The Fowlpox Virus BCL-2 Homologue,FPV039, Interacts with Activated Bax and a Discrete Subset of BH3-Only Proteins To Inhibit Apoptosis

Apoptosis is a potent immune barrier against viral infection, and many viruses, including poxviruses, encode proteins to overcome this defense. Interestingly, the avipoxviruses, which include fowlpox and canarypox virus, are the only poxviruses known to encode proteins with obvious Bcl-2 sequence homology. We previously characterized the fowlpox virus protein FPV039 as a Bcl-2-like antiapoptotic protein that inhibits apoptosis by interacting with and inactivating the proapoptotic cellular protein Bak. However, both Bak and Bax can independently trigger cell death. Thus, to effectively inhibit apoptosis, a number of viruses also inhibit Bax. Here we show that FPV039 inhibited apoptosis induced by Bax overexpression and prevented both the conformational activation of Bax and the subsequent formation of Bax oligomers at the mitochondria, two critical steps in the induction of apoptosis. Additionally, FPV039 interacted with activated Bax in the context of Bax overexpression and virus infection. Importantly, the ability of FPV039 to interact with active Bax and inhibit Bax activity was dependent on the structurally conserved BH3 domain of FPV039, even though this domain possesses little sequence homology to other BH3 domains. FPV039 also inhibited apoptosis induced by the BH3-only proteins, upstream activators of Bak and Bax, despite interacting detectably with only two: BimL and Bik. Collectively, our data suggest that FPV039 inhibits apoptosis by sequestering and inactivating multiple proapoptotic Bcl-2 proteins, including certain BH3-only proteins and both of the critical “gatekeepers” of apoptosis, Bak and Bax.Apoptosis is a highly conserved form of programmed cell death that plays an important role in the immune defense against pathogens. The controlled and deliberate destruction of virally infected cells comprises a potent innate immune barrier against rampant viral replication and infection. As such, many viruses, including poxviruses, encode numerous proteins that inhibit a variety of steps in the biochemical pathways that lead to cell death (29, 69).The mitochondria, and the Bcl-2 family of proteins that preside over them, serve as an important control point in the regulation of apoptosis (87). United by the presence of one to four highly conserved Bcl-2 homology (BH) domains, the Bcl-2 family regulates the integrity of the outer mitochondrial membrane (OMM) and controls the release of apoptogenic molecules from the mitochondrial intermembrane space. Bak and Bax, the two proapoptotic Bcl-2 proteins, possess BH domains 1 to 3 and, upon activation, commit the cell to death (53, 77). Whereas Bak resides constitutively at the OMM, Bax exists in an inactive form in the cytoplasm and, upon apoptotic insult, undergoes a conformational change that exposes its C-terminal transmembrane domain and results in its relocalization to the OMM (10, 34, 41, 56). The attendant exposure of the N termini of both Bak and Bax precedes Bak and Bax homooligomerization, which facilitates mitochondrial damage and, ultimately, the release of cytochrome c (3, 4, 36, 37, 76). Cytochrome c, in turn, triggers the activation of caspases, a group of cysteine proteases responsible for dismantling the apoptotic cell (59). Bak and Bax are therefore crucial for the induction of apoptosis and, because either Bak or Bax alone is sufficient to facilitate the release of cytochrome c, both must be inactivated to effectively inhibit apoptosis (53, 77, 90). The activation of Bak and Bax is counteracted by the antiapoptotic members of the Bcl-2 family, including Bcl-2, Bcl-X_L, and Mcl-1. These three proteins, which possess all four BH domains, reside at the mitochondria and prevent apoptosis by directly interacting with and inhibiting Bak and Bax or the BH3-only proteins (87). The BH3-only proteins, which possess only the BH3 domain, act as sentinels responsive to a variety of cellular stresses, including virus infection (79). Upon receipt of an apoptotic stimulus, BH3-only proteins become activated and subsequently activate Bak and Bax or inhibit the antiapoptotic function of Bcl-2, Bcl-X_L, and Mcl-1 (15). Of the eight BH3-only proteins that are directly involved in the induction of apoptosis—namely, Bim, Bid, Puma, Bik, Bmf, Bad, Noxa, and Hrk—each displays a specific and characteristic ability to bind and inhibit Bcl-2 proteins (79).Like cellular antiapoptotic Bcl-2 proteins, viral inhibitors of apoptosis have evolved especially to interfere with the activation of Bak and Bax (18, 40). For example, E1B 19K, encoded by adenovirus, and M11L, encoded by myxoma virus, bind and inactivate both Bak and Bax to inhibit apoptosis (26, 49, 65, 67, 72). Similarly, ORF125, the antiapoptotic protein encoded by the poxvirus Orf virus, also inactivates Bak and Bax, but exactly how ORF125 mediates this inactivation remains unknown (78). Although interacting with Bak and Bax is ostensibly the most direct way to prevent apoptosis, several viral antiapoptotic proteins appear to inhibit apoptosis by functioning upstream of Bak and Bax at the level of the BH3-only proteins. The vaccinia virus protein F1L, for example, interacts with Bak but not Bax, yet F1L is nonetheless capable of inactivating Bax, likely a result of F1L interacting with the BH3-only protein and Bax activator, Bim (61, 70, 74). Moreover, the Bcl-2 homolog encoded by Kaposi''s sarcoma-associated herpesvirus, and BHRF-1, encoded by Epstein-Barr virus, each interact with a specific and distinct array of BH3-only proteins, yet neither protein interacts detectably with Bak or Bax (14, 27, 44). Thus, to effectively inhibit apoptosis, it may not be necessary for viral proteins to directly target Bak and Bax but, instead, to prevent the activation of Bak and Bax by interfering with the upstream BH3-only proteins (15).Recently, our lab has shown that FPV039, encoded by fowlpox virus, localizes to the mitochondria, where it inhibits apoptosis induced by a variety of stimuli (6). Interestingly, FPV039 is the only characterized poxvirus protein that shares obvious, albeit limited, sequence homology with cellular Bcl-2 proteins (1, 6). FPV039 possesses a highly conserved BH1 and BH2 domain but lacks an obvious BH3 and BH4 domain. Importantly, however, we predicted structural homology between the Bcl-2 BH3 domain and a corresponding region in FPV039, and we validated the prediction by showing that this cryptic FPV039 BH3 domain is functionally important (6). Indeed, the ability of FPV039 to interact with the proapoptotic protein Bak is dependent on this cryptic BH3 domain (6). Thus, despite lacking sequence conservation of a highly conserved BH3 domain, FPV039 is able to interact with, and inactivate, the proapoptotic protein Bak. Nevertheless, to completely inhibit apoptosis, both Bak and Bax must be inactivated.Accordingly, we wanted to determine whether FPV039, in addition to inactivating Bak, could inactivate Bax. We report here that FPV039 inhibited Bax activity and prevented critical steps in Bax activation. FPV039 did not appear to interact with endogenous inactive Bax; however, FPV039 was able to interact with active Bax. Moreover, FPV039 inhibited apoptosis induced by the BH3-only proteins despite interacting with only BimL and Bik. Together, these data strongly suggest FPV039 inhibits apoptosis by inactivating multiple proapoptotic Bcl-2 proteins, including the critical Bak and Bax, as well as a discrete subset of BH3-only proteins.     

Vesicular Stomatitis Virus Induces Apoptosis Primarily through Bak Rather than Bax by Inactivating Mcl-1 and Bcl-XL

Vesicular stomatitis virus (VSV) induces apoptosis via the mitochondrial pathway. The mitochondrial pathway is regulated by the Bcl-2 family of proteins, which consists of both pro- and antiapoptotic members. To determine the relative importance of the multidomain proapoptotic Bcl-2 family members Bak and Bax, HeLa cells were transfected with Bak and/or Bax small interfering RNA (siRNA) and subsequently infected with recombinant wild-type VSV. Our results showed that Bak is more important than Bax for the induction of apoptosis in this system. Bak is regulated by two antiapoptotic Bcl-2 proteins, Mcl-1, which is rapidly turned over, and Bcl-X_L, which is relatively stable. Inhibition of host gene expression by the VSV M protein resulted in the degradation of Mcl-1 but not Bcl-X_L. However, inactivation of both Mcl-1 and Bcl-X_L was required for cells to undergo apoptosis. While inactivation of Mcl-1 was due to inhibition of its expression, inactivation of Bcl-X_L indicates a role for one or more BH3-only Bcl-2 family members. VSV-induced apoptosis was inhibited by transfection with siRNA against Bid, a BH3-only protein that is normally activated by the cleavage of caspase-8, the initiator caspase associated with the death receptor pathway. Similarly, treatment with an inhibitor of caspase-8 inhibited VSV-induced apoptosis. These results indicate a role for cross talk from the death receptor pathway in the activation of the mitochondrial pathway by VSV.The induction of cell death is a major mechanism by which many viruses cause disease in the tissues they infect (23). In addition, the cytolytic activity of viruses has the potential for therapeutic applications, such as the development of oncolytic viruses for the treatment of cancer (27). Vesicular stomatitis virus (VSV) is well studied as a prototype for negative-strand RNA viruses and is an exceptionally potent inducer of apoptosis in a wide variety of cell types (4, 20, 21). Due to its particularly rapid cytopathic effects, VSV is one of the major viruses being developed as an oncolytic agent (27). VSV is capable of inducing apoptosis by activation of multiple apoptotic pathways. It is important to determine how these pathways are activated and the role that they play in apoptosis induced by VSV in order to understand the virulence and oncolytic activity of the virus, as well as to provide a model to which other viruses can be compared.Previous work showed that wild-type (wt) VSV induces apoptosis via the mitochondrial (intrinsic) pathway through the initiator caspase caspase-9 (4, 19). This is due in part to the inhibition of host gene expression by the VSV M protein (19). The inhibition of host gene expression by M protein is the mechanism by which VSV inhibits the host antiviral response (2, 31) and leads to induction of apoptosis, similar to that induced by pharmacologic inhibitors of host gene expression (19). Additionally, M protein mutants of VSV that are deficient in the ability to inhibit new host gene expression are effective inducers of apoptosis (12, 13, 19, 20). However, in contrast to wt VSV, induction of apoptosis by M protein mutant virus occurs primarily via the extrinsic pathway through the initiator caspase caspase-8 (12, 13). Infection with M protein mutant VSV results in the expression of proapoptotic genes that are suppressed during infection with wt VSV (12). Therefore, in the case of VSV with wt M protein, the induction of apoptosis is most likely mediated by proteins already present in the host cell. Since it has previously been shown that wt VSV activates the intrinsic pathway, we focused on the Bcl-2 family of proteins to determine the role of Bcl-2 family members in apoptosis induced by wt VSV.Bcl-2 family proteins function to either suppress or promote mitochondrial outer membrane permeabilization, thereby regulating the release of proapoptotic factors into the cytosol, such as cytochrome c, apoptosis-inducing factor (AIF), and Smac/Diablo (5). Bcl-2 family proteins are subdivided into three groups, depending on the conservation of Bcl-2 homology (BH) domains and function (reviewed in references 8 and 38). The multidomain antiapoptotic Bcl-2 proteins contain BH domains BH1 to BH4 and function to inhibit apoptosis by binding to proapoptotic Bcl-2 family members. Members of this group include Bcl-2, Bcl-X_L, Mcl-1, Bcl-w, and BFL-1/A1. The proapoptotic Bcl-2 proteins are comprised of two groups, the multidomain proteins and the BH3-only proteins. Bax and Bak are the two main members of the multidomain group, containing BH domains BH1 to BH3. These proteins are primarily responsible for the permeabilization of the mitochondrial outer membrane, if their activity is not suppressed by antiapoptotic Bcl-2 family members. The BH3-only proteins contain only one Bcl-2 homology domain (BH3) and include Bid, Bad, Bim, Puma, Noxa, and Bik, among others. These proteins function as upstream sensors of signaling pathways and convey to other Bcl-2 family proteins the signals to initiate apoptosis. These death signals can be transmitted from the BH3-only proteins by either binding to antiapoptotic proteins, causing the release of Bak and Bax, or binding to Bak and Bax, thereby causing their activation (6).The pathways leading to activation of Bak differ from those that activate Bax. Interestingly, only two antiapoptotic Bcl-2 proteins, Mcl-1 and Bcl-X_L, have been shown to interact with Bak, while Bax appears to be able to interact with all of the antiapoptotic proteins, with the exception of Mcl-1 (7, 35). BH3-only proteins have strong binding affinities to the antiapoptotic proteins, suggesting that their primary role may be to derepress Bak and Bax by binding and inhibiting the antiapoptotic proteins (36). In addition, BH3-only proteins may play a role in activation of Bak and Bax by binding and inducing an activated conformation (6, 34). For some stimuli, such as the protein kinase inhibitor staurosporine (SSP), the topoisomerase II inhibitor etoposide, and UV radiation, Bak and Bax appear to be redundant, in that the deletion of both is required to render cells resistant to these agents (33). In contrast, Bak and Bax were nonredundant in the induction of apoptosis by Neisseria gonorrhoeae and cisplatin, such that both were required for apoptosis to occur (18).In the experiments reported here, the silencing of Bak or Bax expression with small interfering RNA (siRNA) showed that Bak is more important than Bax for the induction of apoptosis in HeLa cells infected with wt VSV. Overexpression of both of the antiapoptotic Bcl-2 family proteins known to interact with Bak, Mcl-1 and Bcl-X_L, delayed the onset of apoptosis, while depletion of Mcl-1 or Bcl-X_L by siRNA transfection prior to infection increased the rate of apoptosis. Furthermore, M protein inhibition of new host gene expression led to the depletion of Mcl-1, enabling the rapid activation of apoptosis. However, inhibition of Bcl-X_L was also required for the initiation of apoptosis, indicating a role for one or more BH3-only proteins. Bid, a BH3-only protein that is normally activated by the cleavage of caspase-8, was shown to be important for induction of apoptosis by VSV. Likewise, treatment with an inhibitor of caspase-8 inhibited VSV-induced apoptosis. These results indicate a role for cross talk from the death receptor pathway in the activation of the mitochondrial pathway by VSV.     

Theiler's Murine Encephalomyelitis Virus Leader Protein Is the Only Nonstructural Protein Tested That Induces Apoptosis When Transfected into Mammalian Cells

Theiler''s murine encephalomyelitis virus (TMEV) induces two distinct cell death programs, necrosis and apoptosis. The apoptotic pathway is of particular interest because TMEV persists in the central nervous system of mice, largely in infiltrating macrophages, which undergo apoptosis. Infection of murine macrophages in culture induces apoptosis that is Bax dependent through the intrinsic or mitochondrial pathway, restricting infectious-virus yields and raising the possibility that apoptosis represents a mechanism to attenuate TMEV yet promote macrophage-to-macrophage spread during persistent infection. To help define the cellular stressors and upstream signaling events leading to apoptosis during TMEV infection, we screened baby hamster kidney (BHK-21) cells transfected to express individual nonstructural genes (except 3B) of the low-neurovirulence BeAn virus strain for cell death. Only expression of the leader protein led to apoptosis, as assessed by fluorescence-activated cell sorting analysis of propidium iodide- and annexin V-stained transfected cells, immunoblot analysis of poly(ADP-ribose) polymerase and caspase cleavages, electron microscopy, and inhibition of apoptosis by the pancaspase inhibitor qVD-OPh. After transfection, Bak and not Bax expression increased, suggesting that the apical pathway leading to activation of these Bcl-2 multi-BH-domain proapoptotic proteins differs in BeAn virus infection versus L transfection. Mutation to remove the CHCC Zn finger motif from L, a motif required by L to mediate inhibition of nucleocytoplasmic trafficking, significantly reduced L-protein-induced apoptosis in both BHK-21 and M1-D macrophages.Theiler''s murine encephalomyelitis viruses (TMEV), members of the genus Cardiovirus in the family Picornaviridae, are highly cytolytic RNA viruses. Mice experimentally infected with a low-neurovirulence TMEV, such as BeAn virus, develop persistent infection in the central nervous system (CNS) and an inflammatory demyelinating disease, providing an experimental analogue for multiple sclerosis. BeAn virus persists primarily in macrophages in the CNS of infected mice. Schlitt et al. (34) found that 74% of TUNEL-positive cells in infected spinal cords (primarily in CNS lesions) were T and B lymphocytes and 8% were macrophages, although virus genomes were detected in <1% of apoptotic cells, consistent with infection of only a low percentage of macrophages and the fact that TMEV does not infect T or B lymphocytes in culture. Thus, some means other than direct infection was responsible for apoptosis of most CNS macrophages, including TMEV triggering apoptosis through tumor necrosis factor alpha or tumor necrosis factor alpha-related apoptosis-inducing ligand by binding death receptors on activated macrophages in vitro, as reported elsewhere (17).Infection of mouse macrophages induces apoptosis (16, 26) mediated by Bax through the intrinsic or mitochondrial pathway and severely restricts the yield of progeny virus (37). Thus, apoptosis may be a mechanism to attenuate the virus yet promote macrophage-to-macrophage spread through phagocytosis of infected apoptotic blebs during persistence (37). In contrast, TMEV infection in other rodent cells tested thus far, including baby hamster kidney (BHK-21) cells, produces necrotic cell death with high virus yields. The contrasting outcomes of TMEV infection point to the existence of two distinct virus-induced cell death programs.The genes of an increasing number of RNA viruses have been shown to encode proteins that trigger apoptosis. Among picornaviruses, coxsackievirus B3 1B (VP2) (12, 13), avian encephalomyocarditis virus 1C (VP3) (24) and 2C (25), enterovirus 71 2A (20), and poliovirus 2A (10) and 3C protease (3C^pro) (3) induce apoptosis, mostly through the intrinsic pathway. Coxsackievirus B3 VP2 has been shown to interact with the proapoptotic Siva protein in a yeast two-hybrid screen (12), but exactly how the VP2-Siva interaction or any of the other picornavirus proteins initiates the apoptotic cascade remains unknown.To gain insight into the upstream signaling events that lead to apoptosis, we tested the ability of individual BeAn virus nonstructural genes to induce apoptosis in uninfected BHK-21 cells. Only the leader (L) protein resulted in apoptosis and mutation of the CHCC Zn finger motif in L significantly reduced L protein-induced apoptosis.     

Bid chimeras indicate that most BH3-only proteins can directly activate Bak and Bax,and show no preference for Bak versus Bax

The mitochondrial pathway of apoptosis is initiated by Bcl-2 homology region 3 (BH3)-only members of the Bcl-2 protein family. On upregulation or activation, certain BH3-only proteins can directly bind and activate Bak and Bax to induce conformation change, oligomerization and pore formation in mitochondria. BH3-only proteins, with the exception of Bid, are intrinsically disordered and therefore, functional studies often utilize peptides based on just their BH3 domains. However, these reagents do not possess the hydrophobic membrane targeting domains found on the native BH3-only molecule. To generate each BH3-only protein as a recombinant protein that could efficiently target mitochondria, we developed recombinant Bid chimeras in which the BH3 domain was replaced with that of other BH3-only proteins (Bim, Puma, Noxa, Bad, Bmf, Bik and Hrk). The chimeras were stable following purification, and each immunoprecipitated with full-length Bcl-x_L according to the specificity reported for the related BH3 peptide. When tested for activation of Bak and Bax in mitochondrial permeabilization assays, Bid chimeras were ~1000-fold more effective than the related BH3 peptides. BH3 sequences from Bid and Bim were the strongest activators, followed by Puma, Hrk, Bmf and Bik, while Bad and Noxa were not activators. Notably, chimeras and peptides showed no apparent preference for activating Bak or Bax. In addition, within the BH3 domain, the h0 position recently found to be important for Bax activation, was important also for Bak activation. Together, our data with full-length proteins indicate that most BH3-only proteins can directly activate both Bak and Bax.The Bcl-2 family of proteins controls the mitochondrial pathway of apoptosis, a process often dysregulated in cancer and other diseases.^{1, 2, 3} Apoptotic triggers including DNA damage and oncogene activation cause the synthesis or activation of one or more pro-apoptotic Bcl-2 homology region 3 (BH3)-only proteins,^{1, 2, 3, 4} a subfamily that includes Bid, Bim, Puma, Noxa, Bad, Bik, Bmf and Hrk. These proteins then engage via their BH3 domain with other Bcl-2 family members. BH3-only proteins that can directly bind and activate the Bcl-2 effector proteins Bak or Bax are called ‘activators''.⁵ When Bak or Bax become activated and oligomerize in the mitochondrial outer membrane (MOM), the apoptotic ‘switch'' has flipped and the cell is committed to cell death. The prosurvival members (Bcl-2, Bcl-x_L, Mcl-1, Bcl-w, Bfl-1/A1 and Bcl-B) inhibit apoptosis by specifically binding both the BH3-only proteins and activated Bak and Bax.^{6, 7, 8, 9, 10, 11} Thus, the cell''s complement of prosurvival proteins, Bak, and Bax, determines the sensitivity of that cell to each BH3-only protein, and by extension to each type of pro-apoptotic stimulus.A thorough understanding of BH3-only proteins is crucial for the development of cancer therapeutics such as the new class of anti-cancer molecules called BH3 mimetics that are showing significant promise in clinical trials.^{12, 13} The binding of BH3-only proteins to prosurvival proteins has been well-characterized and revealed significant preferences for engaging different members.^{6, 8, 9} How BH3-only proteins bind and activate Bak and Bax remains less understood for several reasons. First, generating stable recombinant BH3-only proteins is difficult because, except for Bid, they are intrinsically disordered^{14, 15, 16} and because most contain hydrophobic C-terminal membrane anchors.¹⁷ Thus, most in vitro studies of BH3-only proteins have used synthetic peptides corresponding to the BH3 domains, C-terminally truncated recombinant proteins or in vitro translated (IVT) proteins. Second, BH3-only reagents bind poorly to recombinant Bak and Bax in the absence of membranes, although detergents and liposomes may substitute for the MOM.^{18, 19, 20} Third, activation of Bak and Bax on mitochondria can be complicated by the presence of other proteins such as prosurvival proteins. Indeed, genetically altering BH3-only protein levels in mice resulted in complex phenotypes due to multiple interactions between family members, precluding firm conclusions as to which BH3-only proteins are direct activators.^{18, 21, 22}Bid and Bim are direct activators according to a variety of approaches,^{5, 8, 9, 23, 24} and were recently proposed to be specific for Bak and Bax, respectively.²⁵ Early studies using Noxa BH3 peptides^{5, 8} and IVT Noxa⁹ concluded that Noxa was not an activator. However, in more recent studies a Noxa BH3 peptide²³ and purified recombinant NoxaΔC²⁰ were found to be activators of both Bak and Bax. Puma has also been described as both an activator^{26, 27} and not an activator.^{8, 28} Du et al.²³ analyzed the full panel of BH3 peptides and classified Bim as a strong activator, Bid, Noxa and Bmf as moderate activators, and Puma, Bik and Hrk as weak activators. The only BH3-only member that has never been described as an activator is Bad.While BH3 peptides and recombinant truncated BH3-only proteins have been useful for in vitro studies, new reagents that target mitochondria may better reflect the behavior of the parent proteins. As Bid is stable as a recombinant protein, we generated chimeras of Bid in which the BH3 domain of Bid was replaced with that of seven other BH3-only proteins. This is a similar approach to the Bim chimeras used for expression in cells¹⁸ and in mice.²⁹ More recently, truncated Bid (tBid) chimeras containing the BH3 domains of Bim, Bak and Bax as well as those of the prosurvival proteins, have been generated as IVT proteins.¹¹To compare the ability of BH3-only proteins to activate Bak and Bax in vitro, we incubated Bid chimeras and BH3 peptides with mitochondria containing either Bak or Bax. We found that the membrane-targeted Bid chimeras were much more potent activators than their related BH3 peptides, and that all BH3 domains except for Bad and Noxa were activators to some extent. We conclude that activation of Bak and Bax may be underestimated by studies using BH3 peptides, and that even BH3-only proteins such as Bik, Bmf and Hrk that are often considered unable to activate Bak or Bax, may act as activators under certain conditions.     

Context-dependent Bcl-2/Bak Interactions Regulate Lymphoid Cell Apoptosis

The release of cytochrome c from mitochondria, which leads to activation of the intrinsic apoptotic pathway, is regulated by interactions of Bax and Bak with antiapoptotic Bcl-2 family members. The factors that regulate these interactions are, at the present time, incompletely understood. Recent studies showing preferences in binding between synthetic Bcl-2 homology domain 3 and antiapoptotic Bcl-2 family members in vitro have suggested that the antiapoptotic proteins Mcl-1 and Bcl-x_L, but not Bcl-2, restrain proapoptotic Bak from inducing mitochondrial membrane permeabilization and apoptosis. Here we show that Bak protein has a much higher affinity than the 26-amino acid Bak Bcl-2 homology domain 3 for Bcl-2, that some naturally occurring Bcl-2 allelic variants have an affinity for full-length Bak that is only 3-fold lower than that of Mcl-1, and that endogenous levels of these Bcl-2 variants (which are as much as 40-fold more abundant than Mcl-1) restrain part of the Bak in intact lymphoid cells. In addition, we demonstrate that Bcl-2 variants can, depending on their affinity for Bak, substitute for Mcl-1 in protecting cells. Thus, the ability of Bcl-2 to protect cells from activated Bak depends on two important contextual variables, the identity of the Bcl-2 present and the amount expressed.The release of cytochrome c from mitochondria, which leads to activation of the intrinsic apoptotic pathway, is regulated by Bcl-2 family members (1–5). This group of proteins consists of three subgroups: Bax and Bak, which oligomerize upon death stimulation to form a putative pore in the outer mitochondrial membrane, thereby allowing efflux of cytochrome c and other mitochondrial intermembrane space components; Bcl-2, Bcl-x_L, Mcl-1, and other antiapoptotic homologs, which antagonize the effects of Bax and Bak; and BH3-only proteins² such as Bim, Bid, and Puma, which are proapoptotic Bcl-2 family members that share only limited homology with the other two groups in a single 15-amino acid domain (the BH3 domain, see Ref. 6). Although it is clear that BH3-only proteins serve as molecular sensors of various stresses and, when activated, trigger apoptosis (3, 6–11), the mechanism by which they do so remains incompletely understood. One current model suggests that BH3-only proteins trigger apoptosis solely by binding and neutralizing antiapoptotic Bcl-2 family members, thereby causing them to release the activated Bax and Bak that are bound (reviewed in Refs. 9 and 10; see also Refs. 12 and 13), whereas another current model suggests that certain BH3-only proteins also directly bind to and activate Bax (reviewed in Ref. 3; see also Refs. 14–17). Whichever model turns out to be correct, both models agree that certain antiapoptotic Bcl-2 family members can inhibit apoptosis, at least in part, by binding and neutralizing activated Bax and Bak before they permeabilize the outer mitochondrial membrane (13, 18, 19).Much of the information about the interactions between pro- and antiapoptotic Bcl-2 family members has been derived from the study of synthetic peptides corresponding to BH3 domains. In particular, these synthetic peptides have been utilized as surrogates for the full-length proapoptotic proteins during structure determinations (20–22) as well as in functional studies exploring the effect of purified BH3 domains on isolated mitochondria (14, 23) and on Bax-mediated permeabilization of lipid vesicles (15).Recent studies using these same peptides have suggested that interactions of the BH3 domains of Bax, Bak, and the BH3-only proteins with the “BH3 receptors” of the antiapoptotic Bcl-2 family members are not all equivalent. Surface plasmon resonance, a technique that is widely used to examine the interactions of biomolecules under cell-free conditions (24–26), has demonstrated that synthetic BH3 peptides of some BH3-only family members show striking preferences, with the Bad BH3 peptide binding to Bcl-2 and Bcl-x_L but not Mcl-1, and the Noxa BH3 peptide binding to Mcl-1 but not Bcl-2 or Bcl-x_L (27). Likewise, the Bak BH3 peptide exhibits selectivity, with high affinity for Bcl-x_L and Mcl-1 but not Bcl-2 (12). The latter results have led to a model in which Bcl-x_L and Mcl-1 restrain Bak and inhibit Bak-dependent apoptosis, whereas Bcl-2 does not (10).Because the Bak protein contains multiple recognizable domains in addition to its BH3 motif (28, 29), we compared the binding of Bak BH3 peptide and Bak protein to Bcl-2. Surface plasmon resonance demonstrated that Bcl-2 binds Bak protein with much higher affinity than the Bak 26-mer BH3 peptide. Further experiments demonstrated that the K_D for Bak differs among naturally occurring Bcl-2 sequence variants but is only 3-fold higher than that of Mcl-1 in some cases. In light of previous reports that Bcl-2 overexpression contributes to neoplastic transformation (30–33) and drug resistance (34–36) in lymphoid cells, we also examined Bcl-2 expression and Bak binding in a panel of neoplastic lymphoid cell lines. Results of these experiments demonstrated that Bcl-2 expression varies among different lymphoid cell lines but is up to 40-fold more abundant than Mcl-1. In lymphoid cell lines with abundant Bcl-2, Bak is detected in Bcl-2 as well as Mcl-1 immunoprecipitates; and Bak-dependent apoptosis induced by Mcl-1 down-regulation can be prevented by Bcl-2 overexpression. Collectively, these observations shed new light on the role of Bcl-2 in binding and neutralizing Bak.     

Lack of Bax Prevents Influenza A Virus-Induced Apoptosis and Causes Diminished Viral Replication

The ectopic overexpression of Bcl-2 restricts both influenza A virus-induced apoptosis and influenza A virus replication in MDCK cells, thus suggesting a role for Bcl-2 family members during infection. Here we report that influenza A virus cannot establish an apoptotic response without functional Bax, a downstream target of Bcl-2, and that both Bax and Bak are directly involved in influenza A virus replication and virus-induced cell death. Bak is substantially downregulated during influenza A virus infection in MDCK cells, and the knockout of Bak in mouse embryonic fibroblasts yields a dramatic rise in the rate of apoptotic death and a corresponding increase in levels of virus replication, suggesting that Bak suppresses both apoptosis and the replication of virus and that the virus suppresses Bak. Bax, however, is activated and translocates from the cytosol to the mitochondria; this activation is required for the efficient induction of apoptosis and virus replication. The knockout of Bax in mouse embryonic fibroblasts blocks the induction of apoptosis, restricts the infection-mediated activation of executioner caspases, and inhibits virus propagation. Bax knockout cells still die but by an alternative death pathway displaying characteristics of autophagy, similarly to our previous observation that influenza A virus infection in the presence of a pancaspase inhibitor leads to an increase in levels of autophagy. The knockout of Bax causes a retention of influenza A virus NP within the nucleus. We conclude that the cell and virus struggle to control apoptosis and autophagy, as appropriately timed apoptosis is important for the replication of influenza A virus.The pathology of influenza A virus infection usually arises from acute lymphopenia and inflammation of the lungs and airway columnar epithelial cells (23, 38). Influenza A virus induces apoptotic death in infected epithelial, lymphocyte, and phagocytic cells, and apoptosis is a source of tissue damage during infection (3, 22, 33) and increased susceptibility to bacterial pathogens postinfection (31). While the induction of apoptosis by influenza A virus has been well documented (4, 19-21, 28, 33, 37), the mechanisms of this interaction are not well understood. Two viral proteins, NS1 and PB1-F2, have been associated with viral killing of cells. NS1, originally characterized as being proapoptotic (34), was later identified as being an interferon antagonist, inhibiting the activation of several key antiviral responses and restricting the apoptotic response to infection (1, 10, 15, 18, 35, 39, 46). In contrast, PB1-F2 induces apoptosis primarily by localizing to the outer mitochondrial membrane, promoting cytochrome c release, and triggering the apoptotic cascade (43). This effect, however, is typically restricted to infected monocytes, leading to the hypothesis that PB1-F2 induces apoptosis specifically to clear the landscape of immune responders (5, 44). Although PB1-F2 activity does not directly manipulate virus replication or virus-induced apoptosis, PB1-F2 localization to the mitochondrial membrane during infection potentiates the apoptotic response in epithelial and fibroblastic cells through tBID signaling with proapoptotic Bcl-2 family protein members Bax and Bak (22, 43, 44).The Bcl-2 protein family consists of both pro- and antiapoptotic members that regulate cytochrome c release during mitochondrion-mediated apoptosis through the formation of pore-like channels in the outer mitochondrial membrane (12, 16). During the initiation of mitochondrion-mediated apoptosis, cytoplasmic Bid is cleaved to form tBID. This, in turn, activates proapoptotic Bax and Bak (40), which drive cytochrome c release and subsequent caspase activation. Bak is constitutively associated with the mitochondrial membrane, whereas inactive Bax is primarily cytosolic, translocating to the outer mitochondrial membrane only after activation (6). The activation of Bax and Bak results in homo- and heterodimer formation at the outer mitochondrial membrane, generating pores that facilitate mitochondrial membrane permeabilization and cytochrome c release (14, 17), leading to caspase activation and the apoptotic cascade (8). Antiapoptotic members of the Bcl-2 protein family, including Bcl-2, inhibit the activation of proapoptotic Bax and Bak primarily by sequestering inactive Bax and Bak monomers via interactions between their BH3 homology domains (7).Bcl-2 expression has been linked to decreased viral replication rates (26). Bcl-2 overexpression inhibits influenza A virus-induced cell death and reduces the titer and spread of newly formed virions (29). The activation of caspase-3 in the absence of sufficient Bcl-2 is critical to the influenza A virus life cycle. Both Bcl-2 expression and the lack of caspase activation during infection lead to the nuclear accumulation of influenza virus ribonucleoprotein (RNP) complexes, thereby leading to the improper assembly of progeny virions and a marked reduction in titers of infectious virus (26, 41, 42, 45).Here we show that influenza A virus induces mitochondrion-mediated (intrinsic-pathway) apoptosis signaled specifically through Bax and that this Bax signaling is essential for the maximum efficiency of virus propagation. In contrast, Bak expression is strongly downregulated during infection. Cells lacking Bak (while expressing Bax) display a much more severe apoptotic phenotype in response to infection and produce infectious virions at a higher rate than the wild type (WT), suggesting that Bak, which can suppress viral replication, is potentially downregulated by the virus. Our results indicate essential and opposing roles for Bax and Bak in both the response of cells to influenza A virus infection and the ability of the virus to maximize its own replicative potential.     

GLTSCR2/PICT-1, a Putative Tumor Suppressor Gene Product,Induces the Nucleolar Targeting of the Kaposi's Sarcoma-Associated Herpesvirus KS-Bcl-2 Protein

KS-Bcl-2, encoded by Kaposi''s sarcoma-associated herpesvirus (KSHV), is a structural and functional homologue of the Bcl-2 family of apoptosis regulators. Like several other Bcl-2 family members, KS-Bcl-2 protects cells from apoptosis and autophagy. Using a yeast two-hybrid screen and coimmunoprecipitation assays, we identified a novel KS-Bcl-2-interacting protein, referred to as protein interacting with carboxyl terminus 1 (PICT-1), encoded by a candidate tumor suppressor gene, GLTSCR2. Confocal laser scanning microscopy revealed nucleolar localization of PICT-1, whereas KS-Bcl-2 was located mostly at the mitochondrial membranes with a small fraction in the nucleoli. Ectopic expression of PICT-1 resulted in a large increase in the nucleolar fraction of KS-Bcl-2, and only a minor fraction remained in the cytoplasm. Furthermore, knockdown of endogenous PICT-1 abolished the nucleolar localization of KS-Bcl-2. However, ectopically expressed PICT-1 did not alter the cellular distribution of human Bcl-2. Subsequent analysis mapped the crucial amino acid sequences of both KS-Bcl-2 and PICT-1 required for their interaction and for KS-Bcl-2 targeting to the nucleolus. Functional studies suggest a correlation between nucleolar targeting of KS-Bcl-2 by PICT-1 and reduction of the antiapoptotic activity of KS-Bcl-2. Thus, these studies demonstrate a cellular mechanism to sequester KS-Bcl-2 from the mitochondria and to downregulate its virally encoded antiapoptotic activity. Additional characterization of the interaction of KS-Bcl-2 and PICT-1 is likely to shed light on the functions of both proteins.Kaposi''s sarcoma (KS)-associated herpesvirus (KSHV), also referred to as human herpesvirus 8 (HHV-8), is a gamma 2 herpesvirus implicated in several cancers, including KS, primary effusion lymphoma (PEL), and a subset of multicentric Castleman''s disease. Among human viruses, KSHV is most closely related to the Epstein-Barr virus (EBV), a tumorigenic gamma 1 herpesvirus known to be associated with lymphomas and nasopharyngeal carcinoma (10, 12).KSHV open reading frame 16 (orf16) encodes the KS-Bcl-2 protein, which shares sequence and functional homology with the Bcl-2 family (9, 31). Members of the Bcl-2 family are defined by the presence of up to four conserved domains known as the Bcl-2 homology (BH) domains. Several members also possess a carboxy-terminal transmembrane domain that mediates their association with intracellular membranes, such as the endoplasmic reticulum or mitochondria. Bcl-2 proteins are thought to serve primarily as cell death agonists or antagonists that integrate diverse survival and death signals, which are generated outside and within the cell (15, 37), yet Bcl-2 proteins also modulate cell cycle checkpoints, DNA repair/recombination pathways, calcium homeostasis, and cellular bioenergetics.All gammaherpesviruses encode Bcl-2 proteins that generally share 20 to 30% homology with one another and with their cellular counterparts (8, 11). The conservation of Bcl-2 homologues in these viruses indicates their importance for viral infection, with an evolutionarily conserved function of unknown nature. KS-Bcl-2, like most herpesvirus homologues of Bcl-2, contains a transmembrane domain and demonstrates conservation of sequences in both BH1 and BH2 but has only a low degree of homology with other regions of cellular Bcl-2 (18, 22). Still, KS-Bcl-2 shares 3-dimensional structural conservation with Bcl-2 family members and includes the conserved BH3 binding groove and a hydrophobic membrane anchor domain that also contains a mitochondrial outer membrane targeting signal (18). The BH3 binding cleft of KS-Bcl-2 binds with high affinity to peptides encoding BH3 domains present on the proapoptotic proteins Noxa, Bik, PUMA, Bak, Bax, Bid, Bim, and, to a much lesser extent, Bad (13, 18, 22). Based on these characteristics, KS-Bcl-2 has been suggested to have the closest resemblance to the cellular Bcl-2 family member Mcl-1 (13).Previous studies have demonstrated that KS-Bcl-2 protects various cell types from apoptosis mediated by the expression of BAX, tBid, or Bim through Sindbis virus infection or by ectopic expression of KSHV-cyclin-CDK6 (9, 13, 25, 31). However, unlike the cellular Bcl-2, KS-Bcl-2 is not a substrate for KSHV-cyclin-CDK6 phosphorylation (25) and cannot be converted into a proapoptotic protein via caspase cleavage (3). KS-Bcl-2 is able to form a stable complex with the cellular protein Aven, which binds Apaf-1 and is known as a regulator of caspase 9 and ataxia-telangiectasia (ATM) activation (7, 16). Like the cellular and other virus-encoded Bcl-2 proteins, KS-Bcl-2 binds Beclin and disrupts its lysosomal degradation pathway of autophagy (21, 29). However, since KS-Bcl-2 lacks the nonstructured loop located between the BH4 and BH3 domains, its binding to BH3-containing proapoptotic proteins and to the BH3-containing proautophagy protein Beclin is not modulated by phosphorylation (38).KS-Bcl-2 is transcribed during lytic virus infection (30, 31). Thus, inhibition of apoptosis and autophagy by KS-Bcl-2 may provide an attractive mechanism for prolonging the life span of KSHV-infected cells, which in turn enables increased virus production or establishment of latency. Whether the function of KS-Bcl-2 is necessary for KSHV-mediated oncogenesis is still unknown. Nevertheless, the KS-Bcl-2 protein is expressed in late-stage KS lesions but has not been detected in latent or in lytic KSHV-infected PEL cells (39).To explore the role of KS-Bcl-2 in cell signaling, we searched for its potential cellular-protein partners. In the present study, we describe a novel interaction between KS-Bcl-2 and the protein interacting with carboxyl terminus 1 (PICT-1) cellular protein, encoded by a candidate tumor suppressor gene, GLTSCR2. We show that this interaction specifically targets KS-Bcl-2 to the nucleolus and decreases its antiapoptotic activity.(Portions of this work were submitted to Bar Ilan Univeristy, Ramat Gan, Israel, by I. Kalt and T. Borodianskiy-Shteinberg in partial fulfillment of the requirements for the degree of Doctor of Philosophy.)     

Identification of Novel in Vivo Phosphorylation Sites of the Human Proapoptotic Protein BAD: PORE-FORMING ACTIVITY OF BAD IS REGULATED BY PHOSPHORYLATION*

BAD is a proapoptotic member of the Bcl-2 protein family that is regulated by phosphorylation in response to survival factors. Although much attention has been devoted to the identification of phosphorylation sites in murine BAD, little data are available with respect to phosphorylation of human BAD protein. Using mass spectrometry, we identified here besides the established phosphorylation sites at serines 75, 99, and 118 several novel in vivo phosphorylation sites within human BAD (serines 25, 32/34, 97, and 124). Furthermore, we investigated the quantitative contribution of BAD targeting kinases in phosphorylating serine residues 75, 99, and 118. Our results indicate that RAF kinases represent, besides protein kinase A, PAK, and Akt/protein kinase B, in vivo BAD-phosphorylating kinases. RAF-induced phosphorylation of BAD was reduced to control levels using the RAF inhibitor BAY 43-9006. This phosphorylation was not prevented by MEK inhibitors. Consistently, expression of constitutively active RAF suppressed apoptosis induced by BAD and the inhibition of colony formation caused by BAD could be prevented by RAF. In addition, using the surface plasmon resonance technique, we analyzed the direct consequences of BAD phosphorylation by RAF with respect to association with 14-3-3 and Bcl-2/Bcl-X_L proteins. Phosphorylation of BAD by active RAF promotes 14-3-3 protein association, in which the phosphoserine 99 represented the major binding site. Finally, we show here that BAD forms channels in planar bilayer membranes in vitro. This pore-forming capacity was dependent on phosphorylation status and interaction with 14-3-3 proteins. Collectively, our findings provide new insights into the regulation of BAD function by phosphorylation.Apoptosis is a genetically programmed, morphologically distinct form of cell death that can be triggered by a variety of physiological and pathological stimuli (1–3). This form of cellular suicide is widely observed in nature and is not only essential for embryogenesis, immune responses, and tissue homeostasis but is also involved in diseases such as tumor development and progression. Bcl-2 family proteins play a pivotal role in controlling programmed cell death. The major function of these proteins is to directly modulate outer mitochondrial membrane permeability and thereby regulate the release of apoptogenic factors from the intermembrane space into the cytoplasm (for a recent review, see Ref. 4). On the basis of various structural and functional characteristics, the Bcl-2 family of proteins is divided into three subfamilies, including proteins that either inhibit (e.g. Bcl-2, Bcl-X_L, or Bcl-w) or promote programmed cell death (e.g. Bax, Bak, or Bok) (5, 6). A second subclass of proapoptotic Bcl-2 family members, the BH3²-only proteins, comprises BAD, Bik, Bmf, Hrk, Noxa, truncated Bid, Bim, and Puma (4). BH3-only proteins share sequence homology only at the BH3 domain. The amphipathic helix formed by the BH3 domain (and neighboring residues) associates with a hydrophobic groove of the antiapoptotic Bcl-2 family members (7, 8). Originally, truncated Bid has been reported to interact with Bax and Bak (9), suggesting that some BH3-only proteins promote apoptosis via at least two different mechanisms: inactivating Bcl-2-like proteins by direct binding and/or by inducing modification of Bax-like molecules. BAD (Bcl-2-associated death promoter, Bcl-2 antagonist of cell death) was described to promote apoptosis by forming heterodimers with the prosurvival proteins Bcl-2 and Bcl-X_L, thus preventing them from binding with Bax (10). More recently, two major models have been suggested for how BH3-only proteins may induce apoptosis. In the direct model, all BH3-only proteins promote cell death by directly binding and inactivating their specific anti-death Bcl-2 protein partner (11, 12). In this model, the relative killing potency of different BH3-only proteins is based on their affinities for antiapoptotic proteins. Thus, the activation of Bax/Bak would be mediated through their release from antiapoptotic counterparts. Contrary to this model, Kim et al. (13) provided support for an alternative hierarchy model, in which BH3-only proteins are divided into two distinct subsets. According to this model, the inactivator BH3-only proteins, like BAD, Noxa, and some others, respond directly to survival factors, resulting in phosphorylation, 14-3-3 binding, and suppression of the proapoptotic function. In the absence of growth factors, these proteins engage specifically their preferred antiapoptotic Bcl-2 proteins. The targeted Bcl-2 proteins then release the other subset of BH3-only proteins designated the activators (truncated Bid, Bim, and Puma) that in turn bind to and activate Bax and Bak.Non-phosphorylated BAD associated with Bcl-2/Bcl-X_L is found at the outer mitochondrial membrane. Phosphorylation of specific serine residues, Ser-112 and Ser-136 of mouse BAD (mBAD) or the corresponding phosphorylation sites Ser-75 and Ser-99 of human BAD (hBAD), results in association with 14-3-3 proteins and subsequent relocation of BAD (14, 15). Phosphorylation of mBAD at Ser-155 (Ser-118 of hBAD) within its BH3 domain disrupts the association with Bcl-2 or Bcl-X_L, promoting cell survival (16). Therefore, the phosphorylation status of BAD at these serine residues reflects a checkpoint for cell death or survival. Although the C-RAF kinase was the first reported BAD kinase (17), its target sites were not clearly defined. However, there is a growing body of evidence for direct participation of RAF in regulation of apoptosis via BAD (18, 19). In addition, Kebache et al. (20) reported recently that the interaction between adaptor protein Grb10 and C-RAF is essential for BAD-mediated cell survival. On the other hand, numerous reports suggest that PKA (21), Akt/PKB (22), PAK (18, 23, 24), Cdc2 (25), RSK (26, 27), CK2 (28), and PIM kinases (29) are involved in BAD phosphorylation as well. The involvement of c-Jun N-terminal kinase in BAD phosphorylation is controversially discussed. Whereas Donovan et al. (30) reported that c-Jun N-terminal kinase phosphorylates mBAD at serine 128, Zhang et al. (31) claimed that c-Jun N-terminal kinase is not a BAD-serine 128 kinase. On the other hand, it has been shown that c-Jun N-terminal kinase is able to suppress IL-3 withdrawal-induced apoptosis via phosphorylation of mBAD at threonine 201 (32). Thus, taken together, with respect to regulation of mBAD by phosphorylation, five serine phosphorylation sites (at positions 112, 128, 136, 155, and 170) and two threonines (117 and 201) have been identified so far. Intriguingly, only little data are available regarding the role of phosphorylation in regulation of hBAD protein, although significant structural differences between these two BAD proteins exist.During apoptosis, some members of the Bcl-2 family of proteins, such as Bax or Bak, have been shown to induce permeabilization of the outer mitochondrial membrane, allowing proteins in the mitochondrial intermembrane space to escape into the cytosol, where they can initiate caspase activation and cell death (for a review, see Refs. 33 and 34). Despite intensive investigation, the mechanism whereby Bax and Bak induce outer membrane permeability remains controversial (34). Based on crystal structure (35), it became evident that Bcl-X_L has a pronounced similarity to the translocation domain of diphtheria toxin (36), a domain that can form pores in artificial lipid bilayers. This discovery provoked the predominant view that upon commitment to apoptosis, the proapoptotic proteins Bax and Bak also form pores in the outer mitochondrial membrane (37). As expected from the structural considerations, Bcl-X_L was found to form channels in synthetic lipid membranes (38). Since then, other Bcl-2 family members like Bcl-2, Bax, and the BH3-only protein Bid have been reported to have channel-forming ability. These pores can be divided into two different types: proteinaceous channels of defined size and ion specificity (38–42) and large lipidic pores that allow free diffusion of 2-megadalton macromolecules (43, 44). With respect to the BH3-only protein BAD, no pore-forming abilities have been reported so far, although human BAD has been found to possess per se high affinity for negatively charged phospholipids and liposomes, mimicking mitochondrial membranes (14).The RAF kinases (A-, B-, and C-RAF) play a central role in the conserved Ras-RAF-MEK-ERK signaling cascade and mediate cellular responses induced by growth factors (45–47). Direct involvement of C-RAF in inhibition of proapoptotic properties of BAD established a link between signal transduction and apoptosis control (48, 49). However, the early works did not identify the exact RAF phosphorylation sites on BAD (17). Here we demonstrate that hBAD serves as a substrate of RAF isoforms. With respect to hBAD phosphorylation by PKA, Akt/PKB, and PAK1 in vivo, we observed different specificity compared with RAF kinases. hBAD phosphorylation by RAF was accompanied by reduced apoptosis in HEK293 cells (transformed human embryonic kidney cells) and NIH 3T3 cells (a mouse embryonic fibroblast cell line). Furthermore, we show that in vitro phosphorylation of hBAD by RAF at serines 75, 99, and 118 regulates the binding of 14-3-3 proteins and association with Bcl-2 and Bcl-X_L. By use of mass spectrometry, we detected several novel in vivo phosphorylation sites of hBAD in addition to the established phosphorylation sites, serines 75, 99, and 118. Finally, we show here that hBAD forms channels in planar bilayer membranes in vitro. This pore-forming capacity was dependent on phosphorylation status and interaction with 14-3-3 proteins.     

In non-transformed cells Bak activates upon loss of anti-apoptotic Bcl-XL and Mcl-1 but in the absence of active BH3-only proteins

Mitochondrial apoptosis is controlled by proteins of the B-cell lymphoma 2 (Bcl-2) family. Pro-apoptotic members of this family, known as BH3-only proteins, initiate activation of the effectors Bcl-2-associated X protein (Bax) and Bcl-2 homologous antagonist/killer (Bak), which is counteracted by anti-apoptotic family members. How the interactions of Bcl-2 proteins regulate cell death is still not entirely clear. Here, we show that in the absence of extrinsic apoptotic stimuli Bak activates without detectable contribution from BH3-only proteins, and cell survival depends on anti-apoptotic Bcl-2 molecules. All anti-apoptotic Bcl-2 proteins were targeted via RNA interference alone or in combinations of two in primary human fibroblasts. Simultaneous targeting of B-cell lymphoma-extra large and myeloid cell leukemia sequence 1 led to apoptosis in several cell types. Apoptosis depended on Bak whereas Bax was dispensable. Activator BH3-only proteins were not required for apoptosis induction as apoptosis was unaltered in the absence of all BH3-only proteins known to activate Bax or Bak directly, Bcl-2-interacting mediator of cell death, BH3-interacting domain death agonist and p53-upregulated modulator of apoptosis. These findings argue for auto-activation of Bak in the absence of anti-apoptotic Bcl-2 proteins and provide evidence of profound differences in the activation of Bax and Bak.The regulated elimination of cells by apoptosis is a key mechanism of development, tissue homeostasis and defense. In vertebrates, apoptosis is regulated through two pathways, the death receptor-mediated (extrinsic) and the mitochondrial (intrinsic) pathway, which is activated by numerous apoptotic stimuli. Mitochondrial apoptosis is characterized by loss of mitochondrial outer membrane integrity and the release of mitochondrial intermembrane space proteins, most notably cytochrome c, which leads to the activation of the caspase-9 and effector caspases.¹Release of cytochrome c is governed by proteins of the B-cell lymphoma 2 (Bcl-2) family.² The Bcl-2 family consists of three groups, whose expression and interaction decide cell survival. The anti-apoptotic Bcl-2 proteins include Bcl-2, Bcl-X_L (B-cell lymphoma-extra large), Bcl-w (Bcl-2-like protein 2), Mcl-1 (myeloid cell leukemia sequence 1) and A1 (Bcl-2-related protein A1). The pro-apoptotic group of BH3-only proteins (containing a BH3-domain: Bim (Bcl-2-interacting mediator of cell death), Bid (BH3-interacting domain death agonist), Puma (p53-upregulated modulator of apoptosis), Noxa (Phorbol-12-myristate-13-acetate-induced protein 1), Bad (Bcl-2-associated death promoter), Bik (Bcl-2-interacting killer) and Hrk (activator of apoptosis hara-kiri)) activate the pro-apoptotic effectors Bcl-2-associated X protein (Bax) and Bcl-2 homologous antagonist/killer (Bak). Bax and Bak can replace each other in most situations, but the presence of one of them is required for mitochondrial apoptosis. Upon activation Bax and Bak form oligomers in the outer mitochondrial membrane and cause the release of cytochrome c. How Bax and Bak are activated is still under debate. Different activation models have been proposed and investigated.According to the direct activation model BH3-only proteins can directly, by physical interaction activate Bax and Bak.³ The model was derived in studies investigating synthetic BH3-domain peptides in in vitro systems, that is, isolated mitochondria or liposomes, where peptides encompassing the BH3-domains of Bim or Bid (‘activator'' BH3-only proteins) were able to activate Bax. Peptides derived from the BH3-only proteins Bad, Bik, Hrk, Noxa or Puma did not activate Bax directly. However, these peptides can bind to anti-apoptotic Bcl-2 proteins with varying preferences.⁴ As this may neutralize a combination of anti-apoptotic proteins it may facilitate Bax/Bak activation by activator BH3-only proteins. Consequently, this group of BH3-only proteins has been named ‘sensitizer'' or ‘derepressor'' BH3-only proteins.^{3, 5, 6, 7} The direct activation model has received recent support by structural studies of activator BH3-domains bound to Bax.⁸ That study also found that the BH3-only peptides used previously lacked a residue that is important in the activation of Bax, and the previous results may have to be reconsidered. Indeed, a recent study illustrates that placing the BH3-domain from the various BH3-only proteins into intact Bid protein enhances Bax/Bak-activating capacity of the BH3-domains of Bid, Bim, Puma, Bmf (Bcl-2-modifying factor), Bik and Hrk.⁹The displacement (or indirect activation) model on the other hand posits that Bax and Bak are held in check by anti-apoptotic Bcl-2 proteins and auto-activate when this interaction is broken by BH3-only proteins (displacement). BH3-only proteins can bind to anti-apoptotic Bcl-2 proteins and upon apoptotic stimulation may cause the displacement of these proteins from Bax and Bak, which may lead to the activation of effectors. BH3-peptides derived from Bim and Puma can bind to all anti-apoptotic Bcl-2 proteins and its corresponding proteins exert killing upon overexpression, whereas Bad, Bmf, Bid, Bik, Hrk and Noxa display binding patterns restricted to certain anti-apoptotic Bcl-2 proteins.⁴ It was therefore suggested that Bax/Bak activation requires the neutralization/displacement of several anti-apoptotic proteins, which may be achieved by one BH3-only protein with broadly binding characteristics (such as Bim) or by the combination of BH3-only proteins with restricted binding capabilities (for instance Bad plus Noxa).^{10, 11}The models have been further refined; the ‘embedded together'' model additionally considers the dynamic interaction of the proteins with the mitochondrial membrane,¹² and it has been proposed that the models can be unified by taking two ‘modes'' of inhibition into account: anti-apoptotic Bcl-2 proteins have a dual function in inactivating both, BH3-only proteins and effectors. Pro-apoptotic signals cause the release of activator BH3-only proteins from sequestration with anti-apoptotic Bcl-2 proteins. Free BH3-only proteins directly activate effectors, however, cell death may still not be initiated because the effectors are then held in check by anti-apoptotic Bcl-2 proteins. Free activator BH3-only proteins are required to activate effectors.¹³This model unifies the two above models in the sense that it incorporates aspects of both, inhibition and displacement as well as direct activation. However, the core difference between the (direct) activation and the displacement model appears to be irreconcilable: in the activation model Bax and Bak are inactive unless receiving a stimulus from BH3-only proteins whereas in the displacement model they are active unless bound to anti-apoptotic proteins. Thus, in the absence of all other proteins one model predicts that Bax/Bak are active, the other that they are inactive. Obviously they cannot be both.The direct activation model has initially been established with Bax and the displacement model with Bak. The data are very strong that Bax is activated by direct interaction with BH3-only proteins. Recombinant Bak can also be directly activated by recombinant tBid,¹⁴ and Bid/BH3-chimaeras can activate recombinant Bak missing its C terminus.⁹ However, since Bak is normally inserted into the outer mitochondrial membrane where it may be bound to numerous other Bcl-2-family members, it has been difficult directly to test activation of Bak in the physiological situation.One possibility to ‘unify'' the original models may be in a model where Bax is physiologically activated by direct activation (Bax is inactive until receiving a signal through BH3-only proteins) whereas Bak is activated indirectly (auto-activates when the inhibition by Bcl-2-like proteins is relieved). Here we test this possibility of indirect Bak activation. We targeted anti-apoptotic Bcl-2 family proteins using RNAi. In this setting, protein concentrations and conditions are physiological, which avoids some of the problems associated with overexpression or cell-free experiments. Non-malignant cells may respond differently to the loss of anti-apoptotic Bcl-2 proteins compared with tumor cells.¹⁵ In this study, using non-malignant cells, we targeted all anti-apoptotic Bcl-2 molecules in combinations of two. In the absence of apoptotic stimuli we observed that the combined loss of Bcl-X_L and Mcl-1 was sufficient to induce apoptosis. The direct activator proteins Bid, Bim and Puma were not needed. These observations provide evidence for indirect activation of Bak.     

Caspase-9 mediates Puma activation in UCN-01-induced apoptosis

The protein kinase inhibitor 7-hydroxystaurosporine (UCN-01) is one of the most potent and frequently used proapoptotic stimuli. The BH3-only molecule of Bcl-2 family proteins has been reported to contribute to UCN-01-induced apoptosis. Here we have found that UCN-01 triggers Puma-induced mitochondrial apoptosis pathway. Our data confirmed that Akt-FoxO3a pathway mediated Puma activation. Importantly, we elucidate the detailed mechanisms of Puma-induced apoptosis. Our data have also demonstrated that caspase-9 is a decisive molecule of Puma induction after UCN-01 treatment. Caspase-9 mediates apoptosis through two kinds of feedback loops. On the one hand, caspase-9 enhances Puma activation by cleaving Bcl-2 and Bcl-xL independent of caspase-3. On the other hand, caspase-9 directly activated caspase-3 in the presence of caspase-3. Caspase-3 could cleave XIAP in an another positive feedback loop to further sensitize cancer cells to UCN-01-induced apoptosis. Therefore, caspase-9 mediates Puma activation to determine the threshold for overcoming chemoresistance in cancer cells.The apoptosis pathway is closely related to the Bcl-2 family proteins in which antiapoptotic members sequester multidomain proapoptotic proteins, thereby inhibiting their active role in apoptosis. In contrast, BH3-only proteins that are considered stress sensors can dissociate Bax-like proteins from their antiapoptotic sequestrators, and thus leading to apoptosis.¹The expression of Bcl-2 family proteins is regulated during carcinogenesis,¹ and the expression of both the Bcl-2 and Bcl-xL antiapoptotic proteins is associated with resistance to antitumor agents such as cisplatin (CP).² The inhibition of the protective function of antiapoptotic Bcl-2 members can either restore the normal apoptotic process in cancer cells or circumvent resistance to chemotherapy.^3,4 In this regard, enhanced expression of BH3-only proteins can effectively bind the antiapoptotic members and prevent the function of these proteins.Some reports suggest that the BH3-only protein Puma has important roles in p53-dependent and -independent apoptosis in human cancer cells and mediates cell death through the Bcl-2 family proteins Bax/Bak and the mitochondrial pathway.^5,6 Our studies also reveal that Puma upregulation induces cell apoptosis in chemoresistant ovarian cancer cells,^7,8 confirming the requisite role of Puma in chemosensitivity.7-Hydroxystaurosporine (UCN-01) is a protein kinase C-selective inhibitor that is successfully used in phase I and II clinical trials.^9,10 As a modulator, UCN-01 enhances the cytotoxicity of other anticancer drugs such as DNA-damaging agents and antimetabolite drugs by putative abrogation of G2- and/or S-phase accumulation induced by these anticancer agents.¹¹ As a single agent, UCN-01 exhibits two key biochemical effects, namely accumulation of cells in the G1 phase of the cell cycle and induction of apoptosis.¹² Both these effects may be important for its anticancer activity. Previous studies have demonstrated that UCN-01 potently decreased the levels of activated the phosphorylation level of Akt (p-Akt) in in vitro or in in vivo systems.^{12, 13, 14} Some researchers have also approved that UCN-01 can modulate Bcl-2 family members to potentiate apoptosis in cancer cells.^15,16 These reports suggest that Akt and Bcl-2 family proteins may be the potent targets of UCN-01 to trigger cancer cell apoptosis.In this study, we also investigate the role of Puma in UCN-01-induced apoptosis and confirm that p53-independent Puma induction is pivotal for the anticancer effects of UCN-01. Moreover, we first elucidate the detailed mechanism of Puma-induced apoptosis after UCN-01 treatment. We found that Puma expression mediated caspase-9 and caspase-3 activation. Among the caspase proteins, caspase-9 has a key role in Puma-induced apoptosis. Our data demonstrated that caspase-9 could mediate Puma-induced apoptosis through two feedback pathways. On the one hand, activated caspase-9 was initiated followed by caspase-3 activity, and activated caspase-3 cleaved XIAP in a positive feedback loop to strengthen Puma expression. On the other hand, caspase-9 itself cleaved antiapoptotic Bcl-2 and Bcl-xL to positively enhance Puma induction. These results provide the detailed mechanistic insight into therapeutic response to UCN-01 and the theoretical basis for its applications.     

Functional Cooperation of the Proapoptotic Bcl2 Family Proteins Bmf and Bim In Vivo

Bcl2-modifying factor (Bmf) is a member of the BH3-only group of proapoptotic proteins. To test the role of Bmf in vivo, we constructed mice with a series of mutated Bmf alleles that disrupt Bmf expression, prevent Bmf phosphorylation by the c-Jun NH₂-terminal kinase (JNK) on Ser⁷⁴, or mimic Bmf phosphorylation on Ser⁷⁴. We report that the loss of Bmf causes defects in uterovaginal development, including an imperforate vagina and hydrometrocolpos. We also show that the phosphorylation of Bmf on Ser⁷⁴ can contribute to a moderate increase in levels of Bmf activity. Studies of compound mutants with the related gene Bim demonstrated that Bim and Bmf exhibit partially redundant functions in vivo. Thus, developmental ablation of interdigital webbing on mouse paws and normal lymphocyte homeostasis require the cooperative activity of Bim and Bmf.Bmf is a proapoptotic BH3-only member of the Bcl2-related protein family that is implicated in cell death caused by anoikis (23, 26, 27), arsenic trioxide (19), histone deacetylase inhibitors (33, 34), transforming growth factor β (24), and tumor necrosis factor alpha (8). Mice with a loss of Bmf expression exhibit B-cell hyperplasia and increased sensitivity to γ-radiation-induced B-cell lymphoma (14). These observations indicate that Bmf represents an important mediator of cell death signaling pathways.The structure of Bmf includes a BH3 domain that is essential for apoptosis induction. In addition, Bmf contains a sequence motif that is required for interactions with dynein light chain 2 (DLC2), a component of the myosin V motor complex (23). The interaction of Bmf with DLC2 is required for the recruitment of Bmf to the cytoskeleton. The release of Bmf from complexes sequestered on the cytoskeleton may contribute to anoikis (23). Interestingly, this regulatory mechanism is shared by the related proapoptotic BH3-only protein Bim, which interacts via a similar sequence motif with dynein light chain 1 (DLC1), a component of the dynein motor complex (22).The similarities between Bmf and Bim include the presence of a conserved phosphorylation site (Bmf Ser⁷⁴ and Bim Thr¹¹²) that is a substrate for the c-Jun NH₂-terminal kinase (JNK) (15). Data from biochemical studies indicate that the JNK-mediated phosphorylation of Bmf and Bim may increase apoptotic activity (15). Indeed, mice with a germ line point mutation in the Bim gene (Thr¹¹² replaced with Ala) exhibit decreased apoptosis (10). These studies indicate that Bmf and Bim may mediate, in part, proapoptotic signaling by JNK (3, 30).The purpose of this study was to examine the role of Bmf using mouse models with germ line defects in the Bmf gene, including mice with Bmf alleles that disrupt Bmf expression, prevent Bmf phosphorylation, or mimic Bmf phosphorylation. We examined the effects of these mutations in mice with both wild-type and mutant alleles of the related gene Bim. The results of our analysis demonstrate that Bmf and Bim exhibit partially redundant functions, that phosphorylation on Ser⁷⁴ is not essential for Bmf activity, and that phosphorylation on Ser⁷⁴ can contribute to increased levels of Bmf activity in vivo.     

Mcl-1 Integrates the Opposing Actions of Signaling Pathways That Mediate Survival and Apoptosis

Mcl-1 is a member of the Bcl2-related protein family that is a critical mediator of cell survival. Exposure of cells to stress causes inhibition of Mcl-1 mRNA translation and rapid destruction of Mcl-1 protein by proteasomal degradation mediated by a phosphodegron created by glycogen synthase kinase 3 (GSK3) phosphorylation of Mcl-1. Here we demonstrate that prior phosphorylation of Mcl-1 by the c-Jun N-terminal protein kinase (JNK) is essential for Mcl-1 phosphorylation by GSK3. Stress-induced Mcl-1 degradation therefore requires the coordinated activity of JNK and GSK3. Together, these data establish that Mcl-1 functions as a site of signal integration between the proapoptotic activity of JNK and the prosurvival activity of the AKT pathway that inhibits GSK3.Mcl-1 is an antiapoptotic member of the Bcl2 family. Gene knockout studies of mice demonstrate that Mcl-1 is essential for embryonic development and for the survival of hematopoietic cells (28-30). Studies of the stress response have demonstrated that Mcl-1 plays an important role in the sensitization of cells to apoptotic signals (1, 11, 25). Thus, exposure to UV radiation causes the rapid degradation of Mcl-1 and the release of proapoptotic partner proteins from Mcl-1 complexes (e.g., Bim). The mechanism of rapid Mcl-1 destruction is mediated by the combined actions of two different pathways. First, the exposure to stress causes phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2α) on the inhibitory site Ser-51 that prevents translation of Mcl-1 mRNA (1, 11, 25). Second, Mcl-1 is rapidly degraded by the ubiquitin-dependent proteasome pathway (27). Together, these pathways cause a rapid reduction in Mcl-1 expression. This loss of Mcl-1 may be a required initial response for the apoptosis of cells exposed to stress (25).The E3 ubiquitin protein ligase Mule/ARF-BP1 contains a BH3 domain that interacts with Mcl-1 and can initiate ubiquitin-dependent degradation of Mcl-1 (39). Recent studies have demonstrated that rapid stress-induced degradation of Mcl-1 is mediated by an alternative pathway involving the E3 ubiquitin protein ligase β-TrCP, which binds a stress-induced phosphodegron created by the phosphorylation of Mcl-1 by glycogen synthase kinase 3 (GSK3) (7, 21). How the exposure to stress causes GSK3-mediated phosphorylation of Mcl-1 is unclear, but GSK3 has been shown to directly phosphorylate Mcl-1 (7, 21). Mcl-1 phosphorylation and degradation may therefore be controlled by the prosurvival AKT pathway, which can negatively regulate GSK3 (7, 21).Mcl-1 is critically involved in the regulation of cell survival and is therefore subject to regulation by multiple mechanisms (26). Thus, Mcl-1 gene expression is regulated by many growth factors and cytokines (26), and Mcl-1 mRNA is regulated by microRNA pathways (24). The Mcl-1 protein is stabilized by binding TCTP (20) and the BH3-only protein Bim (4). In contrast, the BH3-only protein Noxa binds and destabilizes Mcl-1 (4, 36). Moreover, it is established that Mcl-1 is phosphorylated by several protein kinases on sites that may regulate Mcl-1 function. Phosphorylation of human Mcl-1 (hMcl-1) on Ser-64 (a site that is not conserved in other species) may enhance antiapoptotic activity by increasing the interaction of Mcl-1 with Bim, Noxa, and Bak (18). Phosphorylation on Ser-121 and Thr-163 may inhibit the antiapoptotic activity of hMcl-1 (15), and phosphorylation on Thr-163 may increase hMcl-1 protein stability (9). The conserved GSK3 phosphorylation site Ser-159 (and possibly Ser-155) can initiate rapid proteasomal degradation of hMcl-1 (7, 21). Together, these findings suggest that the function of Mcl-1 is very tightly regulated.The results of previous studies have implicated the c-Jun N-terminal protein kinase (JNK) in the regulation of Mcl-1 (15, 18). The purpose of this study was to test whether Mcl-1 is a target of signal transduction by JNK. We demonstrate that a key function of JNK is to prime Mcl-1 for phosphorylation by GSK3. JNK is required for GSK3-mediated degradation of Mcl-1 in response to stress. Coordinated regulation of the stress-activated JNK pathway and the AKT-inhibited GSK3 pathway is therefore required for stress-induced Mcl-1 degradation.     

Cyclin-Dependent Kinase 1-Mediated Bcl-xL/Bcl-2 Phosphorylation Acts as a Functional Link Coupling Mitotic Arrest and Apoptosis

Despite detailed knowledge of the components of the spindle assembly checkpoint, a molecular explanation of how cells die after prolonged spindle checkpoint activation, and thus how microtubule inhibitors and other antimitotic drugs ultimately elicit their lethal effects, has yet to emerge. Mitotically arrested cells typically display extensive phosphorylation of two key antiapoptotic proteins, Bcl-x_L and Bcl-2, and evidence suggests that phosphorylation disables their antiapoptotic activity. However, the responsible kinase has remained elusive. In this report, evidence is presented that cyclin-dependent kinase 1 (CDK1)/cyclin B catalyzes mitotic-arrest-induced Bcl-x_L/Bcl-2 phosphorylation. Furthermore, we show that CDK1 transiently and incompletely phosphorylates these proteins during normal mitosis. When mitosis is prolonged in the absence of microtubule inhibition, Bcl-x_L and Bcl-2 become highly phosphorylated. Transient overexpression of nondegradable cyclin B1 caused apoptotic death, which was blocked by a phosphodefective Bcl-x_L mutant but not by a phosphomimetic Bcl-x_L mutant, confirming Bcl-x_L as a key target of proapoptotic CDK1 signaling. These findings suggest a model whereby a switch in the duration of CDK1 activation, from transient during mitosis to sustained during mitotic arrest, dramatically increases the extent of Bcl-x_L/Bcl-2 phosphorylation, resulting in inactivation of their antiapoptotic function. Thus, phosphorylation of antiapoptotic Bcl-2 proteins acts as a sensor for CDK1 signal duration and as a functional link coupling mitotic arrest to apoptosis.The cell division cycle is controlled by checkpoints, which ensure the fidelity of chromosome replication and segregation, as well as orderly progression through the cell cycle. If these critical events cannot be completed as scheduled, damaged cells, which might otherwise pose a threat to the organism as precancerous cells, are eliminated (16). The mitotic checkpoint, for example, produces a “prevent anaphase” signal until all the chromosomes are properly attached to kinetochores (22). Microtubule inhibitors (MTIs) and other antimitotic agents prolong the activation of this checkpoint, causing mitotic arrest, which culminates in cell death generally via intrinsic apoptosis, providing a rationale for the use of these agents as antitumor agents (20, 31). Intrinsic or mitochondrial apoptosis is regulated by the Bcl-2 family of proteins, which exhibit either pro- or antiapoptotic properties (17, 37). The BH3-only proapoptotic members act as essential initiators of intrinsic apoptosis, whereas the multidomain proapoptotic members, Bax and Bak, act as essential mediators of mitochondrial membrane permeability. Antiapoptotic Bcl-2 family members, including Bcl-x_L, Bcl-2, and Mcl-1, oppose apoptosis by binding to the proapoptotic members and neutralizing their activity.The molecular mechanisms leading to cell death in response to spindle checkpoint activation have yet to be established. Indeed, how the spindle checkpoint couples to pathways regulating cell survival and death still represents an unresolved issue in cell biology (26, 35). Nonetheless, it seems reasonable to hypothesize that signals generated in response to prolonged mitotic arrest are eventually transduced to the apoptotic machinery. In this regard, it is striking that MTIs consistently induce the phosphorylation of two key antiapoptotic proteins, Bcl-2 and Bcl-x_L, whereas other apoptotic stimuli fail to do so (9, 13, 25). The results of studies with phosphodefective mutants of Bcl-2 and Bcl-x_L indicate that phosphorylation antagonizes their antiapoptotic function (2, 33, 36), but the precise mechanism(s) has yet to be fully clarified.The identity of the kinase responsible for the extensive phosphorylation of Bcl-x_L and Bcl-2 that occurs in response to sustained spindle checkpoint activation is unresolved. Identification of this kinase is considered to be of critical importance, since it will provide insight into the molecular links between mitotic arrest and cell death, as well as the molecular mechanism of action of antimitotic drugs. Several candidates have been proposed, including Raf-1 (3), Jun N-terminal protein kinase (JNK) (2, 11, 36), protein kinase A (PKA) (32), cyclin-dependent kinase 1 (CDK1) (24), and mammalian target of rapamycin (mTOR) (4). In general, however, conclusions have been correlative or have been based on the use of kinase inhibitors tested under conditions that precluded mitotic arrest and thus indirectly blocked the effects of MTIs. Thus, strong experimental evidence supporting identification is lacking.Here we present evidence that the CDK1/cyclin B kinase complex is responsible for mitotic arrest-induced Bcl-x_L/Bcl-2 phosphorylation. Furthermore, we show that CDK1 transiently and incompletely phosphorylates these proteins during normal mitosis. The findings suggest a model whereby a switch in the duration of CDK1 activation, from transient during mitosis to sustained during mitotic arrest, dramatically increases the extent of Bcl-x_L/Bcl-2 phosphorylation, resulting in inactivation of the antiapoptotic function of Bcl-x_L/Bcl-2. Thus, CDK1-mediated phosphorylation of antiapoptotic Bcl-2 proteins acts as a key link coupling mitotic arrest to apoptosis.