abolishes the ability of both SUMO-1 and

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UBC9 to interact with PML (Fig. 1B), implicating the RINGfinger domain in these interactions.

We also tested whether another RING finger-containingprotein, RING1, could interact with UBC9. However, noRING1-UBC9 interaction was observed (data not shown),suggesting that the RING finger alone is not sufficient for the

interaction. Removing the C-terminal 112 amino acids of PMLresults in a loss of the SUMO-1-PML interaction but not in theUBC9-PML interaction (Fig. 1B). Together, these resultsimplicate both the RING finger domain and the C-terminalregion of PML in the interaction of PML with SUMO-1, butonly the RING finger domain in the interaction with UBC9.

Identifying specific residues in the SUMO-1-UBC9-PML interactionTo further investigate the role of the PML RING finger domainin the interactions between PML, SUMO-1 and UBC9, weperformed site-directed mutagenesis of key residues involvedin maintaining the structural integrity of the RING fingerdomain. Mutation of cysteines 57 and 60 to alanine residues(see Fig. 2A) results in a partial or total destabilisation of thethree dimensional structure of the RING finger domain(Borden et al., 1995). This same PML double cysteinemutation also results in a significant reduction in β-galactosidase activity, when assayed for interaction with bothSUMO-1 and UBC9 (75% and 70% decrease respectively;Fig. 2B). These results further demonstrate the importance ofthe RING finger domain in mediating interactions with bothUBC9 and SUMO-1.

Since the covalent modification of target proteins by SUMO-1 occurs via lysine residues (Matunis et al., 1998; Desterro etal., 1998), we were interested in identifying candidate lysinesin PML which might be targeted for SUMO-1 modification.Within the RING finger domain, there are only two lysineresidues at positions 65 and 68 (see Fig. 2A). However,mutation of these lysines to alanine did not significantly alterthe ability of SUMO-1 to interact with PML in the yeast two-hybrid assay (Fig. 2B). Furthermore, this double mutation alsofailed to alter the UBC9-PML interaction (Fig. 2B), suggestingthat these lysines are not major sites for SUMO-1-PMLmodification.

To test the role of the C-terminal region of PML in the UBC9and SUMO-1 interaction, we also performed site-specificmutational analyses of lysine residues within this domain (Fig.2A). Seven lysines are present in this region, but only five ofthem are conserved between mouse and human PML (Goddardet al., 1995). Assuming that the lysine residues which arerequired for SUMO-1 modification are maintained throughoutevolution, we mutated only those lysines that were conservedbetween mouse and human. Considering that some of thecandidate lysine residues are found within the nuclearlocalisation signal (NLS), we substituted the two pairs of NLSlysines to arginine, in an effort to retain some functional andstructural integrity of the NLS signal. The NLS doublemutants, Lys476.478∆Arg and Lys487.490∆Arg, bothsignificantly decrease the PML-SUMO-1 interaction asmeasured by β-galactosidase activity, although they do nottotally abolish the interaction (Fig. 2B). However theLys487.490∆Arg double mutant has a more significant effecton PML-SUMO-1 interaction than the Lys476.478∆Argmutation (75% decrease compared to 45%; Fig. 2B). Mutationof Lys515 to Arg does not affect the PML-SUMO-1 interactionand is comparable to wild type (Fig. 2B). However, none ofthese PML mutations interfere with the UBC9-PMLinteraction as measured using this assay (Fig. 2B).

In summary, these results show that: (1) the structuralintegrity of the RING finger domain is important for the


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Fig. 1.Domains involved in the interaction of PML with SUMO-1and UBC9. (A) Schematic representation of the PML 69 kDaisoform (PML3B) coding for a 560-amino acid protein (Goddard etal., 1991), showing the relative position of the RING finger (RING),the B1 and B2 B-Boxes (B-Box), the coiled-coil domain (Coiled-coil) and the nuclear localisation signal (NLS). The various PMLdeletion constructs used to study the interactions with SUMO-1 andUBC9 are shown: PML∆NH2, PML∆RING and PML∆COOHrepresent the indicated deleted forms of the PML 69 kDa isoform.(B) The interactions of PML 69 kDa (WT) and the various PMLdeletion mutants shown in A with SUMO-1 or with UBC9 weredetermined using the yeast two-hybrid system. Yeast cells were co-transformed with (+) or without (−) SUMO-1 or UBC9 fused to theGAL4 activation domain and PML 69 kDa or the PML deletionmutants fused to the GAL4 DNA-binding domain. Interactionbetween the indicated proteins was assessed by activation of the lacZgene as measured by β-galactosidase activity (arbitrary units). Valuesare the mean ± s.d. of four independent experiments.


interaction between UBC9 and PML, and between SUMO-1and PML; (2) a reduction in PML-SUMO-1 interaction isobserved as a result of two double lysine mutations within theNLS (Lys476.478∆Arg and Lys487.490∆Arg), suggesting thatthese lysines are candidate residues for SUMO-1 modification.

In vitro analysis of PML modification by SUMO-1In order to test whether the potential target lysine residuesidentified using the yeast two-hybrid system are SUMO-1-modified, we utilised an in vitro assay system whichreconstitutes the SUMO-1 ubiquitin-like modification system(Desterro et al., 1998). Using this assay, we observe an alteredelectrophoretic mobility profile of wild-type PML upon theaddition of SUMO-1 (Fig. 3). This altered profile results in theappearance of two additional higher molecular mass PML-specific species (Fig. 3; lanes 11, 12; arrowheads). However,these two additional PML species are present in differingquantities; the more abundant lower protein species (Fig. 3;double arrowheads) representing a larger amount of labelledPML than the less abundant upper species (Fig. 3; singlearrowhead). Given the nature of the in vitro assay used and the

size of the additional PML-specific protein species, weattribute this shift in electrophoretic mobility to a covalentmodification of PML by SUMO-1. The relative abundance ofthe two PML-modified species suggests that, in vitro, themajority of SUMO-1 modification occurs at a single site withinthe PML 69 kDa isoform (Fig. 3; double arrowheads), althoughsome PML molecules are modified at two sites (Fig. 3; singlearrowhead). We assume that each modification representssingle SUMO-1 conjugates at separate lysines, as there is noevidence to suggest that SUMO-1 can from poly-SUMO-1chains like ubiquitin (for a review see Hodges et al., 1998b).

To identify potential lysine residues involved in the PML-SUMO-1 modification, we also tested the ability of the doublemutations within the RING finger and the mutations within theNLS and C-terminal part of the molecule (see Fig. 2A) to bemodified by SUMO-1 in vitro. The Lys65.68∆Ala mutationwithin the RING finger domain appears to be identical to thatof wild-type PML (Fig. 3; lanes 3, 4). However, upon closerexamination an altered electrophoretic mobility profile isobserved, which we attribute to a change in the overall netcharge of PML resulting from the lysine to alanine

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Fig. 2. Identifying critical residues involved in the interaction ofPML with SUMO-1 and UBC9. (A) Site-directed mutagenesis ofthe PML 69 kDa isoform where residues were mutated as pairs inthe RING finger domain (Cys57.60∆Ala and Lys65.68∆Ala) and inthe NLS (Lys476.478∆Arg and Lys487.490∆Arg). An additionalsingle point mutation (Lys515∆Arg) was also made within the C-terminal region of the molecule. The primary amino acid sequencein the areas of the mutated residues (bold) is shown. (B) Theinteractions of PML 69 kDa (WT) and the various PML mutantsshown in (A) with SUMO-1 or with UBC9 were determined usingthe yeast two-hybrid system. Yeast cells were co-transformed with(+) or without (−) SUMO-1 or UBC9 fused to the GAL4 activationdomain and PML 69 kDa or the PML deletion mutants fused to theGAL4 DNA-binding domain. Interaction between the indicatedproteins was assessed by activation of the lacZ gene as measuredby β-galactosidase activity (arbitrary units). Values are the mean ±s.d. of four independent experiments.

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substitutions (Fig. 3, compare lanes 4 and 12). The C-terminalmutants Lys476.478∆Arg and Lys515∆Arg appear to have noeffect on the modification of PML by SUMO-1 in vitro andappear like wild type (Fig. 3, compare lanes 6 and 10 with 12).However, the double lysine NLS mutation Lys487.490∆Arghas a dramatic effect on SUMO-1 modification. This mutantshows only a single minor SUMO-1 modified species (Fig. 3;lane 8), which displays a slightly slower electrophoreticmobility than the major modified wild-type species, and islikely to represent the minor modification site observed forwild-type PML (see Fig. 3; single arrowhead). It is notable thatthe Cys57.60∆Ala RING mutant is efficiently SUMO-1modified in vitro (Fig. 3; lane 2), although in yeast theinteraction between this mutant and SUMO-1/UBC9 issignificantly reduced (Fig. 2B).

In summary, we observe that for the PML 69 kDa isoform,there appear to be two SUMO-1 modification sites in vitro, oneof which is a major site and one a minor site. We demonstratethat either Lys487 or Lys490 are major sites for PML-SUMO-1 modification. Furthermore, the abolition of SUMO-1modification at this site demonstrates that the modification ofthe primary site is not a prerequisite for modification at thesecondary site.

Effect of SUMO-1 modification on PML localisationPML has been shown to localise to large matrix-associatednuclear domains known as PML nuclear bodies. Pointmutations of PML have been shown to abrogate or alter theincorporation of PML in nuclear bodies in vivo (Boddy et al.,1997; Borden et al., 1995). The subcellular localisation ofSUMO-1 is perinuclear, as well as nuclear-diffuse and nuclear-punctate, with SUMO-1-concentrated foci corresponding toPML nuclear bodies (Boddy et al., 1996; Matunis et al., 1996;Fig. 4A, upper panel). Co-transfection of SUMO-1 and PMLresults in a relocalisation of SUMO-1 predominantly into PMLnuclear bodies (Fig. 4A, lower panel).

In order to determine the subcellular localisation of the PMLlysine mutants with reference to SUMO-1, doubleimmunofluorescence staining of transiently co-expressedSUMO-1 and PML was performed. The different PML mutantswere co-transfected with a myc-tagged SUMO-1 in humanfibrosarcoma (2C4) cells and the expressed proteins were

detected by anti-PML and anti-myc antibodies, respectively(Fig. 4B). As described previously, the Cys57.60∆Ala RINGfinger mutant is nuclear diffuse (Borden et al., 1995; Fig. 4Bi).Moreover, the expression of this mutant does not appear toinfluence the localisation of co-expressed SUMO-1, althoughit is difficult to ascertain whether both proteins colocalise, dueto the microparticulate staining pattern observed (see Fig. 4Bi).High magnifications of single sections suggest that bothproteins do not colocalise at least by co-immunofluorescence(data not shown), although this mutant form of PML isefficiently SUMO-1-modified in vitro (Fig. 3, lane 2).Substitution of the two lysine residues present in the RINGfinger domain (Lys65.68∆Ala), has previously been shown toresult in abnormally large exogenous PML nuclear bodies(Boddy et al., 1997). Co-expressed SUMO-1 is sequestered tothese large bodies, which given the in vitro modification data(see Fig. 3, lane 4) and the yeast two-hybrid interaction data(see Fig. 2B) suggests that SUMO-1 modification of the PMLLys65.68∆Ala mutant can occur within these large structures.

Surprisingly, the two NLS double mutants, Lys476.478∆Argand Lys487.490∆Arg, accumulate to differing extents in thecytoplasm, indicating that substitution of these pairs of lysinesto arginines dramatically perturbs the translocation of PMLinto the nucleus (Fig. 4Biii,iv). SUMO-1 transfected witheither of these NLS mutants does not colocalise in thecytoplasm but follows the nuclear distribution pattern forSUMO-1 transfected alone (compare Fig. 4Biii,iv with Fig.4A, top panel). It should be noted that both exogenous andendogenous PML is detected in these experiments, which mayaccount for some apparent nuclear localisation of these NLSmutants (Fig. 4Biii,iv: yellow in merged channels). In ordertherefore, to unambiguously determine whether the NLSmutants can localise to the nucleus, myc-tagged versions ofeach mutant were transiently expressed in 2C4 cells (Fig. 4C).A projection of multiple optical confocal sections shows thatfor the Lys476.478∆Arg NLS mutant, someimmunofluorescent foci are observed both surrounding and/orwithin the nucleus (Fig. 4C). This is more clearly seen in singleconfocal sections, which clearly show that theLys476.478∆Arg PML mutant accumulates at the nuclearenvelope as well as inside the nucleus. In contrast, however,the Lys487.490∆Arg mutant is almost totally excluded from


Fig. 3.The in vitro modification of PML 69kDa and the various PML mutants by SUMO-1. Wild-type (WT) and mutant forms of PML69 kDa were expressed in vitro and labelledwith 35S-methionine. Labelled forms of PMLwere incubated in the presence (+) or absence(−) of the SUMO-1 modification assay mix(described in Materials and methods) for astandard time efficient for in vitro SUMO-1modification. Reaction products were separatedby SDS-PAGE and analysed by phosphor-imaging. The major PML-SUMO-1 conjugateis indicated by double arrowheads, and theminor conjugate by a single arrowhead. Thepostions of molecular mass markers are shown.


the nucleus (see Fig. 4C). Interestingly, the Lys487.490∆ArgPML mutant appears to accumulate at the nuclear envelope,suggesting that the targeting of PML to the exterior of thenucleus is not affected, although nuclear translocation clearlyis. Together with the SUMO-1-PML interaction assays used onthese mutants (Figs 2B, 3), these data would suggest thatSUMO-1 modification of PML is important if not essential forintranuclear localisation. Subsequent experiments, however,failed to support these conclusions (see below).

Western analyses of co-expressed PML mutants andSUMO-1In order to determine whether the PML mutants are SUMO-1-modified in vivo, transient co-expression studies with SUMO-1 in human 2C4 cells were carried out (Fig. 5A). SUMO-1 andPML mono-transfectants are shown in Fig. 5A (lanes 1-2) withresulting protein species detected only with the PML

polyclonal antibody. A PML/SUMO-1 cotransfectionexperiment results in the increase of a PML speciescorresponding to the SUMO-1-modified PML, when comparedto PML expression alone (Fig. 5A, compare lanes 3 with 2;double arrowhead). A large diffuse PML band comprisingseveral closely related protein species is observed (Fig. 5A;labelled PML) as well as a single higher molecular mass bandwhich may be further SUMO-1-modified exogenous PML, dueto its increased appearance upon SUMO-1 cotransfection.Unlike previous reports of three additional SUMO-1-PMLconjugates (Kamitani et al., 1998; Müller et al., 1998; Sternsdorfet al., 1997), we are only able to detect with certainty a singleSUMO-1-modified exogenous PML species, which may reflecteither differences in the PML isoforms used and/or expressionlevels of exogenous PML or SUMO-1. However, using thismethod, we find that co-expression of SUMO-1 with theCys57.60∆Ala, Lys476.478∆Arg and Lys487.490∆Arg PML

Fig. 4.Subcellular localisation of the various PML 69 kDa point mutants and SUMO-1 in human 2C4 fibrosarcoma cells. (A) Human 2C4 cellswere either transfected with myc-tagged SUMO-1, detected with the 9E10 monoclonal antibody (upper panel) or co-transfected with myc-tagged SUMO-1 (red channel) and PML 69 kDa, detected with the PML polyclonal antibody (green channel) (lower panels). A merge of bothsignals shows total immunofluorescence colocalisation of PML and SUMO-1 (yellow). Images represent a projection of multiple opticalconfocal sections. (B) Immunofluorescence localisation of transfected SUMO-1 and the indicated PML mutants in 2C4 cells. Exogenous PMLwas detected using the PML polyclonal antibody (green channel) and exogenous SUMO-1 was detected with the 9E10 monoclonal antibody(red channel). The various transfected PML mutants were (i) Cys57.60∆Ala, (ii) Lys65.68∆Ala, (iii) Lys476.478∆Arg, (iv) Lys487.490∆Arg,(v) Lys516∆Arg. A merge of both signals is shown in the right panels with any overlapping staining appearing yellow. It should be noted thatboth exogenous and endogenous PML is being detected in these experiments. All images represent a projection of multiple optical confocalsections. (C) Confocal analysis of the PML mutants Lys65.68∆Ala and Lys476.478∆Arg, tagged with the myc-epitope and transfected into 2C4cells. Expressed mutants were immunolocalised using the 9E10 myc-monoclonal antibody (red channel), with nuclei counter-stained withDAPI (blue channel). The serial projection (left panels) represent a projection of 22 confocal sections of 0.7µm. Two of these single sections(sections 4 and 13) are shown to better define the nuclear localisation of the mutants. Bars, 10 µm.

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mutants affects the appearance of the SUMO-1-related band(Fig. 5A, lanes 4,6-7). For the Lys487.490∆Arg NLS mutant,this band is almost completely absent (Fig. 5A, lane 7) whereasit is significantly reduced for the Lys476.478∆Arg mutant (Fig.5A, lane 6), possibly reflecting the capacity of these mutantsto localise to the nucleus. The Cys57.60∆Ala RING mutantalso shows a significant reduction in this band when comparedto wild-type PML (Fig. 5A, compare lanes 3 and 4). Thesereductions, however, do not appear to be related to PMLprotein expression levels (Fig. 5A, compare lanes 4 and 5). Theappearance of this SUMO-1-related band is not altered byeither the Lys515∆Arg or Lys65.68∆Ala mutants, whichappear like wild-type (Fig. 5A, compare lanes 5,8 with 3). Itis notable that exogenous PML appears as a ‘smear’ and hasaltered electrophoretic properties depending on the particularPML mutant tested (Fig. 5A, compare PML in lanes 4 and 5with lanes 2 and 6). We attribute this to possiblephosphorylation of exogenous PML, since endogenous PMLhas been shown to be a phosphorylated in vivo (Chang et al.,1995). The altered mobilities of these PML mutants couldeither reflect alterations in the pattern of phosphorylation orchanges in the overall net charge or structure due to mutation(e.g. Lys65.68∆Ala; lane 5).

To verify these observations, stable Chinese hamster ovary(CHO) cell lines were made expressing the PMLCys57.60∆Ala, Lys487.490∆Arg and Lys515∆Arg mutants. InCHO cells, the PML antibody only recognises two proteinspecies, one of which is concurrent with the exogenous PML

69 kDa protein (see Fig. 5B, lane 1). Nonetheless, westernanalyses using the PML antibody in CHO cells enables aclearer assessment of the PML mutant expression pattern thanin human cells (Fig. 5B). The PML Lys515∆Arg mutant, whichin all previous assays displayed a wild-type phenotype,demonstrates an in vivo modification pattern by the appearanceof two additional higher molecular mass species (Fig. 5B, lane4 arrowheads). This modification pattern is identical to thatobtained during the in vitro SUMO-1 modification analysis(compare Fig. 5B, lane 4 with Fig. 3, lane 10), in that the moreabundant lower protein species (Fig. 5B, double arrowhead)represents a larger amount of modified PML than the lessabundant upper species (Fig. 5B, single arrowhead). Thisfurther enforces the idea that the K515 lysine is not a target forSUMO-1 modification. More importantly, however, the PMLLys487.490∆Arg mutant is not SUMO-1-modified at the majormodification site in CHO cells (see Fig. 5B, lane 3), whichcorroborates with both the transient expression results (Fig.5A, lane 7) and the results in vitro (Fig. 3, lane 8).

The contrasting observations for the PML Cys57.60∆Alamutant in vitro (Fig. 3) and in vivo (Fig. 5A) are confusing, inthat a SUMO-1 modification profile similar to wild type isobserved in vitro (see Fig. 3, lane 2), whereas in transientexpression studies a significant reduction in SUMO-1-modification is observed (see Fig. 5A, lane 4). It is also notablein this context that in yeast the interaction between PMLCys57.60∆Ala and SUMO-1 is significantly reduced (see Fig.2B). In stable CHO cells expressing the Cys57.60∆Ala PML


Fig. 5.Western blot analyses of cells co-expressing PML and SUMO-1. (A) Western blot analysis of human fibrosarcoma 2C4 cells transfectedwith SUMO-1 (lane 1), PML 69 kDa (lane 2) or co-transfected with SUMO-1 and PML 69 kDa (lane 3). Additional co-transfections wereperformed with SUMO-1 and the indicated PML mutants (lanes 4-8). Extracts from the various transfection experiments were lysed in SDSsample buffer and separated by SDS-PAGE with the resultant proteins analysed by western blotting using the PML polyclonal antiserum. Theprotein species corresponding to transfected PML is labelled and the major modified PML species indicated by a double arrowhead.(B) Chinese hamster ovary (CHO) cells were stably transfected with three different PML 69 kDa mutants. Expressed proteins were detectedwith a PML antibody which does not recognise much hamster PML. Cell extracts were prepared from non-transfected CHO cells (lane 1); PMLCys57.60∆Ala mutant-expressing cells (lane 2); PML Lys487.490∆Arg mutant-expressing cells (lane 3); and PML Lys515∆Arg mutant-expressing cells (lane 4). Single arrowhead, minor modified PML species; see Fig. 3.


mutant, modification by endogenous SUMO-1 is alsoabrogated (Fig. 5B, lane 2), which corroborates both thetransient expression studies and the yeast data. Taken together,these data strongly suggest that nuclear body localisation isrequired for SUMO-1 modification in vivo. In summary, by invivo western analyses on a number of PML mutants we showthat mutation of cysteines 57 and 60 to alanine, and mutationof lysines 487 and 490 to arginine, dramatically effects themajor modification of PML by SUMO-1.

SUMO-1 modification of PML 97kDa isoform and thetranslocated fusion protein PML-RARAIn order to determine whether SUMO-1 modification is specificto particular PML isoforms, we tested the longer PML 97 kDaisoform (Goddard et al., 1991; Fig. 6A) for SUMO-1modification in vitro. Using the in vitro conjugation assay, weshow that PML 97 kDa is efficiently SUMO-1-modified, withthe appearance of at least three SUMO-1-PML conjugates (Fig.6B; asterisks). The appearance of three SUMO-1-conjugates issimilar to that observed by others in vivo (Kamitani et al.,1998; Müller et al., 1998; Sternsdorf et al., 1997), which mayreinforce the hypothesis that the differing PML isoforms areSUMO-1-modified by differing amounts. In addition, we alsotested the APL-associated translocation fusion protein PML-RARA (brc1) for SUMO-1 modification in vitro. Interestingly,PML-RARA can be SUMO-1-modified minimally at two sites(Fig. 6B; asterisks), but during the course of the modificationreaction, two smaller PML-RARA fragments are observed (seeFig. 6B, arrowheads). It is possible that these are the result ofPML-RARA degradation, which only occurs during theSUMO-1 modification reaction. Nonetheless, the appearanceof these bands after the SUMO-1 reaction is dependent on thepresence of SUMO-1, as they are not present when themodification reaction is reconstituted without the addition ofSUMO-1 and UBC9 (Fig. 6B).

In order to correlate the observed in vitro SUMO-1modifications with subcellular localisation, PML 97 kDa andPML-RARA were co-expressed with SUMO-1 in human 2C4cells. Exogenous PML 97 kDa is exclusively in the cytoplasmwith accumulations around the nuclear envelope, but with noapparent sequestration of exogenous SUMO-1 (Fig. 6C).Although PML 97 kDa can be efficiently SUMO-1-modifiedin vitro, western analyses of exogenous material shows noapparent SUMO-1 modification (data not shown). These datawould suggest that SUMO-1 modification only occurs in thenucleus. In contrast, PML-RARA localises to the nucleus andpartially colocalises with exogenous SUMO-1 (Fig. 6C).Furthermore, PML-RARA can be efficiently SUMO-1-modified in vitro and in vivo (Müller et al., 1998), furthersupporting the notion that SUMO-1 modification of PMLoccurs within the nucleus.

DISCUSSION

Three reports have been published providing evidence for thecovalent modification of PML by SUMO-1, using western blotanalyses and immunoprecipitation (Kamitani et al., 1998;Müller et al., 1998; Sternsdorf et al., 1997). In contrast toRanGAP1 or IκBα, where a single lysine residue is theacceptor site for SUMO-1 modification (Desterro et al., 1998;

Mahajan et al., 1998), PML appears to contain a number ofdifferent lysine residues which can be modified (Kamitani etal., 1998; Müller et al., 1998; Sternsdorf et al., 1997).Furthermore, some experimental evidence has been presentedwhich suggests that SUMO-1 modification is part of a PMLsub-nuclear localisation mechanism, whereby SUMO-1modified PML is specifically targeted to the nuclear matrix(Müller et al., 1998). In this report we have extended theseprevious studies to identify specific lysines in PML which areSUMO-1 modified both in vivo and in vitro. Furthermore, wehave studied the binding of UBC9 to PML and have correlatedthe affects of removing a SUMO-1 modification site and/orUBC9 binding with PML nuclear and sub-nuclear localisation.

It has been known for some time that PML can exist as anumber of distinct isoforms varying in sequence and length atthe C terminus, but with identical N-terminal sequencescomprising the RBCC domain and nuclear localisation signal(Goddard et al., 1991). In the previous PML-SUMO-1 studies,a number of different PML isoforms were used in exogenousexpression studies to show the presence of multiple (at leastthree) SUMO-1 modification sites (Kamitani et al., 1998;Müller et al., 1998; Sternsdorf et al., 1997). In our study usingboth the PML 69 kDa and 97 kDa isoforms (Goddard et al.,1991), we observe alterations in the SUMO-1 modificationpattern that is dependant on the PML isoform (see Figs 3, 5,6B). The 69 kDa isoform exhibits one major and one minorPML-modified species in vitro and at least one major PML-modified species in vivo, whereas the 97 kDa isoform exhibitsmodification at three potential acceptor sites in vitro. It alsoappears that different PML lysines can be modified withvarying efficiencies in vitro, with both isoforms showing onemajor SUMO-1 modification site (see Figs 3, 6B). Thisdifference in PML isoform modification could be explained bythe variability of the C termini of both molecules, although thebiological significance of the different degrees of SUMO-1modification is at present unclear.

Our yeast two-hybrid interaction assays show that the PMLRING finger domain and proline-rich N terminus affect theinteractions between PML and SUMO-1 or UBC9. These datashow that the RING finger is essential for the SUMO-1 andUBC9 interaction with PML in yeast, since deletion of theRING finger abrogates these interactions (see Fig. 1B).Interestingly, removal of the proline-rich N-terminal regionincreases the PML-SUMO-1 interaction, suggesting that thisregion can negatively regulate SUMO-1 interaction. This is ofparticular interest, since the N-terminal proline-rich regioncontains numerous potential phosphorylation sites and it isknown that phosphorylation of both IκBα and PML isinhibitory for SUMO-1 modification (Desterro et al., 1998;Müller et al., 1998). Although speculative, it is possible thatphosphorylation of the N-terminal proline-rich region affectsthe level of PML-SUMO-1 interaction in yeast and perhaps invivo, a hypothesis which we are currently exploring. We alsostudied in yeast the interactions of a number of PML pointmutants with UBC9 and SUMO-1 (see Fig. 2). We find that thedouble Cys mutants in the RING finger, which would affect thestructural integrity of this domain, decrease the interaction withSUMO-1 and UBC9, which is consistent with the RINGdeletion mutant. However a double Lys mutant in the RINGfinger did not affect the SUMO-1 nor the UBC9 interaction,suggesting that these lysines do not contribute to the interaction

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in yeast. However, double point mutants of lysines in the NLS(Lys to Arg) significantly reduce the interaction with SUMO-1 without affecting the interaction with UBC9, suggesting thatthese lysines are important for the PML-SUMO-1 interactionin yeast.

In order to confirm the involvement of these lysines inSUMO-1 modification, we carried out in vitro analyses usingan assay system comprising 35S-labelled PML substrate, apartially purified fraction from HeLa cells containing theSUMO-1 activating enzyme (E1) and recombinant SUMO-1and UBC9 (Desterro et al., 1998). Employing this assay, weclearly demonstrate that either Lys487 or 490 are the majorPML-SUMO-1 modification residues. It is notable that thedouble Cys RING mutant is efficiently SUMO-1-modified invitro and appears like wild-type PML, unlike the observationsin yeast, where this mutant has reduced binding to bothSUMO-1 and UBC9. To further analyse the SUMO-1

modification of PML in vivo, we also carried out expressionstudies of wild-type PML and the PML mutants with SUMO-1. These data also confirmed the in vitro observations, in thatLys487 or 490 are SUMO-1-modified (Fig. 5). This isparticularly seen in the hamster cell expression studies, whichallowed the clear detection of exogenous human PML (Fig.5B). However, both expression studies clearly showed that incontrast to the in vitro assay, the double Cys RING mutant isnot SUMO-1-modified at the major modification site. Sincethis mutant can be efficiently modified in vitro, it is likely thatfurther post-translational modifications and/or subcellularcompartmentalisation play a role in the SUMO-1 modificationof PML in vivo.

Our results point to the major acceptor site for SUMO-1modification as one of the two lysine residues (Lys487 orLys490) that form part of the PML NLS. Mutation of theselysines to arginine dramatically decreases the modification of


Fig. 6.SUMO-1 modification and subcellular localisation of PML 97 kDa and PML-RARA. (A) Schematic representation of PML 97 kDaand the PML/RARA fusion protein. PML 97 kDa is the alternatively spliced isoform (PML1) of 857 amino acids (Goddard et al., 1991),containing 300 amino acids of additional sequence (black box) compared to the PML 69 kDa isoform. PML/RARA is the cancer-predisposingfusion protein generated by the t(15;17) chromosomal translocation at brc1, which generates the longer PML/RARA isoform. (B) PML 97kDa and PML/RARA were assayed for SUMO-1 modification in vitro as described in Fig. 3. The SUMO-1-modified complexes are indicatedby asterisks. For PML/RARA the lower bands, indicated by white single arrowheads, are probably degradation products of PML-RARA.These bands only appear in the SUMO-1-modified PML-RARA (+) and not when PML-RARA is translated in the absence of SUMO-1 andUBC9 (#) For comparison, translated PML-RARA alone is shown (−). (C) Subcellular localisation of myc-tagged SUMO-1 (red channel)when co-expressed in 2C4 cells with the PML 97 kDa isoform (top panels; green channel) and with the PML/RARA fusion protein (lowerpanels; green channel). A merge of both fluorescence signals is shown (right panels) where overlapping immunofluorescence signals appearyellow. Bars, 10 µm.


PML both in vitro and in vivo. IκBα is modified by SUMO-1predominantly on Lys21 (Desterro et al., 1998), whileRanGAP1 is modified at Lys526 (Mahajan et al., 1998).Comparison of the sequences surrounding the two acceptorlysines has allowed the definition of a consensus SUMO-1modification sequence (Desterro et al., 1998). By comparingthis consensus sequence with the sequence of the PML NLS,a remarkable fit to the consensus is observed, stronglysuggesting that Lys490 is the lysine that is SUMO-1-modified(Fig. 7). By further developing this consensus sequence, wecan align a number of other sequences from potential, as wellas known SUMO-1-modified proteins (Fig. 7). There appear tobe two groups with those sequences like PML showingsequence similarity over the full length of the PML NLS.Interestingly, all of these proteins are nuclear, although it is notknown whether this region of similarity is the NLS for theseproteins. Apart from the absolutely conserved SUMO-1acceptor lysine, there appears to be an absolute requirement fora hydrophobic residue and a Glu on either side of the acceptorLys (Fig. 7). Of particular interest is ataxin, a nuclear matrixprotein which, when mutated, is associated withSpinocerebellar Ataxia type I, causing a specific redistributionof PML from PML nuclear bodies (Skinner et al., 1997).However, further studies will be necessary to confirm thevalidity of this PML-SUMO-1 modification consensussequence.

Subcellular localisation of the SUMO-1-target proteinRanGAP1 is affected when modified with SUMO-1, in thatonly modified RanGAP1 localises to the nuclear pore complex(Mahajan et al., 1997; Matunis et al., 1996). Moreover,previous studies have shown that the PML-SUMO-1modification is related to the partitioning of PML within thenucleoplasm and nuclear matrix (Müller et al., 1998;Sternsdorf et al., 1997). Here, we find that the major SUMO-1 modification site is part of the NLS, which prompts thequestion as to whether the SUMO-1 modification of PML isessential for nuclear import and localisation. To address this,

we studied the subcellular localisation of tagged PML mutants.From these results we find that the PML RING Cys mutant isnuclear-diffuse with the RING Lys mutants forming large PMLnuclear bodies (Fig. 4B), similar to our previous observations(Boddy et al., 1997; Borden et al., 1995). However,surprisingly, we find that the conservative replacement of Lysresidues to Arg within the NLS, irrespective of SUMO-1modification (Lys476.478 for example), profoundly affects theefficiency of nuclear import and localisation, although bothPML NLS mutants are targeted to the nuclear envelope (Fig.4C). This is contrary to numerous previous studies, which haveshown that NLS signals comprises both Lys and Arg residuesthat together form a positively charged interface specific fornuclear localisation (Görlich, 1998). However, a recentstructure of karyopherin α bound to an NLS peptide doessuggest specificity for Lys residues at position two (Conti etal., 1998). Nonetheless, the bipartite NLS for PML appears tobe highly specific in that alteration of specific lysine residueson either side of the NLS affect nuclear localisation.

The compartmentalisation of the cell thus makes the in vivosubcellular localisation analysis difficult to interpret, since wecannot effectively address the question of whether SUMO-1modification regulates PML nuclear localisation or whetherPML needs to be in the nucleus to become modified. The factthat we have PML mutants which are differently modified invitro and in vivo allows us to formulate some conclusions. Themutant Lys476.478∆Arg, which accumulates both at thenuclear envelope and inside the nucleus, exhibits a reducedmodification by SUMO-1 in vivo. However, in vitro, thismutant can be modified like wild-type PML. The samephenomenon is also observed for the PML 97 kDa isoformwhich, for unknown reasons, cannot be localised into thenucleus despite having an intact NLS sequence (data notshown) but can be modified in vitro (Fig. 6). Together thesedata suggest that SUMO-1 modification of PML does not occurin the cytoplasm but that PML can only be modified in thenucleus. Further support for this suggestion comes from thelocalisation of the double Cys RING mutant. This mutant isnot significantly SUMO-1-modified in vivo, but it efficientlylocalises to the nucleus albeit not to PML nuclear bodies(diffuse pattern), strongly suggesting that SUMO-1modification per se is not essential for nuclear localisation. Insummary, it appears that, despite the presence of a majorSUMO-1 modification site in the PML NLS, SUMO-1modification of this site is not necessary for nuclearlocalisation and that SUMO-1 modification only occurs in thenucleus. Is SUMO-1 modification therefore necessary fortargeting PML to PML nuclear bodies and/or the nuclearmatrix? At present we are unable to directly address this sincethe PML NLS mutant which contains the major SUMO-1-modified lysine is not localised to the nucleus. However, thenuclear diffuse localisation of the double Cys RING mutant,combined with the lack of in vivo SUMO-1 modification,supports the notion that SUMO-1 modification is part of PMLnuclear body targeting. Furthermore, our results suggest thatthe RING finger is important for this targeting, perhaps bymediating SUMO-1 modification through direct or indirectinteractions with UBC9.

PML-RARA is the fusion protein found in APL, and twoprevious studies have obtained different results in terms ofSUMO-1 modification (Kamitani et al., 1997; Müller et al.,

RanGAP1 GLLKSEDKVIkappaB GL-KKERLLSP100 VDIKKEKPFPML KRKCSQTQCP--RKVIKMESEEMOK2 RRLFVMEESTE-RKVIKGESCSEZH2 KKSEKGPVCW-RKRV-KSEYMRID1 RLKELVPTLPQNRKVSKVEILQLYL-1 RKLLPTHPPD--RKLSKNEVLRSCA1 RKRRWSAPES--RKLEKSEDEP

* *

Fig. 7.Multiple sequence alignment of known and predicted SUMO-1 modification sites. The asterisks mark the SUMO-1-modified Lysas well as the absolutely conserved Glu. PML is aligned so as to fitthe consensus sequence, which suggests that Lys 490 is the primarymodification site in PML. The boxed sequences are human nuclearproteins which fit the PML consensus, based on database searches.MOK2, zinc finger protein (Q16600); EZH2, enhancer of zestehomologue 2 (Q15910); ID1, DNA binding protein inhibitor(P41134); LYL-1, protein P129800; SCA1, spinocerebellar ataxiatype 1 protein / ataxin (P54253). SP100 (Tomas Sternsdorf, personalcommunication).

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1998). When transiently expressed in COS-7 cells PML-RARAdoes not appear to be modified (Kamitani et al., 1998), althoughafter arsenic acid treatment of NB4 cells, SUMO-1 modifiedPML-RARA can be observed (Müller et al., 1998). In order toaddress the ambiguity between these results, we performed invitro SUMO-1 modification of PML-RARA. Considering thatthe major SUMO-1 acceptor site is within the NLS and that thismotif is conserved in all PML-RARA fusion proteins, one mightexpect SUMO-1 modification to occur. Our results clearly showthat PML-RARA can be modified in vitro, and that thismodification seems to induce specific and SUMO-1-dependantdegradation of PML-RARA (see Fig. 6B). Considering thatexogenous expressed PML-RARA disrupts normal PML nuclearbodies (Fig. 6) and that under those conditions PML-RARA isnot SUMO-1 modified (Fig. 6B), it is possible that SUMO-1modification of PML-RARA only occurs when targeted tonormal PML nuclear bodies. During retinoic acid treatment orarsenic acid treatment, this is the case where PML-RARA istargeted to the normal PML nuclear bodies and gets degraded(Zhu et al., 1997). Considering these and previous data, it istempting to speculate that SUMO-1 modification is part of aPML-RARA degradation mechanism upon arsenic treatment, ahypothesis which we are currently exploring.

The authors would like to thank Prof. P. Pellici for providing thePML-RARAexpression clone and D. Grimwade for critical reading ofthe manuscript. E. D. was funded with a TMR fellowship(ERBFMBICT961798). R. T. H. and J. M. P. D. acknowledge thesupport of BBSRC and JNICT-Praxis XXI (Portugal).

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