Review articles
Born to bind: the BTB protein–protein interaction domain Roberto Perez-Torrado, Daisuke Yamada, and Pierre-Antoine Defossez* Summary The BTB domain is a protein–protein interaction motif that is found throughout eukaryotes. It determines a unique tri-dimensional fold with a large interaction surface. The exposed residues are highly variable and can permit dimerization and oligomerization, as well as interaction with a number of other proteins. BTB-containing proteins are numerous and control cellular processes that range from actin dynamics to cell-cycle regulation. Here, we review findings in the field of transcriptional regulation to illustrate how the high variability of the BTB has allowed related transcription factors to evolve different functional abilities. We then report how recent work has showed that, in spite of their high sequence divergence and apparently unrelated functions, many BTB-containing proteins have at least one shared role: the recruitment of degradation targets to E3 ubiquitin ligase complexes. Taken together, these findings illustrate diverse and convergent functions of a versatile protein–protein interaction domain. BioEssays 28: 1194–1202, 2006. ß 2006 Wiley Periodicals, Inc. Introduction The BTB domain was first identified as a sequence motif in genes of DNA virus.(1) It derives its name from the subsequent observation by Laski and colleagues that the Drosophila transcription factors Bric-a-brac, Tramtrack, and Broad Complex display a region of sequence similarity at their N terminus, that they named the BTB domain.(2,3) Concurrently, Bardwell and Treisman realized that some Poxvirus proteins have resemblance to a portion of the Zinc finger proteins ZID, GAGA, and ZF5; they named this region the POZ (Pox virus and Zinc finger) domain.(4) These motifs are one and
CNRS UMR218, Institut Curie, Section Recherche, Paris, France. Funding agencies: Work in the DeFossez lab is supported by CNRS (programme ATIP), and by Association pour la Recherche Contre le Cancer. Roberto Perez-Torrado is supported by a postdoctoral fellowship from CNRS. Daisuke Yamada is supported by a postdoctoral fellowship from Association pour la Recherche Contre le Cancer. *Correspondence to: Pierre-Antoine Defossez, CNRS UMR218, Institut Curie, Section Recherche, Paris, France. E-mail:
[email protected] DOI 10.1002/bies.20500 Published online in Wiley InterScience (www.interscience.wiley.com).
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the same, and define the BTB/POZ domain, often abbreviated simply as ‘‘BTB domain’’ (Fig. 1). The BTB provides a rich example of the evolution and roles of protein–protein interaction domains. It illustrates how a fairly simple protein motif can establish stable and transient interactions simultaneously, how it can evolve to acquire new binding specificities, and how it can be combined to other domains to carry out a wealth of different functions. Here we will review recent findings to illustrate how the structural makeup of the BTB domain allows for its great versatility and for the acquisition of important capacities in proteins, and how BTB-containing proteins, in spite of their apparent diversity, share at least one role in protein degradation. Architecture and evolution of BTB-containing proteins Several resources are useful for the examination of BTBcontaining proteins. Many different computer programs dedicated to the recognition of protein domains are available online and can be used to identify BTB-containing proteins in sequenced genomes. For example, Interpro (http://www.ebi. ac.uk/interpro/) is comprehensive and easy to use. Also, a recent paper by Prive´ and coworkers(5) provides an excellent discussion of the sequence and structural features of the BTB. A search among sequenced genomes shows that the BTB domain is present in viruses and throughout eukaryotes, from fungi to plants to metazoans (Table 1). It is likely that the BTB domain evolved after the origin of eukaryotes. Indeed no BTB is found in archaebacteria, or in bacteria, with one exception: Candidatus Protochlamydia amoebophila.(6) This bacteria is an endosymbiont of an amoeba, from which the BTB-containing genes likely originated. There is a rough correlation between the number of BTB proteins and the complexity of an organism, with H. sapiens having over 350 different BTB-containing proteins. One example that contradicts this trend is the worm C. elegans, which has a disproportionately large number of BTB proteins. It should be pointed out, as a cautionary note, that the high sequence variability of the BTB makes the identification of BTB proteins in databases challenging. As a consequence, the current estimate of BTB proteins may have to be corrected in the future.
BioEssays 28:1194–1202, ß 2006 Wiley Periodicals, Inc.
Review articles
Figure 1. Structure and sequence of a representative BTB domain. A: From left to right, monomer unit of the BTB from the transcription factor BCL6, dimer, and dimer in complex with two SMRT peptides.(16) Each peptide contacts two different regions of a monomer, indicated in gray and green above the sequence of BCL6. The dimer presents a charged groove (arrow at the top of the molecule), which could be another interaction interface. The structures were obtained from the Protein Database (www.rscb.org) B: Sequence alignment of the BTB of BCL6 and the indicated transcription factors. The sequence diversity is high, even between proteins that are fairly closely related, such as Kaiso, ZBTB4 and ZBTB38,(67) and only a few residues are invariant (highlighted). In addition, insertions in the BTB can occur, as in the case of ZBTB4.
Some proteins are made up of just a BTB; this is true of Skp1, which is involved in protein degradation (see following sections), and ElonginC, which controls transcriptional elongation. However, it is more frequent for the BTB to be combined with other domains. Over two dozen different domains are found associated with the BTB in proteins, of which five are much more frequent than the others. They are the MATH, Kelch, NPH3, Ion transport and Zinc finger (ZF) domains (Table 1). Some organism-specific expansions and contractions of the groups have occured: for instance the BTB-NPH3 proteins are present only in Arabidopsis. Conversely, Arabidopsis has no BTB-Kelch or BTB-ZF proteins. Another spectacular expansion took place in C. elegans: this organism,
which lacks BTB-Kelch and BTB-ZF proteins, has a large number of MATH-BTBs. Interestingly, these proteins are under strong positive selection pressure, and are thought to have evolved as a defense against parasites.(7) Of note, the BTB— MATH association has been evolutionarily very succesful: it is among the most-frequent combinations of two domains within all proteins.(8) Just these five groups of the most-common BTB-containing proteins suffice to illustrate the variety of functions that they carry out. The MATH is a subtype of TRAF-like domain, frequently found in proteins involved in cytoplasmic signal transduction, such as the TRAFs, which interact with the TNFalpha receptor.(9) The Kelch domain forms a b-propeller
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Interpro was used to determine the number of BTB in fully sequenced genomes. The number of proteins containing the indicated domains, together with a BTB, is indicated. For instance, H. sapiens contains 80 BTB-Zinc finger proteins. Only the most common associated domains are considered, so the numbers may not add up to the total number of BTB proteins in a given organism. Some especially significant numbers are in bold. TPR: tetratricopeptide.
0 0 0 0 0 0 1 32 27 80 0 0 0 7 0 0 1 1 1 6 0 0 0 0 0 5 0 0 0 0 0 0 0 0 0 29 0 0 0 0 0 0 0 0 0 12 38 4 2 4 0 0 3 0 2 0 8 17 21 95 0 0 0 0 0 0 19 6 5 46 5 1.7 0.2 1.2 12 125 100 180 1500 3400 E. coli M. jannaschi Vaccinia virus Mimivirus S. cerevisiae A. thaliana C. elegans D. melanogaster D. rerio H. sapiens
4600 1700 200 1200 6000 25000 19000 13000 10000 25000
0 0 3 7 9 108 160 136 158 357
0 0 0 0 1 1 0 5 4 9
Kelch Ion Transport Ank Nbr of BTB proteins Organism
Estimated nbr of genes Genome size (Mb)
Table 1. The distribution of BTB and associated domains in selected organisms
MATH
NPH3
TPR
WD-40
Zinc finger
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structure that can interact with actin filaments,(10) and, indeed, several BTB-Kelch proteins have a role in the stability and dynamics of actin filaments.(11,12) The BTB-NPH3 proteins are plant-specific, and at least some of them function in phototropism signalling.(13) An example of BTB-Ion transport protein is the voltage-gated channel for potassium ions.(14) Finally, the BTB-ZF proteins generally function as transcription factors and regulate important processes in cells, including survival and differentiation, as will be illustrated later. In spite of their functional variety, one commonality between these five groups of proteins is that they all use the BTB domain as a protein–protein interface, either for oligomerization or for interaction with other proteins. Specific examples will be given in the following sections. Are there both ancestral functions for the domain and others that have appeared more recently during evolution? The examination of BTB proteins in a simple organism yields some clues to that question. The budding yeast S. cerevisiae only has nine proteins with POZ domains (Table 2). Only some of the functions covered by BTB proteins in higher eukaryotes are represented in yeast: protein degradation (by a Skp1 homolog, but also presumably by the uncharacterized protein YIL001W), transcriptional elongation by an ElonginC homolog, and signal transduction. BTB-containing transcription factors, which are numerous in flies and mammals, are conspicuously absent from yeast, which has a compact genome with rare introns, and simpler transcriptional networks. It is possible that the recruitment of the BTB in transcription factors is a later invention that was selected to help regulate more complex genomes. In contrast, the roles of the BTB in signalling and protein degradation are conserved in yeast and in the other eukaryotes, and can be assumed to be older functions. Structure of the BTB and interaction mode X-ray crystallography has been used to solve the structure of different BTB domains, including those of the transcription factors PLZF and BCL6, the proteins Skp1 and ElonginC, and the voltage-gated potassium channel. Their secondary structure elements and overall arrangement are similar in spite of the low degree of primary sequence conservation.(5) The domain contains a cluster of five alpha-helices capped at one end by a short three-stranded beta-sheet, and its shape is compact and globular (see Fig. 1). The different BTB domains differ in their protein–protein interaction behaviours (Fig. 2). For instance the BTB in ZF proteins can homodimerize and heterodimerize, as well as recruit transcriptional corepressors(15–17) (see Figs 1, 2). In contrast, the BTB domain present in ion channels, which is called ‘‘T1 domain’’ for historical reasons, promotes tetramerization.(18) These characteristics can be explained by two differences. The first is the high degree of sequence variability in the core domain. Of the 95 amino-acids that constitute the
Review articles
Table 2. The BTB proteins of budding yeast Gene name YBR199W YDR132C YDR328C YER132C YGL197W YIL001W YLR108C YOR043W YPL046C
Protein name Ktr4 Unch. Skp1 Pmd1 Mds3 Unch. Unch. Whi2 Elc1
Architecture BTB-Glycosyl-transferase BTB SKP1 only Kelch-BTB Kelch-BTB Ankyrin-BTB-BTB BTB-BTB BTB Elongin only
Localization
Interactions
Golgi Cytoplasm and nucleus Cytoplasm and nucleus Cytoplasm Cytoplasm Cytoplasm Nucleus Plasma membrane Nucleus
? Sxm1, Nmd3 Skp2, Cdc4 Rim1 Sit4, Sap185 Hrt1 ? Psr1 Ela1
Function Protein glycosylation Nuclear export? Protein degradation Signal transduction Signal transduction? Protein degradation? ? Signal transduction Transcription elongation
The yeast S.cerevisiae has 8 BTB-containing proteins. Unch.: uncharacterized protein. Functions followed by a question mark are suggested by the intracellular localization and the nature of the interactors, but have not been proved experimentally.
core domain, about a dozen are conserved, and most of these are hydrophobic residues that are buried within the scaffold of the domain.(19,5) In contrast the exposed interaction surface contains highly variable residues. The nature of these residues determines in part whether the domain can dimerize, tetramerize or interact with other proteins. Another important factor explaining the different behaviors of BTB domains is the existence of class-specific extensions N-terminal or C-terminal of the core domain.(5) In effect, the BTB domains can be divided into four families: T1, Skp1, ElonginC and BTB-ZF (see Fig. 2). The T1 domain contains only the ‘‘core’’ BTB elements. In contrast, BTB domains in the other classes deviate from this canonical form: they have gained or lost additional structural elements. Some BTB-only proteins such as ElonginC lack the last alpha-helix of the domain. But other BTB-only proteins, including Skp1 and its relatives, have gained two additional helices at the C terminus, which make extensive contact with interactors. The BTBs in MATH, BACK-Kelch, Rho and Zinc finger proteins have a 25 amino-acid extension N-terminal of the core domain. It folds into one b-sheet and one a-helix that contribute importantly to dimerization. The binding behaviours of BTB domains have an important influence on the proteins that contain them. To illustrate this point, we will concentrate on two common functions of BTB-containing proteins: transcriptional regulation and protein degradation. BTB-containing proteins in transcriptional regulation There are about 80 BTB-ZF transcription factors in the human genome. This number is large relative to the one observed in invertebrates or even in non-mammalian vertebrates (Table 1) and it seems likely that the BTB-ZF proteins underwent a recent expansion in mammals, along with the rest of the Zinc finger transcription factors.(20) Another conspicuous fact is that the architecture of BTB-ZF proteins is highly stereotypical,
with the BTB domain almost invariably N-terminal to the Zinc fingers. This might reflect structural constraints on the BTB domain for dimerization, or for its functional interaction with the DNA-binding region. Dimerization is frequently observed in transcription factors; it can raise the affinity and specificity of DNA recognition, as well as provide a simple regulatory mechanism rendering the factors inactive under a certain concentration threshold.(21) One function of the BTB in transcription factors is to provide such a dimerization interface. Another is to recruit transcriptional coregulators, as we will illustrate through two examples: PLZF and BCL6, which are oncogenic BTB-ZF transcription factors. PLZF PLZF has originally been identified as a translocation partner of RARa (retinoic acid receptor a) in some cases of acute promyelocytic leukemia (APL).(22) In this context, a transforming PLZF-RARa chimeric protein is produced, in which the BTB domain of PLZF has two effects. First, it drives the homodimerization of RARa, a necessary event for the chimera to have transforming potential.(23) The dimerization interface involves the N-terminal extension of the BTB domain, it is large, and it makes the protein an obligate dimer.(15) A second role of the BTB is to recruit the transcriptional corepressor SMRT, thus inhibiting the target genes of RAR-a and blocking differentiation of myeloid progenitors.(24) The use of the BTB for dimerization is not limited to PLZFRAR, but also occurs in the wild-type PLZF protein. It has been shown in cultured cells that dimerization is absolutely required for the function of PLZF.(25) Some elegant work using mutant mice has strengthened this finding. Mice lacking PLZF show homeotic transformations of the skeleton.(26) Investigation of this phenomenon led to the discovery that PLZF binds target sequences in the Hox regulatory regions, and homodimerizes to create loops of DNA, which permit the correct expression sequence of Hox genes.(27) The creation of DNA loops by
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domain.(30,31,24) These molecules are related but also have some distinguishing features,(32) and their relative roles in vivo have not been clearly defined. The use of the BTB domain as a landing pad for corepressors, in particular NCoR and SMRT, is a common occurrence in BTB-ZF proteins, but it is not systematic, and this function seems to have been lost in some of the transcription factors.(33) The interaction surface maps to a charged pocket that is distinct from the dimerization interface, so that separation-of-function mutants can be generated that still dimerize, but fail to recruit corepressors.(25) These have been useful tools to dissect the respective role of these two functions in vivo.
Figure 2. The families of BTB proteins and their protein— protein interaction abilities. There are four subtypes of BTB domains, with characteristic structures and binding modes. The BTB found in ion channels, called T1 domain, has a basic ‘‘core’’ BTB domain, and is found as a tetramer in the cells. The ElonginC family is characterized by a core BTB domain that lacks the last a-helix. The Skp1 family of proteins has a core BTB domain extended by two C-terminal a-helices. ElonginC and Skp1 form a structural link between adaptor proteins and Cul2 or Cul1, respectively, in E3 ubiquitine ligase complexes. Finally, in proteins with Zinc fingers the core BTB domain has an N-terminal extension of two a-helices. BTB-Kelch, BTB-Rho, and MATH-BTB proteins also have this architecture. Different proteins within the BTB-ZF family can form dimers, oligomers, or complexes with other proteins.
self-association of a BTB-ZF transcription factor is actually a recurrent theme. It has been especially well documented for the Drosophila protein GAGA. In some cases, GAGA binds multiple sequences in the same promoter and organizes its three-dimensional architecture.(28) But GAGA can also bridge different chromosomes, and bring about enhancer activity in trans.(29) All of these effects are dependent on the BTB domain. Like the PLZF–RAR chimera, the wild-type PLZF protein is a transcriptional inhibitor, thanks to the recruitment of the transcriptional corepressors NCoR and SMRT by the BTB
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BCL6 BCL6 is an anti-apoptotic transcription factor. Its expression is normally restricted to the germinal center B-cells that are in maturation. These cells undergo immunoglobulin class switching and somatic hypermutation, which in somatic cells would be sensed as DNA damage and trigger cell death. Among others, BCL6 represses the transcription of p53(34) and p21,(35) and allows the B-cells of the germinal center to bypass apoptosis and proliferate. Consistently, mice lacking BCL6 have no germinal centers.(36) In about 30% of diffuse large B-cell lymphoma, a chromosomal recombination event brings the expression of BCL6 under a heterologous promoter, frequently the immunoglobulin control region. This, along with hypermutation events in the BCL6 gene itself, maintains its expression in circulating B-cells which, as a result, become hyperproliferative and resistant to apoptosis and cause leukemia.(37) Like PLZF, BCL6 illustrates the potential roles that the BTB domain can play in transcription factors: homodimerization, heterodimerization and recruitment of corepressors. First, BCL6 can homodimerize via the BTB domain. This is necessary for the regulation of some direct target genes such as p53.(34) Second, the BTB domain can permit heterodimerization with other BTB-ZF transcription factors, as in the following example. The expression of BCL6 downregulates p21, even though no BCL6-binding sites are apparent in the p21 promoter. This indirect regulation occurs because MIZ-1, another BTB-ZF protein, binds the p21 promoter and recruits BCL6.(35) In addition, BCL6 can also interact with PLZF(38) and the related protein LRF,(39) but the physiological consequence of these interactions is unclear at present. Also, the stoichiometry of these complexes is unknown but, because BCL6 is an obligate dimer, they could be dimers of dimers or other highorder oligomers. Finally, the BTB domain of BCL6 recruits corepressors and inhibits transcription of target genes. BCL6, like PLZF, recruits SMRT. The structure of BCL6 in a complex with a SMRT peptide has been solved(16) and yields interesting insight into the mode of interaction. Each BCL6 dimer binds two SMRT peptides and, even though no cooperative binding is observed,
Review articles
the increased avidity could be important for repression. The BCL6–SMRT interaction region is distinct from the dimerization domain, but overlaps with the region that contributes to oligomer formation in the crystal, and may interfere with the formation of higher-order complexes. While the BCL6 dimerization is obligate, the interaction with SMRT is transient and occurs with a dissociation constant similar to other peptidebinding domains such as the SH3 domains. Unlike an SH3– peptide interaction, though, the interaction with SMRT has little effect on the conformation of BCL6. BCL6 also recruits NCoR and another corepressor, BCoR, which is not bound by PLZF.(40) The binding of BCoR and SMRT to BCL6 is mutually exclusive, and the consequences of the ‘‘choice’’ are unknown. Of note, since the BTB/corepressor interaction is specific and necessary for transcriptional repression, it can be targeted for therapeutic intervention. By rational design, or by genetic screening, peptides have been obtained that interfere specifically with the BCL6–corepressor interaction.(41,42) The administration of these peptides to leukemic cells with BCL6 translocation leads to their apoptosis in vitro, and has some therapeutic potential. BTB-containing proteins in protein degradation. Many cellular processes are regulated by degradation of some key proteins through the ubiquitin system.(43) This pathway is based on the conjugation of a chain of ubiquitin molecules to a selected protein, which is then degraded by the 26S proteasome. Three enzymes have to act in succession to transfer ubiquitin to a substrate: the E1 enzyme activates the ubiquitin, and transfers it to the conjugation enzyme E2. Finally the ubiquitin ligase complex E3 binds the final target and ensures its ubiquitinylation.(44) How is the exquisite specificity of this system achieved? There are only a few different E2s, so each substrate clearly does not have a dedicated E2 enzyme. Instead, the regulation relies on the existence of several E3s, and of numerous adaptor molecules, which bring together the correct E3 and the desired target.(45) This principle is
well illustrated by the subset of E3 ligases that contain a specific kind of scaffolding molecule called ‘‘Cullin’’. There are several different cullins, generating a first layer of complexity. The cullins bind to a second adaptor protein, which itself recruits the substrate, either directly or via yet another adaptor. The BTB-only proteins Skp1 and ElonginC have long been known to be adaptor molecules in E3 ligase complexes. Skp1 is part of a degradation complex called SCF (Skp1, Cul1 and F-box proteins), which was discovered in S. cerevisiae, extensively studied by genetic means, and is now known to be conserved.(46) In the SCF complex, the BTB of Skp1 serves as the bridge between the substrate recognition protein (an F-box protein) and the scaffold protein Cul1 (Fig. 3).(47) In mammals, there are more than 70 F-box proteins.(48) Most of them are still uncharacterized, yet it is likely that they recognize degradation targets. The SCF complex is involved in degradation of many cell cycle regulators, including p21, CDC25 and Cyclin D. It is not surprising, then, that mutations in components of the complex are found in several types of cancers.(49) Another BTB-only protein, ElonginC, plays a role analogous to Skp1 in a different E3 ligase complex known as ECS (ElonginC, Cul2, SOCS-box) or VCB (pVHL, ElonginC, ElonginB). Interestingly, the sequences of ElonginC and Skp1 are only marginally related, and they use unrelated protein interfaces to interact with their respective cullins.(5) In ECS, ElonginC links the Cul2 scaffold protein with a SOCSbox protein that recognizes the substrate (Fig. 3).(50) Again, the large number of SOCS-box proteins in the genome permits the recognition of many different substrates for degradation. One well-understood role of the ECS complex is to direct the degradation of two transcription factors, HIF-1 and HIF-2 (Hypoxia-inducible factor 1and 2), which are critical mediators of the cellular response to hypoxia.(51) Under normoxic conditions, HIF-1 and HIF-2 are continually turned over by ECS. When hypoxia occurs, the transcription
Figure 3. The BTB proteins in protein degradation. In protein degradation complexes, the BTB proteins serve as bridging molecules between the cullin E3 ligases, and the adaptor proteins that recruit the protein that is to be degraded. The BTB protein Skp1 connects Cul1 to different F-box proteins (A), while ElonginC interacts with Cul2 and SOCS-box adapters (B). In Cul3-based complexes (C), the BTB proteins interact with the cullin, and directly recruit the degradation target through their associated domain (MATH, Kelch, ZF and others).
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factors are stabilized and activate a number of transcriptional targets. A series of recent papers have established that the interaction between BTB proteins and cullins is not limited to Skp1–Cul1 and ElonginC–Cul2, but is instead a common and wide-ranging regulatory mechanism. Work in S. pombe,(52) C.elegans(53,54) and human cells(55) led to the realization that many different BTB proteins bind Cul3. Strikingly, in these complexes, the factors involved are not BTB-only proteins. Instead they contain associated domains, such as MATH or ZFs. Because these proteins bind Cul3 with their BTB domain, and the degradation substrate with their associated domain, they combine in one polypeptide the function of two separate proteins in the complexes described above: Skp1 and the F-box proteins in SCF or ElonginC and ElonginB in ECS (Fig. 3). This finding shows that, beyond their apparent diversity, BTB-containing proteins do have at least one widely shared function: the regulation of protein degradation.(56,57) Many BTB proteins are now known to act as adaptors for Cul3based E3 ligases (see Table 3). We will now describe in more detail a few examples taken from different organisms. In Drosophila, Hedgehog (Hh) morphogens control eye development by activating the Cubitus Interruptus (Ci) transcription factor. Zhang and co-workers have recently shown that Hh induces the expression of HIB (Hedgehoginduced BTB-MATH),(58) and that HIB uses its MATH domain to recruit Ci to a Cul3-containing E3 complex. This promotes the ubiquitination and degradation of the transcription factor, providing negative feedback regulation of Hh signalling. In the Wnt signalling pathway, Dishevelled (Dsv) plays a central role of branching point between b-catenin-dependent or -independent pathways, regulating cell differentiation and proliferation or cell polarity and motility, respectively. In the absence of a Wnt signal, the b-catenin cytosolic pool is constitutively degraded via ubiquitinylation by an
SCF complex.(59) Recently it has been shown that Dsv is also regulated by degradation.(60) In a Cul3-based complex, the BTB-Kelch protein KLHL12 binds Dsv and controls Dsv function leading to its degradation. Experiments in Zebrafish and Xenopus embryos have shown that the RNAi knockdown of KLHL12 does negatively regulate Dsvmediated processes. Keap1, a BTB-Kelch protein, can bind to the transcription factor Nrf2 and promote its degradation by ubiquitinylation. Nrf2 is an unstable transcription factor that regulates genes involved in the antioxidant response. In unstressed cells, Keap1 maintains Nrf2 at very low levels but in oxidatively stressed cells, the ubiquitin-directed degradation of Nrf2 is compromised and it activates its target genes. Interestingly, Keap1 is also an oxidative sensor, and modification of certain cysteines seems to be important for the function of the Cul3-based complex.(61) Recently, it has been shown that critical mutations of Keap1 that prevents Nrf2 degradation are found in lung cancer, arguing that constant antioxidant response can help malignant progression.(62) One example of a Cul3 ligase adaptor in Arabidopsis is ETO-1. It contains as an accessory module a tetratricopeptide (TPR) domain. ETO-1 can recruit ACS5, an essential enzyme for ethylene synthesis, to Cul3 ubiquitinylation complex and target it for degradation.(63) By its action on ACS5 levels, ETO-1 is an important regulator of ethylene signalling. Finally, even BTB-containing transcription factors may interact with Cul3 and target some substrates for degradation. PLZF interacts with Cul3 in cells,(55) but the relevant target(s) and the physiological role of this interaction are not yet identified. The structure of a BTB complexed to Cul3 would be valuable to find out what region of the BTB is involved, and whether the mode of interaction with the cullin is related to that of Skp1 or ElonginC, or has evolved independently. These data
Table 3. BTB proteins as adaptors for Cul3-based E3 ligases: targets and functional consequences BTB subgroup
Adaptor
BTB-Kelch
KEAP1 KLHL12 BTBD1
H. sapiens H. sapiens H. sapiens
SPOP MEL-26 C08C3.2 Y105E8B.4 F37A4.9 HIB
H. C. C. C. C. D.
BTB1 BTB3
BTB-Zinc Fingers BTB-TPR
MATH-BTB
BTB-Ankyrin
1200
Function
Reference
Nrf2 DVL1 TOP1
Oxidative response inhibition Wnt signaling control DNA damage response
61 60 55
Daxx MEI-1 unknown unknown unknown Ci
Daxx level regulation Mitotic spindle formation unknown unknown unknown Hh signaling inhibition
55 53 54 54 54 58
S. pombe S. pombe
unknown unknown
unknown unknown
52 52
PLZF
H. sapiens
unknown
unknown
55
ETO1
A. thaliana
ACS5
Ethylen signaling inhibition
63
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sapiens elegans elegans elegans elegans melanogaster
Target
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are not yet available, but structural modeling based on existing data suggests that the interaction surface is distinct from the dimerization surface, suggesting that the POZ domains bind Cul3 as dimers, and may be the driving force for the dimerization of the E3 complexes.(5) Conclusion It has been recently estimated that there may be as many as 4000 different protein domains across the three kingdoms of life.(64) Their distribution follows a power law, with a few domains being present in many proteins, and many domains being present in only a few proteins. The BTB is present in about 2800 proteins, which places it in the former category. The versatility of the domain is the likely reason for its success in evolution. We have focused on two prominent functions of BTBcontaining proteins: transcriptional regulation and protein degradation. The BTB-containing transcription factors illustrate how the sequence variability in the BTB can generate functional diversity. Relatively subtle amino-acid differences, as in BCL6 and PLZF, can cause different proteins to have different affinities for corepressors. Other sequence differences determine whether the BTB domain will drive homo-, hetero-, or oligomerization of a given transcription factor, with important consequences for its function. In contrast, recent work done on protein degradation yields a surprisingly unifying view of the BTB proteins. In spite of their marked sequence divergence, a large number of unrelated BTB-containing proteins perform an analogous task: recruiting target proteins to cullin-based E3 ubiquitin ligases. This realization naturally leads us to wonder whether BTB proteins could have other shared functions. A number of interesting questions remain open for the future. As our knowledge of the BTB domain increases, it may become possible to modify it and use it as a building block for ‘‘designer proteins’’, as has been done for Zinc fingers and Ankyrin domains.(65) Also, a better understanding of the rules that govern BTB homodimerization, heterodimerization and interaction with other proteins will be a great help in the validation of large-scale protein interaction data; it may in the longer term help to predict which proteins could interact with a given BTB domain, and be useful for systems biology.(66) Finally, it seems likely that new interactors of the BTB domain are yet to be discovered. The charged groove that is conserved between BTB transcription factors(15) is a strong candidate as a docking site for peptides, or possibly other types of ligands. Acknowledgments We thank Guillaume Filion for comments on the manuscript. References 1. Koonin EV, Senkevich TG, Chernos VI. 1992. A family of DNA virus genes that consists of fused portions of unrelated cellular genes. Trends Biochem Sci 17:213–214.
2. Godt D, Couderc JL, Cramton SE, Laski FA. 1993. Pattern formation in the limbs of Drosophila: bric a brac is expressed in both a gradient and a wave-like pattern and is required for specification and proper segmentation of the tarsus. Development 119:799–812. 3. Zollman S, Godt D, Prive GG, Couderc JL, Laski FA. 1994. The BTB domain, found primarily in zinc finger proteins, defines an evolutionarily conserved family that includes several developmentally regulated genes in Drosophila. Proc Natl Acad Sci USA 91:10717–10721. 4. Bardwell VJ, Treisman R. 1994. The POZ domain: a conserved proteinprotein interaction motif. Genes Dev 8:1664–1677. 5. Stogios PJ, Downs GS, Jauhal JJ, Nandra SK, Prive GG. 2005. Sequence and structural analysis of BTB domain proteins. Genome Biol 6:R82. 6. Horn M, Collingro A, Schmitz-Esser S, Beier CL, Purkhold U, et al. 2004. Illuminating the evolutionary history of chlamydiae. Science 304:728–730. 7. Thomas JH. 2006. Adaptive evolution in two large families of ubiquitinligase adapters in nematodes and plants. Genome Res 16:1017–1030. 8. Vogel C, Bashton M, Kerrison ND, Chothia C, Teichmann SA. 2004. Structure, function and evolution of multidomain proteins. Curr Opin Struct Biol 14:208–216. 9. Bradley JR, Pober JS. 2001. Tumor necrosis factor receptor-associated factors (TRAFs). Oncogene 20:6482–6491. 10. Adams J, Kelso R, Cooley L. 2000. The kelch repeat superfamily of proteins: propellers of cell function. Trends Cell Biol 10:17–24. 11. Robinson DN, Cooley L. 1997. Drosophila kelch is an oligomeric ring canal actin organizer. J Cell Biol 138:799–810. 12. Hara T, Ishida H, Raziuddin R, Dorkhom S, Kamijo K, Miki T. 2004. Novel kelch-like protein, KLEIP, is involved in actin assembly at cell-cell contact sites of Madin-Darby canine kidney cells. Mol Biol Cell 15:1172–1184. 13. Sakai T, Wada T, Ishiguro S, Okada K. RPT2. 2000. A signal transducer of the phototropic response in Arabidopsis. Plant Cell 12:225–236. 14. Choe S, Cushman S, Baker KA, Pfaffinger P. 2002. Excitability is mediated by the T1 domain of the voltage-gated potassium channel. Novartis Found Symp 245:169–175; discussion 175–177, 261–264. 15. Ahmad KF, Engel CK, Prive GG. 1998. Crystal structure of the BTB domain from PLZF. Proc Natl Acad Sci USA 95:12123–12128. 16. Ahmad KF, Melnick A, Lax S, Bouchard D, Liu J, et al. 2003. Mechanism of SMRT corepressor recruitment by the BCL6 BTB domain. Mol Cell 12:1551–1564. 17. Li X, Peng H, Schultz DC, Lopez-Guisa JM, Rauscher FJ 3rd, Marmorstein R. 1999. Structure-function studies of the BTB/POZ transcriptional repression domain from the promyelocytic leukemia zinc finger oncoprotein. Cancer Res 59:5275–5282. 18. Kreusch A, Pfaffinger PJ, Stevens CF, Choe S. 1998. Crystal structure of the tetramerization domain of the Shaker potassium channel. Nature 392: 945–948. 19. Aravind L, Koonin EV. 1999. Fold prediction and evolutionary analysis of the POZ domain: structural and evolutionary relationship with the potassium channel tetramerization domain. J Mol Biol 285:1353–1361. 20. Babu MM, Luscombe NM, Aravind L, Gerstein M, Teichmann SA. 2004. Structure and evolution of transcriptional regulatory networks. Curr Opin Struct Biol 14:283–291. 21. Marianayagam NJ, Sunde M, Matthews JM. 2004. The power of two: protein dimerization in biology. Trends Biochem Sci 29:618–625. 22. Chen Z, Brand NJ, Chen A, Chen SJ, Tong JH. 1993. Fusion between a novel Kruppel-like zinc finger gene and the retinoic acid receptor-alpha locus due to a variant t(11;17) translocation associated with acute promyelocytic leukaemia. Embo J 12:1161–1167. 23. Kwok C, Zeisig BB, Dong S, So CW. 2006. Forced homo-oligomerization of RARalpha leads to transformation of primary hematopoietic cells. Cancer Cell 9:95–108. 24. Lin RJ, Nagy L, Inoue S, Shao W, Miller WH, Jr. , Evans RM. 1998. Role of the histone deacetylase complex in acute promyelocytic leukaemia. Nature 391:811–814. 25. Melnick A, Ahmad KF, Arai S, Polinger A, Ball H, et al. 2000. In-depth mutational analysis of the promyelocytic leukemia zinc finger BTB/POZ domain reveals motifs and residues required for biological and transcriptional functions. Mol Cell Biol 20:6550–6567. 26. Barna M, Hawe N, Niswander L, Pandolfi PP. 2000. Plzf regulates limb and axial skeletal patterning. Nat Genet 25:166–172.
BioEssays 28.12
1201
Review articles
27. Barna M, Merghoub T, Costoya JA, Ruggero D, Branford M, et al. 2002. Plzf mediates transcriptional repression of HoxD gene expression through chromatin remodeling. Dev Cell 3:499–510. 28. Katsani KR, Hajibagheri MA, Verrijzer CP. 1999. Co-operative DNA binding by GAGA transcription factor requires the conserved BTB/POZ domain and reorganizes promoter topology. Embo J 18:698–708. 29. Mahmoudi T, Katsani KR, Verrijzer CP. 2002. GAGA can mediate enhancer function in trans by linking two separate DNA molecules. Embo J 21:1775–1781. 30. Hong SH, David G, Wong CW, Dejean A, Privalsky ML. 1997. SMRT corepressor interacts with PLZF and with the PML-retinoic acid receptor alpha (RARalpha) and PLZF-RARalpha oncoproteins associated with acute promyelocytic leukemia. Proc Natl Acad Sci USA 94:9028–9033. 31. He LZ, Guidez F, Tribioli C, Peruzzi D, Ruthardt M, et al. 1998. Distinct interactions of PML-RARalpha and PLZF-RARalpha with co-repressors determine differential responses to RA in APL. Nat Genet 18:126–135. 32. Goodson M, Jonas BA, Privalsky MA. 2005. Corepressors: custom tailoring and alterations while you wait. Nucl Recept Signal 3:e003. 33. Deltour S, Guerardel C, Leprince D. 1999. Recruitment of SMRT/N-CoRmSin3A-HDAC-repressing complexes is not a general mechanism for BTB/POZ transcriptional repressors: the case of HIC-1 and gammaFBPB. Proc Natl Acad Sci USA 96:14831–14836. 34. Phan RT, Dalla-Favera R. 2004. The BCL6 proto-oncogene suppresses p53 expression in germinal-centre B cells. Nature 432:635–639. 35. Phan RT, Saito M, Basso K, Niu H, Dalla-Favera R. 2005. BCL6 interacts with the transcription factor Miz-1 to suppress the cyclin-dependent kinase inhibitor p21 and cell cycle arrest in germinal center B cells. Nat Immunol 6:1054–1060. 36. Dent AL, Shaffer AL, Yu X, Allman D, Staudt LM. 1997. Control of inflammation, cytokine expression, and germinal center formation by BCL-6. Science 276:589–592. 37. Pasqualucci L, Bereschenko O, Niu H, Klein U, Basso K, et al. 2003. Molecular pathogenesis of non-Hodgkin’s lymphoma: the role of Bcl-6. Leuk Lymphoma 44 Suppl 3:S5–12. 38. Dhordain P, Albagli O, Honore N, Guidez F, Lantoine D, Schmid M. The HD, Zelent A, Koken MH. 2000. Colocalization and heteromerization between the two human oncogene POZ/zinc finger proteins, LAZ3 (BCL6) and PLZF. Oncogene 19:6240–6250. 39. Davies JM, Hawe N, Kabarowski J, Huang QH, Zhu J, et al. 1999. Novel BTB/POZ domain zinc-finger protein, LRF, is a potential target of the LAZ-3/BCL-6 oncogene. Oncogene 18:365–375. 40. Huynh K, Fischle W, Verdin E, Bardwell VJ. 2000. BCoR, a novel corepressor involved in BCL-6 repression. Genes Dev 14:1810–1823. 41. Polo JM, Dell’Oso T, Ranuncolo SM, Cerchietti L, Beck D, et al. 2004. Specific peptide interference reveals BCL6 transcriptional and oncogenic mechanisms in B-cell lymphoma cells. Nat Med 10:1329–1335. 42. Chattopadhyay A, Tate SA, Beswick RW, Wagner SD, Ferrigno PK. 2006. A peptide aptamer to antagonize BCL-6 function. Oncogene 25:2223– 2233. 43. Ciechanover A. 2005. Proteolysis: from the lysosome to ubiquitin and the proteasome. Nat Rev Mol Cell Biol 6:79–87. 44. Glickman MH, Ciechanover A. 2002. The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction. Physiol Rev 82:373–428. 45. Petroski MD, Deshaies RJ. 2005. Function and regulation of cullin-RING ubiquitin ligases. Nat Rev Mol Cell Biol 6:9–20. 46. Willems AR, Schwab M, Tyers M. 2004. A hitchhiker’s guide to the cullin ubiquitin ligases: SCF and its kin. Biochim Biophys Acta 1695:133–170.
1202
BioEssays 28.12
47. Zheng N, Schulman BA, Song L, Miller JJ, Jeffrey PD, et al. 2002. Structure of the Cul1-Rbx1-Skp1-F boxSkp2 SCF ubiquitin ligase complex. Nature 416:703–709. 48. Jin J, Cardozo T, Lovering RC, Elledge SJ, Pagano M, Harper JW. 2004. Systematic analysis and nomenclature of mammalian F-box proteins. Genes Dev 18:2573–2580. 49. Nakayama KI, Nakayama K. 2006. Ubiquitin ligases: cell-cycle control and cancer. Nat Rev Cancer 6:369–381. 50. Stebbins CE, Kaelin WG Jr, Pavletich NP. 1999. Structure of the VHLElonginC-ElonginB complex: implications for VHL tumor suppressor function. Science 284:455–461. 51. Ohh M, Park CW, Ivan M, Hoffman MA, Kim TY, et al. 2000. Ubiquitination of hypoxia-inducible factor requires direct binding to the beta-domain of the von Hippel-Lindau protein. Nat Cell Biol 2:423–427. 52. Geyer R, Wee S, Anderson S, Yates J, Wolf DA. 2003. BTB/POZ domain proteins are putative substrate adaptors for cullin 3 ubiquitin ligases. Mol Cell 12:783–790. 53. Pintard L, Willis JH, Willems A, Johnson JL, Srayko M, et al. 2003. The BTB protein MEL-26 is a substrate-specific adaptor of the CUL-3 ubiquitin-ligase. Nature 425:311–316. 54. Xu L, Wei Y, Reboul J, Vaglio P, Shin TH, et al. 2003. BTB proteins are substrate-specific adaptors in an SCF-like modular ubiquitin ligase containing CUL-3. Nature 425:316–321. 55. Furukawa M, He YJ, Borchers C, Xiong Y. 2003. Targeting of protein ubiquitination by BTB-Cullin 3-Roc1 ubiquitin ligases. Nat Cell Biol 5: 1001–1007. 56. Pintard L, Willems A, Peter M. 2004. Cullin-based ubiquitin ligases: Cul3BTB complexes join the family. Embo J 23:1681–1687. 57. van den Heuvel S. 2004. Protein degradation: CUL-3 and BTB–partners in proteolysis. Curr Biol 14:R59–61. 58. Zhang Q, Zhang L, Wang B, Ou CY, Chien CT, Jiang J. 2006. A Hedgehog-Induced BTB Protein Modulates Hedgehog Signaling by Degrading Ci/Gli Transcription Factor. Dev Cell 10:719–729. 59. Hart M, Concordet JP, Lassot I, Albert I, del los Santos R, et al. 1999. The F-box protein beta-TrCP associates with phosphorylated beta-catenin and regulates its activity in the cell. Curr Biol 9:207–210. 60. Angers S, Thorpe CJ, Biechele TL, Goldenberg SJ, Zheng N, et al. 2006. The KLHL12-Cullin-3 ubiquitin ligase negatively regulates the Wnt-betacatenin pathway by targeting Dishevelled for degradation. Nat Cell Biol 8:348–357. 61. Zhang DD, Lo SC, Cross JV, Templeton DJ, Hannink M. 2004. Keap1 is a redox-regulated substrate adaptor protein for a Cul3-dependent ubiquitin ligase complex. Mol Cell Biol 24:10941–10953. 62. Padmanabhan B, Tong KI, Ohta T, Nakamura Y, Scharlock M, et al. 2006. Structural basis for defects of Keap1 activity provoked by its point mutations in lung cancer. Mol Cell 21:689–700. 63. Wang KL, Yoshida H, Lurin C, Ecker JR. 2004. Regulation of ethylene gas biosynthesis by the Arabidopsis ETO1 protein. Nature 428:945–950. 64. Orengo CA, Thornton JM. 2005. Protein families and their evolution-a structural perspective. Annu Rev Biochem 74:867–900. 65. Mosavi LK, Cammett TJ, Desrosiers DC, Peng ZY. 2004. The ankyrin repeat as molecular architecture for protein recognition. Protein Sci 13: 1435–1448. 66. Aloy P, Russell RB. 2006. Structural systems biology: modelling protein interactions. Nat Rev Mol Cell Biol 7:188–197. 67. Filion GJ, Zhenilo S, Salozhin S, Yamada D, Prokhortchouk E, Defossez PA. 2006. A family of human zinc finger proteins that bind methylated DNA and repress transcription. Mol Cell Biol 26:169–181.
