New members of the Bcl-2 family and their protein partners

45

New members

of the Bcl-2 fam ily and their protein partners

Stuart N Farrow and Robin Brown Any model of apoptosis must explain the mechanism of action of the Bcl-2 family of proteins. This has proved to be unusually difficult. This review concentrates on some of the newly isolated members of this growing family and attempts to provide an insight into the complexity of interactions through which the Bcl-2 proteins modulate apoptosis.

Address Cell Biology Unit, Glaxo Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SGl 2NY, UK Q Current Biology Ltd ISSN 0959-437X Currant Opinion in Genetics & Development 1666,6:45-49

Introduction Apoptosis is a process as fundamental as proliferation in development and tissue homeostasis. Almost all multicellular organisms maintain an apoptotic program within their cells [l-3]. Indeed, the switch from unicellular to multicellular development may have hinged as much upon the opportunity to remove unwanted cells by apoptosis as it has on the ability to coordinate cell division and differentiation. The most important insights into the control of apoptosis have come from the study of the gene b&Z, which was originally isolated and mapped to the t(14;18) translocation breakpoint common in non-Hodgkin’s lymphomas. Gverexpression of bc/-2 in B cells leads to their prolonged survival and then to tumourigenesis [4,5]. Since then a number of genes that share sequence homology with bcl-2 have been isolated. Members of this family and its interacting partners have been central to models that attempt to explain the mechanism of apoptosis in mammalian cells. In this review, we describe the most recently isolated members of this family and attempt to place them in the context of existing models of apoptosis. Members of the gene family that includes bE/-2 are involved in the control of apoptosis in a range of different cell types ([6”,7 ];Table 11, evidence for which comes principally from experiments in which A&2, or the apoptosis inducer Bux [8] are overexpressed or deleted in cells. Mice in which bc/-2 or btl-X~ [9] has been deleted serve to highlight problems associated with apoptotic dysfunction [lO,ll]. Mice deficient in &C/-XL arrest at El3 and exhibit massive cell death in the nervous and haematopoetic system [ll]. In contrast, animals lacking B&2 do not show this embryonic lethality, but die in adulthood of polycystic kidney disease. Homozygous bcL2 -/-mice also suffer from massive involution of the thymus and spleen due to apoptosis [lo]. Because these two phenotypes are clearly not identical, bc/-2 and bcI-XL may play qualitatively different roles during development.

It is also likely that some cell deaths regulated during development require neither protein. The above transgenic knockout experiments establish that Bcl-2 and B&XL can determine the fate of a cell under conditions in which they are expressed aberrantly (or not at all), but do not necessarily help us to interpret their role in the regulation of apoptosis in normal cells. Similarly, the absence of any measurable enzymatic activity ascribed to any of the known members of the B&2 family prevents measurement of their activation or inactivation. Despite such difficulties inherent in their analysis, members of this protein family do demonstrate a common activity, namely that they are all capable of forming either homodimers, heterodimers or both. Immunoprecipitation of Bax or Bcl-2 using specific antisera demonstrates that these proteins exist in the cell as dimers [l&13]. As Bax induces apoptosis and B&2 protects cells, it has been proposed that Bcl-2 and other members of the family compete for the binding of Bax in order to inactivate it [14,15]. The key set factor is therefore the concentration of Bax homodimers. In such a competitive binding model, additional proteins that compete for binding to either Bax or B&2 would be able to influence the outcome after an apoptotic stimulus.

Dimen

determine

cell fate

The dimerization domains of the B&2 and Bax proteins constitute the regions of greatest homology between family members. Two domains are known to be important for binding and have been called the BHl (residues 138-154 of B&Z) and BH2 domains (residues 188-l%) [12]. Mutations that affect key residues in either of these two domains can abolish the ability of the protein in question to associate or restrict its potential partners. For Bcl-2, mutations that affect either the Gly145 (three-letter amino acid code) residue within the BHl domain or the Trp188 residue in the BH2 domain permit the protein to homodimerize, but prevent its association with Bax. Concomitantly, these Bcl-2 mutants also lose their ability to protect the cell from apoptosis when induced by a number of different stimuli 1121. Although persuasive of a role for a Bcl-2-Bax interaction in apoptosis control, this experiment is open to various interpretations. It is possible that in addition to the elimination of Bax binding, the mutations also prevent the interaction of B&2 with other effector proteins in the cell ; without khowing what these targets might be, the mutants cannot be fully characterized. Indeed, it is becoming clear that B&2 is able to interact with a much wider range of proteins than was originally anticipated (113-151; Table 11). It is also

46

Oncoganos and cell proliferation

Tabk 1 lnluaeHons

of lb0 Bet-2 family.

E&XL

BCI-Xs

Al

MCI-1

Bad

Bag

tt

Intermediate*

Intermediate*

Weak’

-

Weak’

Interaction demonstrated*

tt

tt

Strong’

-

-

Intermediate*

Strong*

-

Bcl-2

BOX

B&2

l-t

Box

tt

Bak’

-

W-XL

-

Bd-xs

-

Al

Wsak’

MCI-1 Bad Baa

Bak

Intermediate* tt

-

Weak*?

Strong*

Intermediate*

Intennediite*

Strong’

we&t

-

-

Weak**

Interaction demonstratedt

(* interaction demonstrated by yeast two hybrid system; t interaction demonstrated by in vitro protein binding assay [GST-fusion proteins]; tt interaotion proven to occur in mammalian cells). While some of these interactions have been demonstrated in mammalian cells, most evidence ‘comes from interactions in yeast or from in viva analyses. Care should be taken in the interpretation of these results. In the yeast experiments, the analysis is of the proteins as fusions with either Gal-4 or LexA, and these are localized in the nucleus. Additionally, interactions in the cell may depend on the presence of a third protein that is neither expressed in yeast nor present in the in vitro binding assays. Yeast may also be incapable of folding and modiiig the protein so that the interactive domains are functional. Nonetheless, the yeast two hybrid system has been very important in the cloning and analysis of these proteins and has proved, in a number of cases, its ability to duplicate interactions that occur in ‘higher’ cells. (Data in thii table have been collated from several sources IQ.1 2,13,14,19,20”,23], including unpublished data from our laboratory.)

possible that these mutants function as dominant negative mutants, preventing the interaction of the endogenous wild-type Bcl-2 with Bax. As yet, no mutant of B&2 has been described that binds to Bax but does not homodimerize and so the dominant negative-hypothesis has yet to be tested. A third domain, comprising the amino-terminal amino acids 11-33, has also been shown to play a role in dimerization. The ability of the first 82 amino acids of Bcl-2 to dimerize with both full-length B&Z, and a mutant lacking these amino acids suggests that the dimers form as ‘head to tail’ complexes [15]. In heterodimers between two mutants of B&2, one of the proteins must contain the amino-terminal domain, whereas the other must have BHl and BH2. Intriguingly, this domain in Bcl-2 seems to be dispensable for binding to Bax in uitru. By contrast, mutations in the BHl, BH2 and carboxy-terminal domains prevent the protein from binding Bax.

Increasing complexity of interactions An important question at this point is do other cellular proteins exist that contain the interaction domains? If so, do these show either ceil-partner or protein-partner specificity? The answer to the first question is yes. During the past few years, the number of characterised Bcl-2 homologues has been steadily increasing. In addition to the identification of the proteins M&l [16], A-l [17], Ced-9 [18”] and BHRF-1 [19], the discovery of the alternatively spliced &XL and bc/-xs [9] has suggested that

the Bax/Bcl-2 heterodimeric complex may not be the only focus of the apoptotic pathway. Very recently, additional proteins have been described that contain BHl and BH2 domains. One of these proteins was discovered almost simultaneously by three different groups [20*,21,22]. The gene was isolated by two of these groups using degenerate PCR primers to the conserved regions of the BHl and BH2 domains. A third group isolated the protein as a binding partner for the adenovirus apoptotic repressor E 1 B 19K protein [20’]. The new gene, called baR (for bc/-2 antagonist/killer), functions as an inducer of apoptosis in all three systems in which it was tested. This finding, coupled with the widespread distribution of the protein, represents an important addition to the family of apoptosis inducers, and has pinpointed a direct link between a viral repressor of apoptosis (ElB 19K) and the bcl-2 family. Using the yeast two-hybrid system it was also possible to demonstrate that Bak binds to Bcl-2 and B&XL, a result that firmly establishes the gene as a member of the B&2 family [20*]. In this new light, the previously described model that attributes the induction/repression of apoptosis to the competition for Bax must be revised. As Bak can bind to both B&2 and B&XL in each cell either a competition exists for both Bax and Bak binding or the two proteins do not operate in the same cell types. Cverexpression studies demonstrate that Bax and Bak can kill the same cells but this may not be relevant as far as normal cells and apoptotic stimuli are concerned (SN Farrow and R Brown, unpublished data).

New members

In addition to Bak, the newly isolated gene Bad is also a member of the family 1131.The homology of the encoded protein is restricted to the dimerization domains and it is a selective binding partner of B&2 and of Bcl-XL. Intriguingly, no evidence exists for homodimerization of Bad, which suggests that its function is to compete for the two apoptosis protectors, Bcl-2 and Bcl-XL, and thus increase the concentration of both Bak and Bax. In this context, Bad functions as an inducer of apoptosis, although with a different mode of action to the ‘homodimeric killers’ Bak and Bax. Despite the sequence homology in the BHl and BH2 domains, these domains do confer specificity as not all heterodimeric combinations are possible. In Table 1, the data are summarized from a number of experiments that use different systems to study interactions between Bcl-2-like proteins. The widest set of interacting partners exist for Bcl-2, which can form heterodimers with Bcl-XL, Bcl-Xg Bax, Bak, and MCI-1 [14,20’]. In contrast, Bax only appears to interact with Bcl-2 and Bcl-XL. Although evidence exists for at least some of these interactions in ‘higher’ cells (Table 1) not all have been demonstrated. In addition, interactions might be demonstrated in yeast, when the proteins are expressed as fusion proteins and localized to the nucleus, that do not occur in mammalian cells; thus care must be taken in the interpretation of these data.

Models of apoptosis At this stage it is instructive to consider three possibilities for the function of the death-inducing or death-protecting members of the Bcl-2 family. First, Bax and Bak might be death-inducers in their own right and need not interact with apoptosis protectors such as Bcl-2/Bcl-XL, in order to function. In this case, identification of the cellular target(s) for Bax and Bak becomes a crucial question. Second, it is possible that Bax and Bak have no function other than to bind to Bcl-2/Bcl-XL and block their protective function. If this were the case, we would require knowledge of the ‘real’ Bcl-Z/B&XL targets to explain the pathway. The third possibility is that both apoptosis protectors and death-inducers have interrelated, but distinct, functions and that relative protein levels in the heterodimeric complex govern the overall response (i.e. protection versus death). Thus far we have concentrated on proteins that have BHl and BH2 domains, but recently a number of proteins have been identified that bind to members of the Bcl-2 family, but which do so through unknown protein interaction domains. This class of proteins are encoded by the genes Bag [23], NBK/BIK (K-T Pun tr al., unpublished data) and Nipl, Nip2, Nip3 and Nip4 [24]. In the case of Bag, the protein functions as a Bcl-2, binding protein which, if expressed in cells, increases the resistance of the cell to apoptosis. Bag/Bcl-2 heterodimers therefore appear to increase the level of protective species in

of the Bei-2 family and their protein partnen

Farrow and Brown

47

the cell. The Bag protein, though lacking the Bcl-2 dimerization domains, does contain a number of sequence motifs of interest. Principally, a region of the protein from residue 37 to residue 73 has a 66% identity with ubiquitin and ubiquitin-like proteins. Future experiments will be required to demonstrate that this domain has a ubiquitin-like function, but the sequence similarity suggests that Bag may be degraded in the cell by a ubiquitin-mediated pathway. This mechanism of action is seen in the case of the ubiquitin-mediated degradation of ~53 by the papilloma virus protein E6 [25,26]. It is conceivable that Bag may be stabilized by its interaction with Bcl-2. This would be interesting from the point of view of regulation of apoptosis and would suggest that Bcl-2 might not only sequester Bax and Bak but also stabilize proteins that protect the cell. A more speculative hypothesis would be that this domain is responsible for both the dimerization of Bag with Bcl-2 and for bringing the heterodimer into proximity with a protease complex involved in cell-death regulation [23]. Taken together with the known involvement of proteases in apoptosis [27”], this might provide an attractive link between members of the Bcl-2 family and proteases. The Elb 19K interacting proteins Nipl, Nip2 and Nip3 and the NbR product (which has a sequence identical to Nip4) are all Bcl-2-binding and/or Bcl-XL-binding proteins which, like Bag, have no sequence homology to Bcl-2. In the case of Nbk, this protein, if overexpressed in cells, functions as an inducer of apoptosis. This induction of cell death may be mediated by inactivation of the protective members of the family, in contrast to the likely mechanism of action of Bag. It is also possible that, in addition to binding to Bcl-2 and Bcl-XL, Nbk and the Nip proteins have other targets in cells not related to Bcl-2. As with Bad, Nbk does not appear to form homodimers in yeast two-hybrid experiments. The fact that new members of the bc/-2 family are still being identified suggests that a unified model of apoptosis may prove to be more complex than was previously thought. In particular, it does not seem possible to account for the cell specificity of apoptosis by considering only the known members of the bc/-2 family. A wide range of proteins, some of which are discussed in this review, compete directly or indirectly for AC/-Z family members in the cell. This raises the question as to whether apoptosis regulating pathways exist in the normal cell that do not depend on Bcl-e-like proteins. It has been reported that overexpression of Bcl-2 does not protect a number of cytokine-dependent cell lines from apoptosis after cytokine withdrawal [28-301. Also, the role of Bcl-2 in protecting cells from cytotoxic T lymphocyte induced apoptosis, which involves both granule and Fas-mediated pathways, is still unclear. Bcl-2 family proteins are known to prevent Fas-mediated apoptosis [31]. In a myeloid cell line, Bcl-2 has no effect on the granule-mediated pathway [32]; however, it is now necessary to rule out the possibility

40

Flgure

Oncogenes

and cell proliferation

1

Signal

Cytoplasmic effecters: cysteine proteases

0 1996 Current Opinion

in Genettcs

8 Developmenl

A schematic diagram of the apoptotic pathway and the M-2 gene family. Signals, such as growth factors, DNA damage and inhibitors of protein or RNA synthesis, affect the ratio of one or more of the dimer pairs through an unknown mechanism. By interaction with their cellular targets some of these dimers are capable of activating a class of cysteine proteases that include interleukin-1 f&converting enzyme (ICE) and CPP-32 1331. Once activated, the targets for the proteases (in the mitochondria 1341, nucleus and endoplasmic reticulum [ER]) are cleaved and the cell death program is complete. Specificity at each of these stages is likely to be achieved by heterogeneity of the binding proteins, their response to a given stimulus and the protease which is activated. In addition, differences in the subcellular localization of the Bcl-2 proteins will provide a further level of specificity. For simplicity, not all the binding proteins of the family are shown ((33,341; see text for further details).

that this failure is due to these cells being dependent upon another member of the family. Therefore, the question may now be reformulated to ask are there any routes to apoptosis that do not involve a member of the bcl-2 family?

Conclusions In this review we have tried to reflect growing knowledge of the complexity of interactions that occur between members of the bc/-2 family. It should be clear, however, that interpreting this complexity is not the same as understanding the mechanism by which these proteins

induce cell death. We still do not understand how signals generated upstream (e.g. from the Fas or tumour necrosis factor receptor-l or effecters downstream such as members of the cysteine protease family) interact with, or are regulated by, Bcl-Z-like proteins. Because Bcl-2 and other members of the family are found in three distinct sites in the cell (Fig. 1) it is conceivable that in each location, the cellular targets are different, but are coupled to a common effector. The search for a single apoptotic mechanism of action for Bcl-2 and its homologues may, therefore, prove to be fruitless.

New members

References and recommended

reading

Papers of particular interest, published within the annual period of review, have been highlighted ae: . l

.

of special interest of outstanding interest

1.

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2.

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3.

of the Bd-2 family and their protein partner9 Far~ow and Brown

49

The contribution of Caenorhebdifis elegans to the understanding of apopte sis cannot be unclereetimated. Together with the identification of ad-3 aa a cysteine protease, this paper underlines the functional similarity of wll death pathwaye in this nematode and mammale. But what does Ced-4 do? 19.

Henderson S, Huen D, ROWBM, Dawson C, Johnaon G, Riineon A: Epstein-Barr vln~s-coded BHRFl protein, a vlml homologue of M-2, protects human Bcdls from progmmmed wll death. Proc Nat/ Aced Sci USA 1993,90:8479-0483.

20. .

Farrow SN, White JHM, Martinou I, Raven T, Pun K-T, Grinham CJ, Martinou J-C, Brown R: Cloning of a bd-2 homologw by lntemdion with adenovlrus El B 1 SK Nature 1995, 374:731-733. Together with [24] this paper provides evidence that the mechanism of acticn of El B 1 QK depends upon its interaction with proteins of the bd-2 fmnily. 21.

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22.

Keifer MC, Brauer MJ, Powers VC, Wu JJ, Umanaky SR, Torwi LD, Barr PJ: Modulation of apoptosls by the widely dlstrlbuted w2 homologw Be&. N&n-e 1995, 374~736-739.

Oltvai ZN, Korsmeyer SJ: CheckpoInts of duellnQ dlmers foll de&h wlshes. Cell 1994, 79:189-l 92. gne of the best short reviews on current models of apoptwis.

23.

Takayama S, Sato T, Krajewski S, Kochel K, lrie S, Millan 14 Reed JC: Clonln9 and functlonal analysis of Baa-l: a novel f&2 bIndIng protein with anttall death ectfvlty. cell 1 BBS, 80~279-204.

24.

Boyd JM, Malstrom S, Subramanian T, Venkateah LK, Schaeper U, Elangovan 8, D’Sa-Eipper, Chinnadumi G: Adenovlrus El B 19 KDa and bd-2 proteins lntemct wtth a common set of wllular protelna Cell 1994,79:341-351.

25.

Sdwffner M, Wetness 84 Huibregtae JM, Levine AJ, Hawley PM: The E6 oncooroteln encoded bv human ~~lllomavlrus type8 16 and 18 promotes the dq#&detton oi p53. Cell 1QQO, 63:1129-l 136

26.

Scheffner M, Huibregtee JM, Vira RD, Howtey PM: The HW16 E6 end E6-AP complex fundfons as a ublqulttn-protdn IlQase In the ublqultinatton of p53. Cell 1993,7%495-505.

4.

Tsujimoto Y, Crow C: Analysis of the structure, tmnscrlpts, and protein products of b&2, the Qene Involved In human follkuler lymphoma. Proc Nat/ Acad Sci USA 1966,83:5214-5218.

5.

6.

7.

8.

Korsmeyer SJ, Shutter JR, Veis DJ, Merry DE, Oltvai ZN: Bd~/SEX: a rheostat that m~ulates an antl-oxidant pathway and wll death. Semin Cancer Biol 1993, 4~327-332. Oltvai ZN, Milliman CL, Kommeyer SJ:Bd-2 heterodlmerlses In v/vo with a conserved homolog, BAX, that accelerates

9.

10.

11.

12.

13.

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29.

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14.

Sedlak TW, Oltvai ZN, Yang E, Wang K, Boise LH, Thompson CB, Korameyer SJ: Multiple bcl-2 family members demonstrate selecttve dlmerlsatlons with bax. Proc Nat/ Acad Sci USA 1995, 92:7034-7038.

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32.

17

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16. ..

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fsctordeprlvad

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34.

Niison DW, Ali 4 Thombeny NA, Vaillancourt JR Ding CK, Gallant M, Gareau Y. Griffin PR. Inbelle M. Lazebnik YA: ldenttfiwtion end lnhlbitlon &-the ICE&D-3 protww nawssary for mammalian apoptosls. Naturn 1995,370:37-43.