RING domains: master builders of molecular scaffolds?1

Article No. jmbi.1999.3429 available online at http://www.idealibrary.com on

J. Mol. Biol. (2000) 295, 1103±1112

REVIEW ARTICLE

RING Domains: Master Builders of Molecular Scaffolds? Katherine L. B. Borden Department of Physiology & Biophysics, Mt. Sinai School of Medicine, New York, NY 10029, USA

Intense interest in the RING domain has arisen because of its widespread occurrence and involvement in human disease. Several intriguing characteristics evident from the study of this cysteine-rich, zinc-binding domain have made it dif®cult to establish a single de®ning biochemical function for RINGs. These proteins are found throughout the cell and mediate diverse cellular processes, e.g. oncogenesis, apoptosis, development and viral infection. Recent developments indicate that RING-mediated protein interactions are critical for transcriptional repression and for ubiquitination. These data are in addition to previously established functions for RINGs in RNA processing, cell-cycle control and peroxisomal biogenesis, to name a few. At ®rst glance, there appears to be little to link such disparate actions. Collectively, these results suggest that RINGs function in formation and architecture of large protein complexes that contribute to diverse cellular processes. Here, new developments, in the context of previous results, are discussed in an attempt to establish a unifying theory for RING function. # 2000 Academic Press

Keywords: RING; zinc-®nger; KAP1; ubiquitination

Introduction Intense interest has arisen in RING domains (for Really Interesting New Gene) because of their link to human disease and their widespread occurrence. There have been over 200 RING proteins reported to date. Establishing a function for these domains has been problematic, because proteins containing them are found throughout the nucleus and cytoplasm. Further, RINGs function in a range of cellular processes, including development, oncogenesis, apoptosis and viral replication. At the molecular level, the domains are involved in a variety of functions, from transcription and RNA processing to organelle transport and peroxisomal biogenesis. To make the problem even worse, RINGs are notoriously dif®cult to work with biochemically because of their legendary ability to aggregate and precipitate. The only unifying theme appears to be the ability of RINGs to mediate protein-protein interactions, particularly those involved in formation of large macromolecular scaffolds. E-mail address of the corresponding author: [email protected] 0022-2836/00/051103±10 $35.00/0

Several RING proteins are implicated in human disease. For instance, many of the familial mutations in the breast cancer gene product, BRCA1, are found in its RING. Other examples of RING proteins implicated in human disease include the promyelocytic leukemia protein PML, which is disrupted in acute promyelocytic leukemia, the melanoma 18 protein Mel18, and the newly reported parkin protein, which is disrupted in autosomal recessive familial juvenile Parkinsonism (Morett & Bork, 1999; Saurin et al., 1996). Further, many viruses contain RING proteins that are critical for viral survival and replication (Saurin et al., 1996). In general, disease-related mutations that alter the integrity of the RING result in disruption of the macromolecular scaffolds that the RINGs form, and subsequently the processes that these scaffolds participate in. Frequently, these disruptions have disastrous effects on the fate of the cell. Recent studies demonstrate new functions for the RING. Rauscher and colleagues report in this issue that the RING of KAP1 is necessary for multimerization of KAP1 and for KAP1 to mediate transcriptional repression. Other studies ®nd that RINGs are involved in the process of ubiquitina# 2000 Academic Press

1104

Review. RING Domains: Builders of Molecular Scaffolds?

tion. The only link between these disparate processes appears to be the ability of RINGs to form large assemblages. Thus, RING functions still appear to focus on the ability to form large macromolecular scaffolds, thereby setting the stage for complex enzymatic and molecular processes. Several thorough reviews of structure and function of RINGs have been reported since the domain was ®rst described by Freemont and co-workers (Borden, 1998; Borden & Freemont, 1996; Freemont, 1993; Kentsis & Borden, 2000; Saurin et al., 1996). Thus, this review focuses on recent advances in the understanding of RING function and how these new functions relate to previous results. Defining features of RINGs RINGs are cysteine-rich, zinc-binding domains characterized by several de®ning features, which are described below and reviewed by Kentsis & Borden (2000). (1) RINGs are de®ned by a pattern of conserved cysteine and histidine residues (Figure 1(a)). The consensus sequence incorporates regions of high and low conservation where in some places spacing between ligands varies from nine to 39 residues and in others four to 48. Conserved metalbinding residues can be substituted for other metal-binding amino acids. For instance, one subfamily of RINGs is known as RING-H2 because Cys5 is substituted by histidine. In other cases, metal-binding residues can be substituted by Asp or Thr. (2) RINGs bind two atoms of zinc, using a unique cross-brace arrangement. In this arrangement, the ®rst and third pair of zinc ligands share zinc 1 and the second and fourth pair of zinc ligands share zinc 2 (Figure 1(b)). In archetypal zinc ®nger domains such as TFIIIA, the ®rst and second pair of zinc ligands share a zinc atom (Figure 1(c)). Interestingly, the af®nity for zinc is higher for site 1 than for site 2 in the RING. This leads to the possibility that RINGs can bind one set of protein partners through site 1 and another through site 2. In cases of low zinc concentrations, it is likely that only site 1 is folded and able to associate with its protein partners (Kentsis & Borden, 2000). (3) Zinc ligation is required for folding of the domain and for subsequent biological action(s). There have been three atomic-resolution structures determined thus far: RINGs from human promyelocytic leukemia protein, PML; equine herpes virus protein, IEEHV; and the human recombination protein, RAG1 (Barlow et al., 1994; Bellon et al., 1997; Borden et al., 1995a). Extensive structural comparisons (Borden, 1998) have been reported and therefore will only be summarized here. All three structures indicate the use of the cross-brace zinc-binding arrangement to bind two zinc atoms. The structure around the ®rst zinc-binding site is

Figure 1. (a) Consensus sequence for the RING and for comparison, TFIIIA. Parenthetical numbers refer to spacing; dots indicate any residue. (b) A representation of metal binding in the RING domain and (c) for the classical zinc ®nger TFIIIA. Hexagons represent histidine residues and octagons represent cysteine residues. Ovals represent zinc atoms that are labeled according to site. Spacing in variable regions of RINGs are shown in (b). The asterisk (*) by x(4-48) indicates that there appears to be new members of the RING family, UIP28 and UIP48, that have inserts longer than 48 residues.

highly conserved amongst all three structures, as is Ê inter-zinc distance and the fold of core the 14 A residues along the central strand. The conserved core residues for all three structures are just Nterminal to C5 and C-terminal to C6. The variable spacing in the consensus sequence (4-48 and 9-39) leads to differences in three-dimensional fold around site 2 in all three proteins. For instance, RAG1 and IEEHV both have a four-residue insert between C6 and C7 relative to PML, which forms a helix. RAG1 is missing b-strand 3, which is present in PML and IEEHV. These observations indicate that RING domains form a conserved structural element and yet possess substantial plasticity, undoubtedly re¯ecting the breadth of their biological functions. The RING is often associated with another unique type of cysteine-rich, zinc-binding domain, the B-box. The RING and either one or two Bboxes are followed by a leucine coiled-coil forming the tripartite or RBCC motif (reviewed by Saurin et al., 1996). The spacing between the RING, Bboxes and the coil is typically highly conserved, with 38-40 residues between the RING and ®rst Bbox, and less than ten residues between the second B-box and the coil. There is no apparent sequence homology in these intervening regions. In proteins that contain two B-boxes, these domains are not always structurally equivalent (Borden et al., 1995b, 1996). These differences presumably afford domain speci®city and allow the use of these molecular building blocks in various places throughout

1105

Review. RING Domains: Builders of Molecular Scaffolds?

the cell. Members of this class of RING proteins include PML, TIF1a, KAP1, Rfp, RET, XNF-7 and SSA-Ro (reviewed by Saurin et al., 1996). RINGs are involved in diverse cellular processes In contrast to classical zinc ®nger proteins, which are typically limited to the nucleus and bind nucleic acids, RINGs are found throughout the cell mediating diverse protein-protein interactions (Borden & Freemont, 1996; Saurin et al., 1996). RINGs are involved in transcription, RNA transport, signal transduction, organelle transport and recombination, to name a few (Saurin et al., 1996). However, there have been few underlying themes that enable one to assign a particular function to RING domains in general. One consistent recurring theme is the ability of RINGs to mediate protein associations that lead to formation of large multiprotein complexes, seemingly acting as molecular building blocks (Borden & Freemont, 1996; Saurin et al., 1996). RINGs appear to have the ability to bind protein partners through different zinc-binding regions independently, where the integrity of zinc-binding site 2 is not necessary for some protein associations through zinc binding site 1 (Roehm & Berg, 1997; reviewed by Kentsis & Borden, 2000). This allows RINGs to bind a wide array of protein partners where some associations may rely on the zinc occupancy of the second zincbinding site, allowing an additional level of regulation of these proteins and the processes in which they function (Roehm & Berg, 1997; Kentsis & Borden, 2000). RINGs form macromolecular assemblages RING proteins appear to function in formation of macromolecular assemblages where the integrity of the RING domain is intimately linked with the ability of these complexes to form. We have catalogued those RINGs involved in formation of macromolecular assemblages previously; thus, these results will only be summarized here (Borden & Freemont, 1996; Saurin et al., 1996). The RING of the PML protein is a well-characterized example. PML forms multiprotein complexes in the nucleus, referred to as PML nuclear bodies. These bodies are approximately 1 mm in diameter and heterogeneous in nature (Melnick & Licht, 1999). Mutagenesis studies of the PML RING designed to abrogate zinc binding and therefore cause unfolding of the RING result in disruption of nuclear body formation in transfected cells (Borden et al., 1995a). Mutations that similarly disrupt other components of the RBCC, the B-boxes and leucine coil, also result in disruption of nuclear bodies, suggesting that the RBCC domain may act as an integral unit (Borden et al., 1996). In order to understand further the contribution of the RING to nuclear body formation, mutations were designed to alter the surface charge of the RING without

altering its ability to fold (Boddy et al., 1997). Mutations around zinc site 1 resulted in formation of fewer but extremely large bodies at the same level of protein expression. Mutation in other areas did not have this affect. Thus, both the structure and the surface charge character of the RING appear to be important for macromolecular assemblage of PML nuclear bodies (Boddy et al., 1997). For PML, there appears to be a requirement for both an intact RING and intact nuclear bodies for its functions in growth suppression, transformation suppression, apoptosis and RNA transport (Borden et al., 1995a, 1997; Mu et al., 1994; H.-K. Lai & K.L.B.B., unpublished results). Thus the links between metal binding, folding, macromolecular assemblage and physiological function are intertwined in this RING. Additional examples for RING involvement in macromolecular assemblages include the MSL proteins that participate in gene dosage compensation in Drosophila. These proteins exist in 1 MDa complexes as observed by gel ®ltration, and require all members, MSL1 to MSL3, for proper localization to the X chromosome (Lucchesi, 1996). MSL2, a RING protein, is required for complex formation and localization to chromosomes (Copps et al., 1998). Similar dependency on the RING is seen in the Polycomb group protein Bmi1, a RING protein that exists in a 2-5 MDa complex (Hemenway et al., 1998). Mutation of conserved residues in the RING leads to anterior posterior transformations of vertebrae (Alkema et al., 1997). At the cellular level, Bmi1 is no longer found in Polycomb complexes, but is located diffusely throughout the nucleus (Alkema et al., 1997). Thus both the assembly of Polycomb complexes and their homeotic function is RING-dependent. New insights from the RING of KAP1 Exciting biochemical studies by Rauscher and colleagues (in this issue) demonstrate the role of the RING in high-order protein associations and the importance of these associations to function (Peng et al., 2000). Using the RBCC domain from KAP1 and reconstituting the interactions from puri®ed components, they demonstrate that the RBCC acts as an integral unit that is required for association with the KRAB domain of KOX-1, one of the most numerous DNA-dependent transcriptional repression domains (Margolin et al., 1994). The RBCC of KAP1 appears to regulate transcriptional repression through protein associations with the KRAB domain. The relevance of these interactions in terms of KAP function and biochemistry are discussed below. KAP1, also known as TIF1b, was initially identi®ed as a co-repressor that binds to the KRAB domain (Friedman et al., 1996). Besides the RING domain within a RBCC motif, KAP1 contains a bromodomain and a PHD ®nger. Like other RINGcontaining proteins, KAP1 is observed by bandshift

1106 assays to be in 1‡ MDa complexes in mammalian nuclear extracts (Moosmann et al., 1996). In vitro, the RING domain is required for complex formation (Peng et al., 2000), and in vivo, the RING domain signi®cantly potentiates the interaction with KRAB, as well as its repression of transcription (Agata et al., 1999). KAP1 binds the HP1 family of proteins, which exert dose-dependent effects on heterochromatin-mediated gene silencing (Cryderman et al., 1998; Ryan et al., 1999). Although no RING mutagenesis data exist to determine the dependence of epigenetic repression by KAP1 through its RING in vivo, observation of KAP1 in a ternary complex with DNA-bound KRAB domain (Friedman et al., 1996), a granular pattern in the nucleus (Moosmann et al., 1996; Ryan et al., 1999), coupled with the above observations strongly implicate KAP1 and its RING in the assembly of epigenetic macromolecular complexes that play a role in transcriptional repression by chromatin remodeling. Rauscher and colleagues are the ®rst to demonstrate, using puri®ed components, that the RING and RBCC mediates the KAP/KRAB/DNA interactions in vitro (Peng et al., 2000). Initially, they indicate that to be functionally active, the RBCC must exist as a high-order multimer and describe requirements for RBCC self-assembly. These extensive biochemical characterizations of the KAP1 protein indicate that the RBCC is an integral structural unit (Peng et al., 2000). A battery of biochemical and biophysical techniques including gel ®ltration, analytical ultracentrifugation and analytical gel electrophoresis on the puri®ed RBCC domain from KAP1 show that the RBCC forms monomers, trimers and hexamers which appear to co-exist in complex equilibria (Peng et al., 2000). Mutagenesis studies point to rules of RBCC assembly (Peng et al., 2000). Interestingly, mutation of conserved cysteine residues to alanine, which disrupts the structural integrity of the RING, or mutations that disrupt coil formation in the RBCC, support the association of mutants with wild-type KAP1 (Peng et al., 2000). However, association with full-length KAP1 occurs only upon coexpression of KAP1 and mutant RBCC; simple addition of two puri®ed proteins was not suf®cient to reconstitute the interaction. This is similar to results that show that wild-type PML can associate with PML RING mutants, B-box mutants or small coil deletion mutants only if these proteins are simultaneously expressed in reticulocyte lysate. It is possible that separately synthesized wild-type and mutant RBCCs form homo-wild-type and homomutant oligomers that do not tend to dissociate and form hetero-oligomers. Like KAP1, separate addition of mutant and wild-type proteins after production was not suf®cient to reconstitute the interaction between PML and PML RBCC mutants (unpublished results). Collectively, these results suggest that RBCC homo- and hetero-association is dependent on a combination of folding kinetics and dynamic reorganization of these domains.

Review. RING Domains: Builders of Molecular Scaffolds?

Rauscher and colleagues extended their studies to demonstrate that the RBCC region of KAP1, as a multimer, associates with the KRAB domain of KOX-1 (Peng et al., 2000). This complex association is required for KAP1 and KOX-1 to associate with DNA. Interestingly, the TIF1a RBCC domain, which is highly homologous to KAP1, does not interact with KOX-1 (Peng et al., 2000). Mutations in the RBCC that destroy the structural integrity of the RING, B-boxes or leucine coiled-coil result in the inability to bind the KRAB domain. Extensive swapping experiments indicate that the RING, Bboxes and coiled-coil regions are speci®c to KAP1. For instance, the RING domain from MID1 is unable to functionally substitute for the RING of KAP1. Separate swapping of coiled coils and Bboxes also abolishes the association with KOX-1. The entire MID-1 RBCC could not reconstitute the interaction with KOX-1. Thus, each component of the RBCC appears to impart speci®city, and the RBCC exists as an intact functional unit. RINGs in ubiquitination complexes Another set of reports in the last few months present novel ®ndings that RINGs can function in ubiquitination. E3 enzymes or ubiquitin ligases comprise four classes, Ubr1, HECT, SCF and APC, all of which contain RING proteins, and possess similar architectures (Tyers & Willems, 1999; Ciechanover, 1998). The enzymatic pathway for ubiquitin conjugation and modi®cation is classically considered as follows: activation of ubiquitin by an E1 activating enzyme leading to the formation of an E1-ubiquitin thioester, transfer of this ubiquitin thioester to an E2 conjugating enzyme, and promotion of ubiquitin onto target proteins by the E3, or in some cases E2, enzyme. The substrate recognition step and subsequent ubiquitin ligation are typically mediated by E3 ligases. Three E3 enzymes, APC (anaphase promoting complex), SCF (Skp1/cullin/F-box) and VCB (Von Hippel, Elongin BC), contain RING-H2 proteins in their assemblies (Seol et al., 1999; Skowyra et al., 1999; Tyers & Willems, 1999). The E3 complexes are subdivided into three distinct functional domains, e.g. the F-box subunit recognizes substrates through speci®c protein interaction domains, the Skp1 subunit links the F-box to the remaining subunits and the cullin subunits recruit E2 and comprise the ubiquitin ligase core. It has been proposed that the F-box mediates speci®c targeting of the ligase to proteins to be degraded, and the RING domain is necessary for this function. The basic ubiquitin ligase complexes can be assembled from the RINGH2/cullin module. Thus, RINGs are part of complexes involved in catalyzing poly-ubiquitination and, as such, contribute to the function of E3 ligases. These complexes can interact with different adapter proteins, thereby recruiting different binding partners through the F-box, and the proteinprotein interaction of the RING may be important for this speci®city.

Review. RING Domains: Builders of Molecular Scaffolds?

1107

E3 ubiquitin ligases are unique in that they utilize the RING-H2 subclass of the RING family. It has been proposed that these RING-H2 domains are speci®c for ubiquitin ligase targeting. Observation of ubiquitination activity of proteins that contain classical RING domains (see below) undermines the uniqueness of RING-H2 domains in E3 ligase function. Further, RING-H2 proteins can function outside of the ubiquitin pathway (Kentsis & Borden, 2000; and see below). In the SCF complex, RING-H2 proteins referred to as ROC1 and ROC2 associate directly with cullins 1-5 (Ohta et al., 1999). APC11, also known as Cdc53 (Seol et al., 1999), contains a RING domain and potently activates auto-ubiquitination of Cdc34 E2 ubiquitin-conjugating enzyme, constituting a minimal ubiquitin ligase (Seol et al., 1999). The speci®city of these complexes is demonstrated by the inability of RINGs from one E3 ligase complex to substitute functionally for RINGs found in the other complexes. For example, the APC homologue to ROC1 is APC11; however, APC11 does not bind cullins 1-4 but binds a yeast homologue APC2 (Ohta et al., 1999). Conversely, ROC1 cannot bind APC2. Thus, although there is extensive sequence homology between ROCs and APC proteins, their interactions are distinct, presumably conferring some level of speci®city to the complexes. For both complexes, the RING components of ROC1 and of APC11 are required for ubiquitin ligase activity. Interestingly, mutations in site 1 of ROC1's RING reduce ubiquitin ligase activity whereas mutations in site 2 destroy them altogether (Ohta et al., 1999). Thus, protein interactions made by site 2 appear to be critical for normal functioning of this complex. In a recent discovery, the VHL protein from the VHB E3 ligase complex, which is mutated in most kidney cancers, was found to be part of an E3 ubiquitin ligase complex (Skowyra et al., 1999). Here, the RING-H2 homologue is referred to as Rbx (for RING box). Rbx directly binds Cdc53/cullin, Cdc34, but not Skp, again emphasizing the speci®city of RING complex formation. The RING-H2 domains involved in these complexes are small proteins, of the order of 100 residues with no other apparent motif. The only common features among these complexes are the RING-H2 protein and its associated cullin. For SCF, APC and VCB E3 ligase complexes, the RING-H2 component is required for formation of active complexes, presumably by mediating crucial protein interactions in the construction of a macromolecular scaffold. Another example of a RING involved in ubiquitination is the Ubr1 protein, which contains a RING-H2 and classical zinc ®nger, functions as a ubiquitin ligase similar to the above RINGs (Kwon et al., 1998, 1999). The ability to promote poly-ubiquitination is not limited to the RING-H2 family. The ubiquitin conjugating enzyme UbcM4, which is necessary for mouse development, interacts with a family of UbcM4 interacting proteins (UIPs) that belong to

the RING ®nger family (Martinez-Noel et al., 1999). UIP48 and UIP28 are RBCC proteins and may recruit UbcM4 to SCF-like complexes. Both of these proteins contain a classical RING. Another example of a classical RING having this activity is found in Cbl, which is involved in the ubiquitination of growth factor receptors through its RING domain (Waterman et al., 1999; Yokouchi et al., 1999; and see below). In summary, E3 ubiquitin ligases exist as large macromolecular assemblies (Seol et al., 1999), which critically depend upon the RING domain for their organization and function. RING-containing proteins speci®c to E3 ligases are of the order of 100 residues containing no other conventional sequence motif. A likely explanation is that thioester-ubiquitin transfer needs spatial coupling of E2 and E3 catalytic systems. It appears that these systems require a surface or scaffold, which is provided by the oligomerization of the RING domain. Further, it is possible that there is some level of directive recruitment of proteins to be degraded by the RING in conjunction with the F-box. Some RINGs can recapitulate E3 function in vitro Recent studies reconstitute ubiquitination activity from puri®ed components. These studies indicate that, in vitro, some RING proteins, both classical RINGs and RING-H2s, enhance the polyubiquitination activity of E2. A recent report from Lorick and colleagues (Lorick et al., 1999) indicates that a new member of the RING-H2 family, AO7, binds directly to UbcH5B, an E2 enzyme. GSTAO7 is able to associate with puri®ed preparations of UbcH5B and undergo ubiquitination. The RING is required for the association with UbcH5B but not for ubiquitination itself. Interestingly, the AO7 protein is not modi®ed in these studies, while GST is ubiquitinated. RING-mediated association of AO7 is necessary for interaction with UbcH5B but is not suf®cient for ubiquitination. Lorick and colleagues extended this work to other RING proteins and showed that as GST fusions Praja1, NF-X1, kf1, TRC8, Siah-1 and BRCA1 can all bind UbcH5B and support ubiquitination (Lorick et al., 1999). In each case, the integrity of the RING is required for association with UbcH5B. For example, treatment of proteins with zinc chelators destroys this activity, as do mutations of conserved zinc-ligating residues. It is important to note that the ubiquitination activity of RINGs in these assays is substantially lower than that found with RINGs that are established components of ubiquitination complexes such as ROC1 (Z.-Q. Pan, personal communication). Independent studies show that the RING of Cbl acts in poly-ubiquitination similarly to the above proteins in vitro (Joazeiro et al., 1999). Further, in vivo, Cbl acts in an ancillary capacity in ubiquitination involving UbcH7 (Yokouchi et al., 1999). It is unlikely that RINGs bind E2 enzymes and form catalytic thiol ester intermediates with

1108 ubiquitin (Lorick et al., 1999). Furthermore, it is unlikely that the RING provides a site of interaction with E2 for direct ubiquitin transfer from E2 to an available target lysine residue. Lorick and colleagues favor the hypothesis that the RING and surrounding regions associate with E2-ubiquitin and provide a favorable environment for the transfer of the ubiquitin from E2 to the targeted lysine (Lorick et al., 1999). Collectively, these results are intriguing. The behavior of RINGs in these in vitro studies lacks the speci®city inherent in other RING-mediated interactions. In general, RINGs typically cannot substitute for each other with regard to other biological functions in vivo or in vitro. Even RINGs found in dedicated ubiquitination complexes such as ROC1 and Rbx are unable to substitute for the action of each other or other closely related RINGs found in other E3 ligase complexes. In contrast, the above results suggest a generality of function where several RINGs can mediate ubiquitination. These results raise several intriguing questions related to RING function and speci®city. One common feature is that the RING itself never appears to be ubiquitinated. In spite of the fact that a subset of RINGs can catalyze ubiquitin transfer reactions, it is unlikely that this is the de®ning function of RING domains. For instance, RINGs from PML, LCMV Z and MAT1 are unable to support catalysis of ubiquitin transfer by UbcH5B (Gordon, Chen & Pan, unpublished results). In order to assess if there were any obvious sequence determinants for RINGs involved in ubiquitination and RINGs that are not, a sequence analysis using CLUSTALW1.5 was carried out. RINGs in dedicated ubiquitination complexes such as APC11 and ROC1 are much more closely related to each other than with other RINGs having a high level of conservation of residues outside of the conserved zinc-ligating residues. The UIP proteins were strikingly similar, in that they had unusually long inserts between C6 and C7 (82-84 amino acid residues). Sequence analysis indicated that the RINGs that do not function in ubiquitination (at least when using UBC5HB) clustered together. Additionally, the RINGs from NF-X1, A07, kf-1 and Praja1 clustered together. Surprisingly, BRCA1 and Siah-1 cluster away from the other ubiquitination-positive RINGs. Aside from general sequence similarity, at present, no clear-cut sequence determinants could be deduced for the likelihood of RINGs being able to function in these in vitro assays. It is clear that some RINGs participate in ubiquitin transfer reactions, as evident from in vitro assays, and more importantly, physiologic observations (Tyers & Willems, 1999). It is the former, however, that demonstrate the interchangeability of RINGs in this function. This activity appears to be non-physiologic, since there is no speci®city, demonstrated by the transfer of ubiquitin even onto GST (Lorick et al., 1999), which is presumably simply positioned in spatial proximity to the ubi-

Review. RING Domains: Builders of Molecular Scaffolds?

quitin ligase. In general, such non-physiologic behavior can be attributed to two reasons: (1) substituted RINGs are physiologically irrelevant and as such do not confer speci®city; or (2) unde®ned factor(s) are absent from the assay that would make ubiquitin transfer target-speci®c. In either scenario, the function of diverse RINGs in supporting albeit non-speci®c ubiquitin transfer emphasizes the more universal and characteristic function of RINGs as molecular building blocks providing a scaffold for this and other processes. The fact that RINGs can participate in ubiquitin transfer to other proteins as well as themselves could represent a novel regulatory mechanism. The RING may target its own or associated proteins for ubiquitin-dependent degradation or for modi®cation of biological activity such as intracellular targeting that occurs with modi®cation by SUMO-1 (small ubiquitin-like modi®er) of RanGAP (Matunis et al., 1998). In future, it will be exciting to see if any of the RINGs that are able to catalyze poly-ubiquitination in vitro interact with ubiquitin machinery in vivo. One RING protein, PML, is modi®ed by SUMO1/PIC1/Sentrin (Boddy et al., 1996). SUMO-1 allows modi®cation of protein function and localization of both PML and RanGAP. For PML, the RING is required for binding the ubiquitin conjugating 9 protein (Ubc9), which is speci®c to SUMO-1, but SUMO-1 modi®cation occurs approximately 400 residues C-terminal to the RING (Duprez et al., 1999). However, another RING, RING1, cannot substitute for this activity of PML, indicating speci®city for RING Ubc9 interaction (Duprez et al., 1999). It is noteworthy that non-RING proteins, e.g. RanGAP, can be SUMO-1modi®ed and thus a RING domain is not a requirement for productive association with Ubc9. Determinants of molecular recognition and specificity Clearly, it is critical to understand the determinants of molecular recognition of any domain. These are the ®rst steps forward in assessing what determinants are necessary for RING association and, ultimately, for predicting partner proteins on the basis of sequence analysis. Several studies have been conducted showing that RINGs are immensely good at interacting with themselves, resulting in a frustrating propensity for aggregation when carrying out biochemical, structural and biophysical studies. RING interactions can be loosely classed into two categories, those that bind other RINGs and those that bind non-RING domains. A list of RINGs that bind directly to given domains (other then themselves) reveals only a few wellcharacterized proteins (Table 1; Kentsis & Borden, 2000). RING-RING interactions have been well characterized for BRCA1. Elegant biochemical and biophysical studies by Klevitt and co-workers indicate that the BRCA1 RING forms homodimers and heterodimers with another RING protein, BARD1

1109

Review. RING Domains: Builders of Molecular Scaffolds? Table 1. Characteristics of known RING-binding domains Protein

RBD

Assembly

Comments/Method

PML Z BRCA1

PRH PRH BRCA1

NBs/CBs NBs/CBs NBs/CBs

BARD1

NBs

BAP1 ATF RAG1 Ste4p Ste5p CIP8 MSL1 DinG Bmi1 CUL1-5 APC2 CUL5 Cullins

NBs NBs V(D)J, NBs

Binds proline region/Y2H/IP/IF (Topcu et al., 1999) Binds proline region/Y2H/IP (Topcu et al., 1999) R/R/Gel fil/x-link (Brzovic et al., 1998; Roehm & Berg, 1997; Scully et al., 1997a,b) R/R/Y2H/IP/IF/Gel fil/x-link (Scully et al., 1997a,b;Brzovic et al., 1998; Roehm & Berg, 1997; Wu et al., 1996) Ub hydrolase/Y2H/IP/IF (Jensen et al., 1998) Transcription factor/Y2H/IP/GST (Houvras, 1999) V(D)J, R/R/MBP/UC/Xray (Bellon et al., 1997; Rodgers et al., 1996) MAPK signaling, Z1 required/Y2H/IP (Inouye et al., 1997) MAPK signaling, RH2, Z1 required, RH2/RH2/Y2H (Inouye et al., 1997) R/RH2/Y2H/IP/Gel fil (Miyake et al., 1998; von Arnim & Deng, 1993) Z1, but not Z2 required/Y2H/IP/Gel fil (Copps et al., 1998) Heterochromatin remodeling, R/R/Y2H (Hemenway et al., 1998) R/R/Y2H (Hemenway et al., 1998) RH2, Z2 necessary, Z1 sufficient/Y2H (Ohta et al., 1999) RH2/Y2H (Ohta et al., 1999) RH2/Y2H (Ohta et al., 1999) RH2/Y2H (Kwon et al., 1999)

RAG1-ZFA Step5p Ste5p COP1 MSL2 Bmi1 DinG/RING1b ROC1 APC11 Ubr1

X bodies PcG PcG E3 ligase E3 ligase E3 ligase E3 ligase

Proteins containing RINGs and those containing RING-binding domains (RBDs) are listed. Assembly refers to high-order (MDa) assembly. Comments includes the region of the RBD that is bound to the RING or general information about the RBD domain including biological functions. Further, the part of the RING required for binding is included in this section. Also included are the methods used to determine the association and appropriate references. Abbreviations are as follows: RBD, RING-binding domain; Y2H, yeast 2-hybrid; IP, immunoprecipitation; IF, immuno¯uorescence; Gel ®l, gel ®ltration; GST, glutathione S-transferase pulldown; MBP, maltose-binding protein pulldown; UC, ultracentrifugation; x-link, glutaraldehyde cross-linking; R/R, RING/RING; RH2, RING-H2; PcG, polycomb group; NBs, nuclear bodies; CBs, cytoplasmic bodies; X bodies, X chromosome bodies. Z1 and Z2 refer to RING zinc-binding sites.

(Brzovic et al., 1998; Meza et al., 1999). Further, BARD1 also forms homodimers. Interestingly, the BRCA1/BARD1 heterodimers are more resistant to proteolysis than either homodimer, suggesting a structural rearrangement upon heterodimer formation (Meza et al., 1999; Brzovic et al., 1998). Two illustrative examples of speci®city in RINGbinding domains (RBDs) are discussed below. (1) The BRCA1 RING is known to interact with a ubiquitin hydrolase molecule, BAP1 (Jensen et al., 1998). BAP1 is able to target BRCA1 and/or associated proteins for ubiquitin-dependent degradation. This action is signi®cant for BRCA1, which plays a role in cell growth. The RING from Rpt1, which has more than 90 % sequence identity with the RING of BRCA1, cannot support binding of BAP1 when substituted for BRCA1's domain (Jensen et al., 1998). In this case, the major difference between the two RINGS occurs C-terminal to site 2, where Rpt1 has an 11-residue insert relative to BRCA1. (2) Another example of speci®city in molecular recognition of RINGs is demonstrated by swapping experiments utilizing the RINGs of TRAF3 and TRAF5. Constructs containing mid and C-terminal fusions of the two RINGs switches speci®city of signaling (Dadgostar & Cheng, 1998; Brink & Lodish, 1998). These data demonstrate that, in general, even closely related RINGs cannot substitute for physiological or biochemical function. The importance of the RING to PML function is well documented. Disruption of the RING through mutations in conserved cysteine residues results in a loss of PML nuclear bodies (Borden et al., 1995a) and this correlates with a loss of growth suppression, transformation suppression (Mu et al., 1994)

and apoptotic activities (Borden et al., 1997). A viral protein, Z, is known to directly interact with the region just N-terminal to the RING of PML (Borden et al., 1998a). This 90-residue viral protein represents the smallest RING reported to date. This allows the study of RING function without modulation by other domains. In general, Z mimics functions attributed to the PML RING (Borden et al., 1997, 1998a,b). The functional importance of the RING led to studies into possible protein partners for this domain. Using the yeast 2hybrid system, the proline-rich homeodomain protein PRH, also known as Hex, was found to associate with the RINGs of PML and of Z (Topcu et al., 1999). In both cases, the integrity of site 1 is required for association. Further, 65 residues in the proline-rich region of PRH are required for association with both PML and Z RINGs (Topcu et al., 1999). This result is novel, in that, outside of cullins, this is the ®rst non-RING RBD that recognizes more than one RING. Analysis of sequence determinants for RING binding to non-RING-containing proteins has been carried out with limited success (Kentsis & Borden, 2000). Using strict criteria to establish direct proteinprotein interactions, there have been only eight nonRING RBD domains reported with sequences spread from plants to man (Kentsis & Borden, 2000; Table 1). Three proteins that bind directly to RINGs through non-RING-containing sequences exhibit a proline-rich region of high similarity. These proteins are PRH, MSL1, and Ste4p (Kentsis & Borden, 2000). This consensus sequence is de®ned by PXBXPJXP, where B and J are Leu/Val and Ala/Ser, respectively (Figure 2). In all three cases, it appears that an

1110

Review. RING Domains: Builders of Molecular Scaffolds?

References Figure 2. Alignment of a proline-rich sequence shared by the RING-binding regions of PRH, MSL, and Ste4p. Only these FRODOs containing this sequence have been observed to bind the RING region around zinc-binding site 1 (Kentsis & Borden, 2000).

intact zinc-binding site 1, but not site 2, is required for association. The sequence is referred to as a FRODO, named after the most famous RING bearer (Kentsis & Borden, 2000; Tolkien, 1954). Clearly, there will be additional classes of non-RING RBDs; some may have the putative FRODO consensus sequence and some will most likely represent new families of RBDs. Undoubtedly, the sequence determinants for non-RING RBDs will be further re®ned as more direct protein partners are identi®ed. Ultimately, a greater number of non-RING binding sequences will be necessary, as will further experimentation, to assess if FRODOs will be major players in RING interactions. Conclusions As yet, no de®ning biochemical process has been identi®ed for RING domains. Exciting new ®ndings have pointed directly to novel functions for RINGs in transcriptional repression and ubiquitination. However, it is unlikely that RING function will be limited to these two activities. RINGs have been implicated in several other cellular processes such as recombination and peroxisomal biogenesis. There is little to link such disparate actions. A common theme in all these processes is the requirement for high-order architecture and scaffolding. This architecture may set the biochemical stage, allowing essential spatial positioning for participants in complex processes and for imparting speci®city. Thus, RINGs may act as both molecular building blocks, forming scaffolds for these complex biological processes, and as molecular modi®ers, mediating spatial and perhaps temporal positioning and speci®city. It is essential to understand how RINGs participate in formation of these complexes, and how disruption of RING complexes contribute to the pathogenesis of human disorders such as breast cancer, acute promyelocytic leukemia and Parkinson's disease.

Acknowledgments I am very grateful for helpful discussions and the sharing of unpublished data from Z-Q. Pan. I am grateful for discussions of the manuscript with Graeme Carlile, Alex Kentsis and Allan Capili. Financial support was provided by NIH RO1 CA80728-01.

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Edited by P. E. Wright (Received 2 December 1999; accepted 3 December 1999)