PML Protein Isoforms and the RBCC/TRIM Motif

PML protein isoforms and the RBCC/TRIM motif

Kirsten Jensen1, Carol Shiels1 and Paul S Freemont*,1

1Centre for Structural Biology, Imperial College of Science, Technology and Medicine, Flowers Building, Armstrong Road, London SW7 2AZ, UK

PML is a component of a multiprotein complex, termed reciprocal chromosomal translocation of chromosomes nuclear bodies, and the PML protein was originally 15 and 17. This translocation results in a fusion protein discovered in patients suering from acute promyelocytic comprising PML and the retinoid acid receptor a leukaemia (APL). APL is associated with a reciprocal (RARa) (de TheÂ et al., 1991; Goddard et al., 1991; chromosomal translocation of chromosomes 15 and 17, Kakizuka et al., 1991; Kastner et al., 1992). The which results in a fusion protein comprising PML and expression of the fusion protein PML-RARa is the retinoic acid receptor a. The PML genomic locus is sucient for transformation in APL cells and the approximately 35 kb and is subdivided into nine exons. A development of leukaemia (Altabef et al., 1996; Brown large number of alternative spliced transcripts are et al., 1997; Grisolano et al., 1997). APL is character- synthesized from the PML gene, resulting in a variety ized by a block in dierentiation of promyelocytes, and of PML proteins ranging in molecular weight from 48 ± unlike normal cells, PML is dispersed into hundreds of 97 kDa. In this review we summarize the data on the tiny dots in the nucleus and the cytoplasm in APL cells known PML isoforms and splice variants and present a (Dyck et al., 1994; Koken et al., 1994; Weis et al., new unifying nomenclature. Although, the function/s of 1994). the PML variants are unclear, all PML isoforms contain One striking feature of APL cells is that treatment an identical N-terminal region, suggesting that these with all-trans retinoid acid (ATRA) restores the normal sequences are indispensable for function, but dier in pattern of PML nuclear bodies (Daniel et al., 1993) their C-terminal sequences. The N-terminal region and the dierentiation block of the promyelocytes is harbours a RING-®nger, two B-boxes and a predicted released (Huang et al., 1988). Treatment of APL a-helical Coiled-Coil domain, that together form the patients with ATRA often leads to complete remission, RBCC/TRIM motif found in a large family of proteins. which is however, mostly short-lived (see review by In PML this motif is essential for PML nuclear body Degos and Wang in this issue). More recently, it has formation in vivo and PML-homo and hetero interac- been demonstrated that treatment with low doses of tions conferring growth suppressor, apoptotic and anti- arsenic trioxide also represents a successful alternative viral activities. In APL oligomerization mediated by the treatment for APL, inducing apoptosis in leukaemic RBCC/TRIM motif is essential for the transformation cells (Chen et al., 1996; Zhu et al., 1997). Disintegra- potential of the PML-RARa fusion protein. Oncogene tion of PML nuclear bodies is believed to play a key (2001) 20, 7223 ± 7233. role in the development of APL, which is consistent with the observation that the normal PML protein has Keywords: PML isoforms; splice variants; RBCC; growth- and transformation-suppressing properties SUMO; TRIM (Ahn et al., 1995; Koken et al., 1995; Le et al., 1996; Mu et al., 1994; Quignon et al., 1998). Other studies on PML and PML nuclear bodies in cell growth control Introduction have been carried out using cell lines derived from PML knockout mice, revealing tumour suppressor and PML is a part of a multiprotein complex called PML pro-apoptotic functions for PML (Wang et al., 1998). nuclear bodies, ND10 or PODs (PML oncogenic Recently, it has been shown that PML might be domains) (reviewed in Hodges et al., 1998; Sternsdorf involved in p53-dependent apoptosis (Fogal et al., et al., 1997a). It is a heterogenous group of nuclear 2000; Guo et al., 2000) and also in the regulation of multi-protein complexes (Brasch and Ochs, 1992) gene expression (Wu et al., 2001; Zhong et al., 2000). which do not colocalize with any of the known nuclear More extensive reviews on PML function are presented substructures (Ascoli and Maul, 1991; Dyck et al., in this volume. 1994; Raska et al., 1992; Stuurman et al., 1992; Weis et A large number of proteins are described to localize al., 1994). PML was originally discovered in patients to PML nuclear bodies. Some of these namely, PML, suering from the haematopoietic malignancy acute Sp100 and ISG20 are inducible by IFN a/b (Type I) promyelocytic leukaemia (APL), associated with a and IFN g (Type II) (Chelbi-Alix et al., 1995; Gongora et al., 1997; GroÈ tzinger et al., 1996; Lavau et al., 1995; Stadler et al., 1995) suggesting that PML nuclear *Correspondence: PS Freemont; E-mail: [email protected] bodies could be part of a cellular defence mechanism. PML isoforms and the RBCC/TRIM motif K Jensen et al 7224 In support of this notion, PML nuclear bodies are RBCC/TRIM family, TIF1 and Rfp, also become often targeted and disrupted by various viral regulatory oncogenic as a result of chromosomal translocations proteins (reviewed in Seeler and Dejean, 1999; (Hasegawa et al., 1996; Klugbauer and Rabes, 1999; Le Sternsdorf et al., 1997a), possibly leading to an Douarin et al., 1995). inactivation of these defence systems and subsequent expression of viral genes (see review by Regad and Chelbi-Alix; and Everett in this issue). Another Identi®cation of alternative spliced PML transcripts important PML nuclear body constituent is the small ubiquitin-related protein SUMO-1 (Boddy et al., 1996), The PML gene consist of nine exons and several found covalently linked to PML (Kamitani et al., alternative spliced PML transcripts have been de- 1998a; Muller et al., 1998; Sternsdorf et al., 1997b). scribed (de TheÂ et al., 1991; Fagioli et al., 1992; Modi®cation of proteins by SUMO-1 is proposed to Goddard et al., 1991; Kakizuka et al., 1991; Kastner et either, regulate speci®c protein ± protein interactions, al., 1992). Interestingly, all of the PML isoforms target proteins to speci®c subcellular compartments, or contain the N-terminal region comprising the RBCC/ to prevent degradation by the ubiquitin-dependent TRIM motif, but dier either in the central region or pathway (Desterro et al., 1998; Everett et al., 1998; in the C-terminal region, due to alternative splicing Mahajan et al., 1997; Matunis et al., 1996; Muller et (Figure 1 and Table 1a,b). In addition to the RBCC/ al., 1998; Saitoh et al., 1997). It has also been proposed TRIM, all PML isoforms (excluding the b/c variants) that SUMO modi®cation has a role in protein contain the three characterized SUMO-1 modi®cation degradation (Lallemand-Breitenbach et al., 2001; see sites (at position 65 in the RING ®nger, 160 in the B1- review by de TheÂ in this issue). The functional Box and 490 in the NLS) and a bipartite nuclear implications of PML SUMO modi®cation is discussed localization signal, suggesting that the functionality of by Seeler and Dejean in this volume. these motifs are relevant to most PML isoforms. It is The PML protein itself contains three cysteine-rich notable that in all of the cell lines examined to date, zinc-binding domains, a RING-®nger, two B-boxes (B1 the diering PML splice variants are more or less and B2) and a predicted a-helical Coiled-Coil domain, equally expressed (Fagioli et al., 1992), although total which together form the RBCC motif (Borden, 1998; cell extracts blotted with the PML 5E10 mAb show Saurin et al., 1996) or recently termed TRIpartite some variation in protein levels (H de TheÂ , personal Motif or TRIM (Reymond et al., 2001). The RBCC/ communication). TRIM motif is present in all PML isoforms and in the The PML isoforms can be divided into seven groups, PML/RARa fusion protein and is discussed extensively which we are designating as PML I ± VII, on the basis below. It is notable that two other members of the of sequence dierences at the C-terminus. (Figure 1

Figure 1 Exon assembly and structural domains of the PML isoforms. All PML isoforms share the N-terminal region containing the RBCC motif. The seven dierent C-termini are generated by alternative usage of 3' exons, resulting in PML isoforms of varying sizes (total length of each isoform is given on the right). SUMOylation sites (S) at amino acid positions 65, 160 and 490 are indicated. *Retained intron

Oncogene PML isoforms and the RBCC/TRIM motif K Jensen et al 7225 Table 1A Summary and exon assembly of PML isoforms Isoforms Exons References

PML 1 1-2-3-4-5-6-7a-8a-9 . . . PML4 (Fagioli et al., 1992) PML-1 (Goddard et al., 1991) TRIM19 alpha (Reymond et al., 2001) PML II 1-2-3-4-5-6-7a-7b . . . PML2 (Fagioli et al., 1992) PML-3 (Goddard et al., 1991) TRIM19 gamma (Reymond et al., 2001) TRIM19 delta (Reymond et al., 2001) TRIM19 kappa (Reymond et al., 2001) PML III 1-2-3-4-5-6-7a-7ab retained intron-7b . . . PML-L (de TheÂ et al., 1991) PML IV 1-2-3-4-5-6-7a-8a-8b . . . PML3 (Fagioli et al., 1992) Myl (Kastner et al., 1992) TRIM19 zeta (Reymond et al., 2001) PML V 1-2-3-4-5-6-7a-7ab retained intron . . . PML1 (Fagioli et al., 1992) PML-2 (Goddard et al., 1991) TRIM19 beta (Reymond et al., 2001) PML VI 1-2-3-4-5-6-intron seq.-7a . . . PML-1 (Kakizuka et al., 1991) PML-3B (Goddard et al., 1991) TRIM19 epsilon (Reymond et al., 2001) PML VIIb 1-2-3-4-7b . . . TRIM19 theta (Reymond et al., 2001)

Table 1B Nomenclature for PML isoforms and alternative spliced variants Isoforms References Accession

PML I 882aa PML4 (Fagioli et al., 1992) 882aa PML-1 (Goddard et al., 1991) M79462 882aa TRIM19 alpha (Reymond et al., 2001) AF230401 PML II 829aa PML2 (Fagioli et al., 1992) 824aa PML-3 (Goddard et al., 1991) M79464 824aa TRIM19 gamma (Reymond et al., 2001) AF230403 854aa TRIM19 delta1 (Reymond et al., 2001) AF230404 829aa TRIM19 kappa (Reymond et al., 2001) AF230410 PML III 641aa PML-L (de TheÂ et al., 1991) S50913 PML IIIa 593aa PML-S (de TheÂ et al., 1991) PML IV 633aa PML3 (Fagioli et al., 1992) 633aa Myl (Kastner et al., 1992) X63131 633aa TRIM19 zeta (Reymond et al., 2001) AF230406 PML IVa 585aa TRIM19 lambda (Reymond et al., 2001) AF230411 PML V 611aa PML1 (Fagioli et al., 1992) 611aa PML-2 (Goddard et al., 1991) M79463 611aa TRIM19 beta (Reymond et al., 2001) AF230402 PML VI 560aa PML-1 (Kakizuka et al., 1991) M73778 560aa PML-3B (Goddard et al., 1991) M80185 560aa TRIM19 epsilon (Reymond et al., 2001) AF230405 PML VIb 423aa TRIM19 iota2 (Reymond et al., 2001) AF230409 PML VIb 423aa TRIM19 eta2 (Reymond et al., 2001) AF230407 PML VIIb 435aa TRIM19 theta (Reymond et al., 2001) AF230408 adenotes isoform without exon 5. bdenotes isoform without exon 5 and 6. cdenotes isoform without exon 4, 5 and 6. 1Same as PML-3 but contains an extra 30aa repeat. 2Missing intron seq. GTAGGGAG before exon 7a. Note: There is evidence to suggest that all isoforms can exist as a/b/c variants (Fagioli et al., 1992)

and Table 1a). A further division into sub-groups a/b/c new PML isoform (PML VIIb/TRIM19 theta) and one re¯ects the alternative splicing of exons 4, 5 and 6 new alternative spliced variant (PML VIb/TRIM19 (Table 1b). It has been shown that PML III and PML iota/eta) were identi®ed. There is some evidence to IV exist without exon 5 (de TheÂ et al., 1991; Fagioli et suggest that all PML isoforms can exist as a/b/c al., 1992) and that PML V can exist without exons 5 variants (Fagioli et al., 1992), although further studies and 6 or 4, 5 and 6 (Fagioli et al., 1992), although the are needed to con®rm this. Since the b and c variants sequences for the latter two isoforms are not available. are missing the NLS, they are likely to be cytoplasmic. Recently a comprehensive study of all human proteins, It should be noted that there are two proposed belonging to the RBCC/TRIM-family, has been initiation methionines resulting in PML translation described (Reymond et al., 2001). In this study, one products that dier by 22 amino acids. The functions

Oncogene PML isoforms and the RBCC/TRIM motif K Jensen et al 7226 of the PML splice variants are not known, alternative repress transcription by interacting with histone splicing could add new functional domains to the deacetylase (HDAC) (Wu et al., 2001). In contrast protein or may be an important mechanism for PML VI does not interact with HDAC (Li et al., 2000) generating diverse PML-binding interfaces for a variety and PML/RARa interacts poorly, suggesting that the of factors. C-terminal region of PML IV protein is required for HDAC interaction. In APL this transcription-silencing function of PML might be disrupted (by the expression Isoform speci®c interactions of PML-RARa) and may lead to altered chromatin remodelling and gene expression patterns and subse- An extensive number of studies have been carried out quent onset of leukaemia (Saha et al., 1998). aimed at identifying speci®c PML interacting proteins The retinoblastoma protein (pRB), a key protein in (Table 2). However, careful analysis of the particular regulating cell growth, also colocalizes with PML PML isoform used in these studies often reveals that nuclear bodies (Alcalay et al., 1998). PML IV interacts only one isoform has been studied. It still remains to with the nonphosphorylated fraction of the retinoblas- be shown if the observed PML-protein interactions are toma protein (pRB), with the RBCC/TRIM motif speci®c to individual PML isoforms. Here we focus on required for optimal stability of the complex (Alcalay several PML isoform-speci®c interactions. et al., 1998). However, the PML C-terminus is also Recently, an isoform speci®c interaction between p53 important for pRB interaction, as PML II exhibited a and PML IV has been demonstrated (Fogal et al., weaker interaction compared to PML IV (Alcalay et 2000). These data showed p53 recruited to PML al., 1998). Surprisingly, the non-phosphorylated form nuclear bodies by PML IV or by co-expression of of pRB also interacts with PML/RARa despite the SUMO-1 and its E2 conjugating enzyme hUBC9. The absence of the PML IV C-terminus, although other p53-PML IV interaction is mediated by the p53 core cellular proteins might be involved in the formation of domain and the C-terminal part of PML IV, as p53 a PML/RARa-pRB complex in vivo. Interestingly, does not interact with PML-RAR a or PML III (Fogal PML, but not PML/RARa, has an inhibitory eect et al., 2000). Furthermore overexpression of PML IV on pRB transactivation, suggesting that an interaction in cells expressing wild type p53 results in a reduction between PML and pRB might be important in the of cell survival, suggesting that recruitment of p53 into regulation of cell dierentiation and proliferation PML nuclear bodies (by PML IV) modulates the (Alcalay et al., 1998). PML/RARa could antagonize survival functions linked to PML nuclear bodies. this eect and thereby contribute to a deregulation of Interestingly, relocalization of p53 to PML nuclear dierentiation and proliferation in APL cells. bodies enhances p53 transactivation in a promotor speci®c manner only when PML IV is SUMO-1 modi®ed, suggesting that p53 activity is modulated by Breakpoints in APL PML nuclear body relocalization (Fogal et al., 2000). PML also plays a role in transcriptional regulation. Two major breakpoint positions have been described The PML IV isoform has been shown to speci®cally in APL (de TheÂ et al., 1991; Goddard et al., 1991;

Table 2 PML interacting proteins and their function Function PML interacting proteins PML isoform Reference

Tumour suppression and cell growth control p53* PML IV Fogal et al., 2000 PML IV Guo et al., 2000 pRB* PML IV Alcalay et al., 1998 Transcriptional regulation CBP PML II Zhong et al., 1999 ? Doucas et al., 1999 Sp 1 ? Vallian et al., 1998a Transcriptional repression HDAC* PML IV Wu et al., 2001 Tas PML III Regad et al., 2001 Apoptosis Daxx PML VI Li et al., 2000 ? Ishov et al., 1999 Cell growth control AP-1 PML III Vallian et al., 1998b TIF1a PML II Zhong et al., 1999 RFP PML VI Cao et al., 1998 PRH PML VI Topcu et al., 1999 GATA-2 PML VI Tsuzuki et al., 2000 Ubiquitination Ubc9 PML VI Duprez et al., 1999 Unknown SUMO-1/PIC 1 PML VI Boddy et al., 1996 Z protein (LCMV) PML VI Borden et al., 1998a IE1 (HCMV) PML VI Ahn et al., 1998 P proteins (ribosomal) ? Borden et al., 1998b eIF-4E ? Lai and Borden, 2000

*Isoform speci®c interaction

Oncogene PML isoforms and the RBCC/TRIM motif K Jensen et al 7227 Kakizuka et al., 1991; Kastner et al., 1992; Pandol® et The RBCC/TRIM family al., 1991). These breakpoints mark exon ± exon boundaries and are located between exons 3 and 4 PML belongs to a family of proteins that contain a (amino acid 394; Figure 1) and between exons 6 and 7 RBCC or recently termed TRIM motif (Borden, 1998; (amino acid 552; Figure 1). It is interesting to note Reymond et al., 2001), (Table 3). The RBCC/TRIM that the breakpoints are positioned before and after motif was ®rst identi®ed in the Xenopus transcriptional the central region in the PML protein (exons 4, 5 and regulator XNF7 (Reddy et al., 1992) and comprise 6) that is alternatively spliced, although the relevance several cysteine-rich zinc binding domains (RING of this observation is unclear. In APL, similar splicing ®nger and B-box) followed by a predicted a-helical occurs and PML-RARa and RARa-PML transcripts Coiled-Coil domain (reviewed by Borden, 1998; Saurin have been identi®ed without exon 5 (de TheÂ et al., et al., 1996), (Figure 2). A recent and comprehensive 1991; Kastner et al., 1992). All the PML-RARa fusion study of the RBCC/TRIM family, using a functional proteins contain the RBCC/TRIM motif, but not the genomics approach, has identi®ed and characterized a variable C-terminal domain. The dierent breakpoints large number of novel family members (Reymond et also result in PML fusion proteins that dier in the al., 2001). The genes encoding each of the RBCC/ presence of the PML NLS, SUMOylation sites and TRIM family members are scattered throughout the the caspase proteolytic cleavage site implicated in human genome, but there are two distinct clusters on PML-RARa degradation (Nervi et al., 1998). Only the chromosomes 11p15 and 6p21 ± 23 suggesting that RARa-PML transcripts contain the C-terminal do- duplication of an ancestral RBCC/TRIM gene at these main of the PML protein (from exon 4 or exon 7, genomic locations may have occurred (Reymond et al., depending on the breakpoint). As the function of the 2001). The order of the sub-domains is conserved, with C-terminal domain of the PML protein is not known the RING ®nger, B-box and coiled-coil domains it is dicult to predict the eect of the RARa ± PML arranged from the N to C-terminus. However, the fusion protein (Alcalay et al., 1992; Li et al., 1997), number of B-boxes can vary between dierent family although the RARa-PML fusion has been shown to members (Table 3) and the number of RBCC/TRIM accelerate APL in transgenic mice (Pollock et al., domains is not ®xed since RNF21 has two RBCC/ 1999). TRIM domains in tandem (Orimo et al., 2000a). The

Table 3 Summary of RBCC-containing proteins* Name Structure References

AFP (acid finger protein) R B2 CC RFP Chu et al., 1995 ARD1 (ADP-ribosylation factor domain protein) R B1 B2 CC ARF Mishima et al., 1993; Vitale et al., 1996; Vitale et al., 1998 EFP (estrogen-responsive finger protein) R B1 B2 CC RFP Inoue et al., 1993; Inoue et al., 1999 GERP (glioblastoma expressed RING finger protein) R B1 B2 CC Vincent et al., 2000 HERF1 (haematopoietic RING finger 1) R B2 CC RFP Harada et al., 1999 HT2A R B1 CC NHL Fridell et al., 1995 Leu5 R B2 CC Kapanadze et al., 1998 lin-41 R B1 B2 CC NHL Slack et al., 2000 MID1 (midline defect 1)/FXY (RING finger on X and Y) R B1 B2 CC RFP Quaderi et al., 1997; Palmer et al., 1997; Cainarca et al., 1999; Cox et al., 2000 MID2/FXY2 R B1 B2 CC RFP Buchner et al., 1999; Perry et al., 1999 MUL (Mulibrey nanism) R B2 CC Avela et al., 2000 PML (Promyelocytic leukaemia protein) R B1 B2 CC Kakizuka et al., 1991; de TheÂ et al., 1991; Goddard et al., 1991; Fagioli et al., 1992; Kastner et al., 1992; Reymond et al., 2001 PwA33 R B2 CC RFP Bellini et al., 1993; Bellini et al., 1995 RFP (RET finger protein) R B2 CC RFP Takahashi et al., 1988; Cao et al., 1997 Shimono et al., 2000 TRIM31/RING R B2 CC Reymond et al., 2001 RNF16/TERF (testis RING finger protein) R B2 CC RFP Ogawa et al., 1998 RNF21/ifp1 (interferon responsive finger protein 1) R B CC R B CC RFP Orimo et al., 2000a RNF22/BERP (brain expressed RING finger protein) R B CC NHL El-Husseini and Vincent, 1999; El-Husseini et al., 2000, 2001 RNF23/Tfp (testis-abundant finger protein) R B CC RFP Orimo et al., 2000b RPT1 (regulatory protein, T lym, 1) R B2 CC RFP Patarca et al., 1988 SS-A/Ro (nuclear antigen A of Sjogren's syndrome) R B2 CC RFP Chan et al., 1991 Staf 50 (stimulated transacting factor of 50 kD) R B2 CC RFP Tissot and Mechti, 1995; Tissot et al., 1996 TIF1a R B1 B2 CC BROMO Le Douarin et al., 1995 TIF1b/KAP1/KRIP1 R B1 B2 CC BROMO Moosmann et al., 1996; Kim et al., 1996 Friedman et al., 1996; Peng et al., 2000 TIF1g R B1 B2 CC BROMO Venturini et al., 1999 XL43, XL75 R B1 B2 CC RFP Perrin and Lacroix, 1998 XNF7 (Xenopus nuclear factor 7) R B2 CC RFP Reddy et al., 1991; Shou et al., 1996

*A number of new RBCC/TRIM members have recently been reported in Reymond et al., 2001

Oncogene PML isoforms and the RBCC/TRIM motif K Jensen et al 7228

Figure 2 Schematic of the PML RBCC/TRIM motif. The insert pictures are three-dimensional structural representations of the three sub-domains; the PML RING ®nger domain (Borden et al., 1995a), the XNF7 B-box domain (Borden et al., 1995b) and an idealized heterodimeric alpha-helical coiled-coil. Zinc atoms are represented as grey/white spheres, beta-strands as arrows and alpha- helices as ribbons. The spatial arrangement of three sub-domains is unknown as is the stoichiometry of the predicted coiled-coil, although in PML the B2 B-box leads directly into this region

C-terminal regions are variable, but subfamilies sharing with each sub-domain contributing towards oligomer- domains such as the ret proto-oncogene-encoded ®nger ization and/or ligand binding. protein (RFP)-like domain (also known as the B30.2 domain) and the NHL domain can be de®ned. A shared C-terminal domain between RBCC/TRIM The RBCC/TRIM motif and PML function members may also indicate common functions/activities, for example, the chromatin-associated TIF1 The RING ®nger motif is a cysteine-rich zinc-binding family share a C terminal bromodomain (Jacobson et module found in many functionally diverse proteins al., 2000; Venturini et al., 1999). from plants to man, and was ®rst identi®ed in the The RBCC/TRIM proteins can homo-interact human protein RING1 (Freemont, 1993). The family is through their coiled-coil domains, and in the case of large (fourteenth most common motif in the human RFP, this interaction requires an intact B-box, but not genome), and a number of RING-speci®c functions the RING ®nger (Cao et al., 1997). The correct folding have been postulated. The most cited is for RING of the B-box may be required to help orient the coiled- domains acting as protein ± protein interaction modules coil prior to homomultimerization and to maintain the (Borden, 1998; Kentsis and Borden, 2000) and in correct overall structure of the protein (Cao et al., particular to mediate E3 ubiquitin ligase activities by 1997). The coiled-coil domain of RFP is also required interacting and promoting E2-dependent ubiquitin for interaction with Enhancer of polycomb (EPC) conjugation (Freemont, 2000). The PML RING (Shimono et al., 2000) and for heterodimerization with domain has been subjected to extensive structural and PML (Cao et al., 1998). In the latter case, this functional analyses. A three-dimensional solution interaction is mediated through the distal two helices structure has revealed the conserved RING structural of the coiled-coil domain, whereas the proximal helix elements, namely two zinc atoms ca 14 AÊ apart bound mediates homo-RFP interactions (Cao et al., 1997, via a cross-brace arrangement of Cys and single His 1998). In contrast to these ®ndings, the entire RBCC/ ligands (Borden, 1998; Borden et al., 1995a). Mutations TRIM domain is required for multimer formation and in PML of these conserved zinc-binding ligands (Cys) interaction of TIF1b to the KRAB (KruÈ ppel-associated disrupt PML nuclear body formation in vivo (Borden et box) transcriptional repressor domain. This interaction al., 1995a; Kastner et al., 1992). Furthermore, these is entirely speci®c for the TIF1b RBCC/TRIM domain mutations are correlated with a loss of PML growth since substitution of RBCC/TRIM motifs from related suppression (Fagioli et al., 1998; Mu et al., 1994), proteins such as TIF1a or MID1 do not bind the apoptotic and anti-viral activities (Borden et al., 1997; KRAB domain (Peng et al., 2000). In addition, Regad et al., 2001), see review by Regad and Chelbi- chimaeric RBCC/TRIM proteins containing either the Alix in this issue. RING, B-box or coiled-coil motifs from MID1 fail to The PML RING structure also prompted a series of bind KRAB (Peng et al., 2000). The RBCC domain mutagenesis studies aimed at testing surface residues can therefore function as a single integrated structure and PML nuclear body formation in vivo (Boddy et

Oncogene PML isoforms and the RBCC/TRIM motif K Jensen et al 7229 al., 1997). Several striking mutations that localize to a have shown the B-box to be important for facilitating particular surface region (Glu63DArg; Lys65DGlu; homo-interactions of the coiled-coil region (Cao et al., Glu63Lys65DAla; Lys65Lys68DAla) result in abnor- 1997). Molecular modelling suggests that the position mally large PML nuclear aggregates in vivo that are and orientation of the B-box (adjacent to the coiled- not linked to aberrant PML protein turnover (Boddy coil) would be critical for the correct alignment of the et al., 1997). Co-transfection studies of these mutants a -helices that form the coiled-coil (see below). with PML-RARa however, prevents the formation of Interestingly, unlike the RING and coiled-coil motifs, PML-RARa speckles (E Duprez and P Freemont, the B-box is only found in RBCC/TRIM family unpublished observations), suggesting a dominant members suggesting that it is a critical determinant mutant phenotype in terms of sub-nuclear localization. of the overall motif and its function (Reymond et al., The dependence on an intact RING ®nger for PML 2001). nuclear body formation in vivo may relate to speci®c Coiled-coil structures are formed from inter-wound protein ± protein interactions mediated by the RING. a-helices which produce rod-like structures and their Several studies have shown PML RING-speci®c presence in protein sequences can be predicted (Lupas, interactions with the SUMO-1 E2 enzyme UBC9 1996). Coiled-coils mediate both homo and hetero- (Duprez et al., 1999), the proline rich homeodomain dimeric interactions, as well as higher order multimer PRH (Topcu et al., 1999), and the viral proteins Z interactions, for example trimers/hexamers (Peng et al., (Borden et al., 1998a), IE1 (Ahn et al., 1998) and Tas 2000). The weakly predicted coiled-coil region for PML (Regad et al., 2001). Interestingly, the PML RING is comprises residues 229 ± 323. Several studies have SUMO modi®ed (Kamitani et al., 1998b), although shown that these regions (without the RING and B- other studies are in disagreement (Duprez et al., 1999; boxes) are essential for PML multimerization as well as Lallemand-Breitenbach et al., 2001). Whether PML heterodimerization with PML-RARa (Grignani et al., nuclear body formation in vivo is directly linked to 1996; Kastner et al., 1992; Le et al., 1996; Perez et al., ecient SUMO-1 modi®cation of PML is still a 1993). The coiled-coil region is also essential for PML matter of considerable debate (Lallemand-Breitenbach nuclear body formation and full growth suppressor et al., 2001; see reviews by de TheÂ ; and Seeler and activity in vivo (Fagioli et al., 1998). More importantly, Dejean in this issue), although it is clear that the the formation of high molecular weight complexes RING is essential for PML nuclear body formation in through the coiled-coil is essential for the PML-RARa vivo. retinoic acid response (Grignani et al., 1999; see review Adjacent to the RING domain in PML are two by Evans in this issue). These studies are in general distinct cysteine-rich motifs, termed B-boxes (B1 and agreement with other RBCC/TRIM proteins where B2). Both motifs are small (42 and 46 residues removal of the coiled-coil region prevent the formation respectively) and bind zinc but dier in the number of high molecular complexes (Peng et al., 2000; and spacing of conserved Cys and His ligands (Borden Reymond et al., 2001). et al., 1996). A solution structure of a related B-box In summary the RBCC/TRIM motif in PML is (XNF7) showed B-boxes to be small elongated essential for PML nuclear body formation in vivo as domains with a single well-ordered zinc-binding site well as PML growth suppressor, apoptotic and anti- (Borden et al., 1995b). One surface of the XNF7 B-box viral activities. These and other PML functions appear is hydrophobic and a general model for B-box dependent on higher-order homo or hetero protein ± dimerization (or hetero protein interaction) has been protein interactions as mediated by the RBCC/TRIM proposed that utilizes this surface and a second zinc- motif. binding site (Borden et al., 1995b). In terms of PML, substitution of conserved zinc ligands in B1 and/or B2 disrupt PML nuclear body formation in vivo but do Disease linked RBCC/TRIM mutations not prevent PML-PML oligomerization (Borden et al., 1996). Together with the RING ®nger, the PML B- Mutations within certain RBCC/TRIM proteins have boxes also exert biological activity in terms of PML been described that lead to genetically inherited growth suppressor function (Fagioli et al., 1998). disorders. The gene encoded by MID1 is a micro- Interestingly the B1 SUMOylation site is critical for tubule-associated protein and mutations within this recruitment of the 11S proteosomal subunit to PML gene have been shown to underlie the X-linked form of nuclear bodies (Lallemand-Breitenbach et al., 2001). Opitz Syndrome and correlate with a disruption in the In other RBCC/TRIM proteins only one copy of the microtubule-associated pattern (Cainarca et al., 1999; B-box motif is present, but inspection of the whole Quaderi et al., 1997). Although mutations in the C- family reveals a conserved residue spacing between the terminus of the protein are predominant and lead to RING, B-box and coiled-coil domains (Table 3 and the accumulation of large cytoplasmic aggregates, a Reymond et al., 2001). This strongly suggests that the number of N-terminal mutations have now been overall architecture of the RBCC/TRIM motif is described, including one that is predicted to truncate highly conserved, perhaps relating to the motif acting the protein before the B-box motifs (Cox et al., 2000). as a scaold for higher-order protein ± protein interac- The MID1 protein can homo-interact through its tions (Peng et al., 2000). In support of this idea, RBCC/TRIM motif and forms large protein complexes deletion studies of the RBCC/TRIM protein RFP, within the cell (Cainarca et al., 1999). N-terminal

Oncogene PML isoforms and the RBCC/TRIM motif K Jensen et al 7230 mutations aect the intracellular location of the tions and PML nuclear body formation and is essential protein in a manner quite distinct from the C-terminal for PML growth suppressor, apoptotic and anti-viral mutations, since these mutated proteins do not form activities in vivo. In APL, the motif confers the cytoplasmic aggregates but are instead found diuse transformation potential of the PML-RARa fusion throughout the cytoplasm (Cox et al., 2000). MID1 protein. Although a number of pleotropic cellular mutants missing the B-box domain are also found in functions have been assigned to PML, less is known the nucleus, consistent with the idea that the B-box about PML molecular function and little about the may be required for MID1 cytoplasmic retention (Cox functional roles of the PML isoforms. Given that all et al., 2000), as has also been found for RFP (Cao et the isoforms appear to be expressed at similar levels (in al., 1997). Similarly, mutations in the RBCC/TRIM cell lines studies to date), it will be important to de®ne protein MUL result in Mulibrey nanism (Avela et al., precisely the sub-nuclear localization patterns for each 2000). All four of the MUL mutations described cause isoform, identify isoform-speci®c protein partners and a frameshift and predict a truncated protein, and in test isoform hetero-oligomerization and interactions three of them, as in Opitz Syndrome, the disease with known PML-targeted viral proteins. Our current phenotype does not seem to arise directly from loss of understanding of PML splice variants would suggest function related to the RBCC/TRIM motif (Avela et overlapping but distinct functions for each isoform. al., 2000). One of the big challenges ahead therefore, will be to identify speci®c functions attributable to individual PML isoforms and to understand the functional cross- Summary and future prospects talk between them, given that they can likely heterodimerize. In terms of APL, it will be increasingly The large number of alternatively spliced PML important to study the reciprocal RARa-PML fusion transcripts generates a variety of PML proteins that transcripts and their contribution to the APL pheno- dier in their C-terminal sequences (from 8 to 330 type, given that they would re¯ect alternative PML residues). The observation that particular PML iso- splice variants. forms mediate speci®c protein interactions suggests that PML splice variants could have potentially diverse cellular functions. All PML isoforms contain identical Acknowledgements N-terminal sequences (552 residues) that comprise the We thank Kathy Borden and Thomas Sternsdorf for RBCC/TRIM motif, a motif that is also found in a critical reading of the manuscript and helpful discussions. large family of unrelated proteins. The RBCC/TRIM We also thank all our colleagues for contributing to the domain mediates homo/hetero PML-protein interac- proposed nomenclature for the PML isoforms.

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Oncogene