death executioner Bcl-2 homologue

EVOLUTIONARY HOMOLOGS

Table of contents

Domain structure and function of proapoptotic Bcl-2 family members

The Bcl-2 related protein Bad is a promoter of apoptosis and has been shown to dimerize with the anti-apoptotic proteins Bcl-2 and Bcl-XL. Overexpression of Bad in murine FL5.12 cells demonstrates that the protein not only can abrogate the protective capacity of coexpressed Bcl-XL but can accelerate the apoptotic response to a death signal when it is expressed in the absence of exogenous Bcl-XL. Using deletion analysis, the minimal domain able to dimerize with Bcl-xL in the murine Bad protein has been identified. A 26-amino-acid peptide within this domain, which shows significant homology to the alpha-helical BH3 domains of related apoptotic proteins like Bak and Bax, is found to be necessary and sufficient to bind Bcl-xL. To determine the role of dimerization in regulating the death-promoting activity of Bad and the death-inhibiting activity of Bcl-xL, mutations within the hydrophobic BH3-binding pocket in Bcl-xL that eliminate the ability of Bcl-xL to form a heterodimer with Bad were tested for the ability to promote cell survival in the presence of Bad. Several of these mutants retain the ability to impart protection against cell death, regardless of the level of coexpressed Bad protein. These results suggest that BH3-containing proteins like Bad promote cell death by binding to antiapoptotic members of the Bcl-2 family and thus inhibiting their survival promoting functions (Kelekar, 1997).

Bax is a proapoptotic member of the Bcl-2 family of proteins which localizes to and uses mitochondria as its major site of action. Bax normally resides in the cytoplasm and translocates to mitochondria in response to apoptotic stimuli, and it promotes apoptosis in two ways: (1) by disrupting mitochondrial membrane barrier function by formation of ion-permeable pores in mitochondrial membranes and (2) by binding to antiapoptotic Bcl-2 family proteins via its BH3 domain and inhibiting their functions. A hairpin pair of amphipathic alpha-helices (alpha5-alpha6) in Bax has been predicted to participate in membrane insertion and pore formation by Bax. Several charged residues in the alpha5-alpha6 domain of Bax were mutagenized, changing them to alanine. These substitution mutants of Bax constitutively localize to mitochondria and display a gain-of-function phenotype when expressed in mammalian cells. Furthermore, substitution of 8 out of 10 charged residues in the alpha5-alpha6 domain of Bax results in a loss of cytotoxicity in yeast but a gain-of-function phenotype in mammalian cells. The enhanced function of this Bax mutant was correlated with increased binding to Bcl-X(L), through a BH3-independent mechanism. These observations reveal new functions for the alpha5-alpha6 hairpin loop of Bax: (1) regulation of mitochondrial targeting and (2) modulation of binding to antiapoptotic Bcl-2 proteins (Nouraini, 2000).

BNIP3 (formerly NIP3) is a pro-apoptotic, mitochondrial protein classified in the Bcl-2 family based on limited sequence homology to the Bcl-2 homology 3 (BH3) domain and COOH-terminal transmembrane (TM) domain. BNIP3 expressed in yeast and mammalian cells interacts with survival promoting proteins Bcl-2, Bcl-X(L), and CED-9. Typically, the BH3 domain of pro-apoptotic Bcl-2 homologues mediates Bcl-2/Bcl-X(L) heterodimerization and confers pro-apoptotic activity. Deletion mapping of BNIP3 excluded its BH3-like domain and identified the NH(2) terminus (residues 1-49) and TM domain as critical for Bcl-2 heterodimerization, and either region is sufficient for Bcl-X(L) interaction. Additionally, the removal of the BH3-like domain in BNIP3 does not diminish its killing activity. The TM domain of BNIP3 is critical for homodimerization, pro-apoptotic function, and mitochondrial targeting. Several TM domain mutants were found to disrupt SDS-resistant BNIP3 homodimerization but did not interfere with its killing activity or mitochondrial localization. Substitution of the BNIP3 TM domain with that of cytochrome b(5) directs protein expression to nonmitochondrial sites and still promotes apoptosis and heterodimerization with Bcl-2 and Bcl-X(L). It is proposed that BNIP3 represents a subfamily of Bcl-2-related proteins that functions without a typical BH3 domain to regulate apoptosis from both mitochondrial and nonmitochondrial sites by selective Bcl-2/Bcl-X(L) interactions (Ray, 2000).

The Bcl-2 homology 3 (BH3) domain is crucial for the death-inducing and dimerization properties of pro-apoptotic members of the Bcl-2 protein family, including Bak, Bax, and Bad. Synthetic peptides corresponding to the BH3 domain of Bak bind to Bcl-xL, antagonize its anti-apoptotic function, and rapidly induce apoptosis when delivered into intact cells via fusion to the Antennapedia homeoprotein internalization domain. Treatment of HeLa cells with the Antennapedia-BH3 fusion peptide results in peptide internalization and induction of apoptosis within 2-3 h, as indicated by caspase activation and subsequent poly(ADP-ribose) polymerase cleavage, as well as morphological characteristics of apoptosis. A point mutation within the BH3 peptide that blocks its ability to bind to Bcl-xL abolishes its apoptotic activity, suggesting that interaction of the BH3 peptide with Bcl-2-related death suppressors, such as Bcl-xL, may be critical for its activity in cells. While overexpression of Bcl-xL can block BH3-induced apoptosis, treatment with BH3 peptides resensitizes Bcl-xL-expressing cells to Fas-mediated apoptosis. BH3-induced apoptosis is blocked by caspase inhibitors, demonstrating a dependence on caspase activation, but is not accompanied by a dramatic early loss of mitochondrial membrane potential or detectable translocation of cytochrome c from mitochondria to cytosol. These findings demonstrate that the BH3 domain itself is capable of inducing apoptosis in whole cells, possibly by antagonizing the function of Bcl-2-related death suppressors (Holinger, 1999).

Bcl-2 family members as regulators of the cell death hierarchy: Bcl-2 interacts with Apaf-1

The C. elegans Bcl-2-like protein CED-9 prevents programmed cell death by antagonizing the Apaf-1-like cell-death activator CED-4. Endogenous CED-9 and CED-4 proteins localize to mitochondria in wild-type embryos, in which most cells survive. By contrast, in embryos in which cells have been induced to die, CED-4 assumes a perinuclear localization. CED-4 translocation induced by the cell-death activator EGL-1 (EGL-1 protein contains a Bcl-2 homology 3 domain and can physically interact with CED-9) is blocked by a gain-of-function mutation in ced-9 but is not dependent on ced-3 function, suggesting that CED-4 translocation precedes caspase activation and the execution phase of programmed cell death. Thus, a change in the subcellular localization of CED-4 may drive programmed cell death (Chen, 2000).

The death-promoting proteins Bax and BAD, which like EGL-1 contain BH3 domains, translocate to mitochondria and bind anti-apoptotic Bcl-2 family members in response to apoptotic signals. Whether and how this translocation promotes cell death is unknown. The results presented here suggest that Bax and BAD may act to release Apaf-1 or another CED-4-like protein, allowing it to activate caspase processing. Some caspase precursors, specifically procaspases-2, and -3, are present in mitochondria and upon activation translocate to nuclei. It is possible that this movement of caspases involves the translocation of a complex that includes a CED-4-like protein. By analogy, the translocation of a CED-4-CED-3 complex from mitochondria to the nuclear envelope could provide access for the active caspase to both the nucleus and the cytosol, thereby fulfilling the roles of the multiple, differentially localized mammalian caspases (Chen, 2000).

The Bcl-2 family of proteins regulates apoptosis, the cell death program triggered by activation of certain proteases (caspases). An attractive model for how Bcl-2 and its closest relatives prevent caspase activation is that they bind to and inactivate an adaptor protein required for procaspase processing. That model has been supported by reports that mammalian prosurvival Bcl-2 relatives bind the adaptor Apaf-1, which activates procaspase-9. However, the in vivo association studies reported here with both overexpressed and endogenous Apaf-1 challenge this notion. Apaf-1 can be immunoprecipitated together with procaspase-9, and the Apaf-1 caspase-recruitment domain is necessary and sufficient for their interaction. Apaf-1 does not bind, however, to any of the six known mammalian prosurvival family members (Bcl-2, Bcl-x(L), Bcl-w, A1, Mcl-1, or Boo), or their viral homologs adenovirus E1B 19K and Epstein-Barr virus BHRF-1. Endogenous Apaf-1 also fails to coimmunoprecipitate with endogenous Bcl-2 or Bcl-x(L), or with two proapoptotic relatives (Bax and Bim). Moreover, apoptotic stimuli do not induce Apaf-1 to bind to these family members. Thus, the prosurvival Bcl-2 homologs do not appear to act by sequestering Apaf-1 and probably instead constrain its activity indirectly (Moriishi, 1999).

In the initiation of apoptosis, Apaf-1, homologous to C. elegans CED-4, functions downstream of bcl-2 but upstream of caspase-3. Bcl-2 may function upstream of Apaf-1 by regulating the release of cytochrome c from mitochondria. Cytochrome c is a required cofactor for Apaf-1. Another protein factor, Apaf-3, has been identified that participates in caspase-3 activation in vitro. Apaf-3 was identified as a member of the caspase family, caspase-9. Caspase-9 and Apaf-1 bind to each other via their respective NH2-terminal CED-3 homologous domains in the presence of cytochrome c and dATP, an event that leads to caspase-9 activation. This N-terminal region of caspase-9 is termed a caspase recruitment domain (CARD). Activated caspase-9 in turn cleaves and activates caspase-3. Depletion of caspase-9 from S-100 extracts diminished caspase-3 activation. Mutation of the active site of caspase-9 attenuates the activation of caspase-3 and cellular apoptotic response in vivo, indicating that caspase-9 is the most upstream member of the apoptotic protease cascade that is triggered by cytochrome c and dATP (Li, 1997).

Bcl-2 family members are targets of caspases

There is a direct interaction between caspases and Bcl-xL. The loop domain of Bcl-xL is cleaved by caspases in vitro and in cells induced to undergo apoptotic death after Sindbis virus infection or interleukin 3 withdrawal. Mutation of the caspase cleavage site in Bcl-xL in conjunction with a mutation in the BH1 homology domain impairs the death-inhibitory activity of Bcl-xL, suggesting that interaction of Bcl-xL with caspases may be an important mechanism for inhibiting cell death. The BH1 and BH2 domains of Bcl-2 and Bcl-xL are important for heterodimerization with other Bcl-2 family members. Once Bcl-xL is cleaved, the C-terminal fragment of Bcl-xL potently induces apoptosis. Taken together, these findings indicate that the recognition/cleavage site of Bcl-xL may facilitate protection against cell death by acting at the level of caspase activation and that cleavage of Bcl-xL during the execution phase of cell death converts Bcl-xL from a protective to a lethal protein (Clem, 1998).

Caspases comprise a family of cysteine proteases implicated in the biochemical and morphological changes that occur during apoptosis (programmed cell death). The loop domain of Bcl-2 is cleaved at Asp34 by caspase-3 (CPP32) in vitro, in cells overexpressing caspase-3, and after induction of apoptosis by Fas ligation and interleukin-3 withdrawal. The carboxyl-terminal Bcl-2 cleavage product triggers cell death and accelerates Sindbis virus-induced apoptosis, which is dependent on the BH3 homology and transmembrane domains of Bcl-2. Inhibitor studies indicate that cleavage of Bcl-2 may further activate downstream caspases and contribute to amplification of the caspase cascade. Cleavage-resistant mutants of Bcl-2 have increased protection from interleukin-3 withdrawal and Sindbis virus-induced apoptosis. Thus, cleavage of Bcl-2 by caspases may ensure the inevitability of cell death (Cheng, 1997).

Caspases are cysteine proteases that mediate apoptosis by proteolysis of specific substrates. Although many caspase substrates have been identified, for most substrates the physiologic caspase(s) required for cleavage is unknown. The Bcl-2 protein, which inhibits apoptosis, is cleaved at Asp-34 by caspases during apoptosis and by recombinant caspase-3 in vitro. Endogenous caspase-3 is a physiologic caspase for Bcl-2. Apoptotic extracts from 293 cells cleave Bcl-2 but not Bax, even though Bax is cleaved to an 18-kDa fragment in SK-NSH cells treated with ionizing radiation. In contrast to Bcl-2, cleavage of Bax is only partially blocked by caspase inhibitors. Inhibitor profiles indicate that Bax may be cleaved by more than one type of noncaspase protease. Immunodepletion of caspase-3 from 293 extracts abolishes cleavage of Bcl-2 and caspase-7, whereas immunodepletion of caspase-7 has no effect on Bcl-2 cleavage. Furthermore, MCF-7 cells, which lack caspase-3 expression, do not cleave Bcl-2 following staurosporine-induced cell death. However, transient transfection of caspase-3 into MCF-7 cells restores Bcl-2 cleavage after staurosporine treatment. These results demonstrate that in these models of apoptosis, specific cleavage of Bcl-2 requires activation of caspase-3. When the pro-apoptotic caspase cleavage fragment of Bcl-2 is transfected into baby hamster kidney cells, it localizes to mitochondria and causes the release of cytochrome c into the cytosol. Therefore, caspase-3-dependent cleavage of Bcl-2 appears to promote further caspase activation as part of a positive feedback loop for executing the cell (Kirsch, 1999).

Bcl-2 family members: transcriptional regulation

Binding of the proinflammatory cytokine tumor necrosis factor (TNFalpha) to its receptor triggers competing signaling pathways that determine whether a cell lives or dies. Whereas one pathway is conducive to cell death, the other leads to activation of Rel/NF-kappaB transcription factors and the coincident inhibition of apoptosis. Accumulating evidence supports a proactive role for NF-kappaB in the inhibition of cell death induced by TNFalpha and other death-causing agents. Whereas the activation of NF-kappaB blocks cell killing, its inhibition enhances the cytotoxicity of TNFalpha and promotes apoptosis in various cell systems, demonstrating the need for NF-kappaB function for cell survival. Bcl-2-family proteins are key regulators of the apoptotic response. The pro-survival Bcl-2 homolog Bfl-1/A1 is a direct transcriptional target of NF-kappaB. bfl-1 gene expression is dependent on NF-kappaB activity and it can substitute for NF-kappaB to suppress TNFalpha-induced apoptosis. bfl-1 promoter analysis has identified an NF-kappaB site responsible for its Rel/NF-kappaB-dependent induction. The expression of bfl-1 in immune tissues supports the protective role of NF-kappaB in the immune system. The activation of Bfl-1 may be the means by which NF-kappaB functions in oncogenesis and promotes cell resistance to anti-cancer therapy (Zong, 1999).

Activation of CD40 is essential for thymus-dependent humoral immune responses and rescuing B cells from apoptosis. Many of the effects of CD40 are believed to be achieved through altered gene expression. In addition to Bcl-x, a known CD40-regulated antiapoptotic molecule, a related antiapoptotic molecule, A1/Bfl-1, has been identified as a CD40-inducible gene. Inhibition of the NF-kappaB pathway by overexpression of a dominant-active inhibitor of NF-kappaB abolishes CD40-induced up-regulation of both the Bfl-1 and Bcl-x genes and also eliminates the ability of CD40 to rescue Fas-induced cell death. Within the upstream promoter region of Bcl-x, a potential NF-kappaB-binding sequence was found to support NF-kappaB-dependent transcriptional activation. Furthermore, expression of physiological levels of Bcl-x protects B cells from Fas-mediated apoptosis in the absence of NF-kappaB signaling. Thus, these results suggest that CD40-mediated cell survival proceeds through NF-kappaB-dependent up-regulation of Bcl-2 family members (Lee, 1999).

The Brn-3a POU family transcription factor has been shown to strongly activate expression of the Bcl-2 proto-oncogene and thereby protect neuronal cells from programmed cell death (apoptosis). This activation of the Bcl-2 promoter by Brn-3a is strongly inhibited by the p53 anti-oncogene protein. This inhibitory effect of p53 on Brn-3a-mediated transactivation is observed with nonoverlapping gene fragments containing either the Bcl-2 p1 or p2 promoters but is not observed with other Brn-3a-activated promoters such as in the gene encoding alpha-internexin or with an isolated Brn-3a binding site from the Bcl-2 promoter linked to a heterologous promoter. In contrast, p53 mutants, which are incapable of binding to DNA, do not affect Brn-3a-mediated activation of the Bcl-2 p1 and p2 promoters. Moreover, Brn-3a and p53 have been shown to bind to adjacent sites in the p2 promoter and to directly interact with one another, both in vitro and in vivo, with this interaction being mediated by the POU domain of Brn-3a and the DNA binding domain of p53. The significance of these effects is discussed in terms of the antagonistic effects of Bcl-2 and p53 on the rate of apoptosis and the overexpression of Brn-3a in specific tumor cell types (Budhram-Mahadeo, 1999).

Nerve growth factor (NGF) and other neurotrophins support survival of neurons through processes that are incompletely understood. The transcription factor CREB is a critical mediator of NGF-dependent gene expression, but whether CREB family transcription factors regulate expression of genes that contribute to NGF-dependent survival of sympathetic neurons is unknown. To determine whether CREB-mediated gene expression is necessary for NGF-dependent neuronal survival, this study monitored survival of sympathetic neurons after expression of either of two distinct inhibitors of CREB. One CREB inhibitor, A-CREB, is a potent and selective inhibitor of CREB DNA binding activity. The other, CREBm1, binds to CREB binding sites in DNA but is not activated because the transcriptional regulatory residue, serine 133, is mutated to alanine. CREB-mediated gene expression is both necessary for NGF-dependent survival and sufficient on its own to promote survival of sympathetic neurons. Moreover, expression of Bcl-2 is activated by NGF and other neurotrophins by a CREB-dependent transcriptional mechanism. A region of the bcl-2 gene between 1640 and 1337 relative to the translation start site is required for NGF-sensitive transcription. This region contains a near-perfect consensus CRE. Activated CREB can bind to this region of the bcl-2 promoter, and this interaction is critical for expression of Bcl-2 in a B lymphocyte cell line. Thus, a test was performed to see if the integrity of the bcl-2 CRE is necessary for the NGF-induced expression of bcl-2. A bcl-2 reporter construct harboring a two-base pair mutation of the CRE, rendering it unable to bind CREB, is impaired in its responsiveness to NGF. Overexpression of Bcl-2 reduces the death-promoting effects of CREB inhibition. Together, these data support a model in which neurotrophins promote survival of neurons, in part through a mechanism involving CREB family transcription factor-dependent expression of genes encoding prosurvival factors (Riccio, 1999).

The ETS family transcriptional repressor TEL is frequently disrupted by chromosomal translocations, including the t(12;21) in which the second allele of TEL is deleted in up to 90% of the cases. Consistent with its role as a putative tumor suppressor, TEL expression inhibits colony formation by Ras-transformed NIH 3T3 cells and hinders proliferation of a variety of cell types. Although no alteration is observed in the cell cycle of TEL-expressing cells, a marked increase in apoptosis of serum-starved TEL-expressing NIH 3T3 cells was found. This decrease in cell survival requires the DNA binding domain of TEL, suggesting that TEL represses an anti-apoptotic gene. These observations prompted a search for genes regulated by ETS family proteins that regulate apoptosis. The anti-apoptotic molecule Bcl-XL contains multiple ets-factor binding sites within its promoters, and TEL represses a Bcl-XL promoter-linked reporter gene. Moreover, the enforced expression of TEL decreases the endogenous expression of both Bcl-XL mRNA and protein. TEL-mediated repression of Bcl-XL likely affects cell survival via regulation of the apoptotic pathway (Irvin, 2003).

The pattern of programmed cell death was studied in the neural crest and how it is controlled by the activity of the transcription factors Slug and msx1 was examined. The results indicate that apoptosis is more prevalent in the neural folds than in the rest of the neural ectoderm. Through gain- and loss-of-function experiments with inducible forms of both Slug and msx1 genes, it was shown that Slug acts as an anti-apoptotic factor whereas msx1 promotes cell death, either in the neural folds of the whole embryos, in isolated or induced neural crest and in animal cap assays. The protective effect of expressing Slug can be reversed by expressing the apoptotic factor Bax, while the apoptosis promoted by msx1 can be abolished by expressing the Xenopus homologue of Bcl2 (XR11). Furthermore, Slug and msx1 control the transcription of XR11 and several caspases required for programmed cell death. In addition, expression of Bax or Bcl2 produced similar effects on the survival of the neural crest and on the development of its derivatives as those produced by altering the activity of Slug or msx1. Finally, it was shown that in the neural crest, the region of the neural folds where Slug is expressed, cells undergo less apoptosis, than in the region where the msx1 gene is expressed; this region corresponds to cells adjacent to the neural crest. The expression of Slug and msx1 controls cell death in certain areas of the neural folds, and how this equilibrium is necessary to generate sharp boundaries in the neural crest territory and to precisely control cell number among neural crest derivatives is discussed (Tribulo, 2004).

Signaling upstream of Bcl-2 family members

BAD is a distant member of the Bcl-2 family that promotes cell death. Phosphorylation of BAD prevents this. BAD phosphorylation induced by interleukin-3 (IL-3) is inhibited by specific inhibitors of phosphoinositide 3-kinase (PI 3-kinase). Akt, a survival-promoting serine-threonine protein kinase, is activated by IL-3 in a PI 3-kinase-dependent manner. Active, but not inactive, forms of Akt are found to phosphorylate BAD in vivo and in vitro at the same residues that are phosphorylated in response to IL-3. Thus, the proapoptotic function of BAD is regulated by the PI 3-kinase-Akt pathway (del Paso, 1997).

Growth factors can promote cell survival by activating the phosphatidylinositide-3'-OH kinase and its downstream target, the serine-threonine kinase Akt. However, the mechanism by which Akt functions to promote survival is not understood. Growth factor activation of the PI3'K/Akt signaling pathway culminates in the phosphorylation of the BCL-2 family member BAD, thereby suppressing apoptosis and promoting cell survival. Akt phosphorylates BAD in vitro and in vivo, and blocks the BAD-induced death of primary neurons in a site-specific manner. These findings define a mechanism by which growth factors directly inactivate a critical component of the cell-intrinsic death machinery (Datta, 1997).

The initiation of apoptosis often transpires in the presence of agents that regulate cell survival. This study evaluated the effects of stress-induced ceramide on the anti-apoptotic activity of the phosphoinositide-3 kinase [PI(3)K] pathway. PI(3)K activity is directly down-regulated by stress-induced ceramide in a dose-dependent manner with rapid kinetics and high specificity. Ceramide inhibition of PI(3)K is dependent on acid-sphingomyelinase. Down-regulation of PI(3)K by ceramide results in inhibition of the kinase Akt and decreased phosphorylation of the death effector, Bad. Thus, ceramide levels could act as a general apoptotic rheostat controlling cell survival by regulating PI(3)K anti-apoptotic effector mechanisms. Ceramide contributes to apoptosis not only by regulating effector mechanisms such as caspases and c-Jun, but by deregulation of the anti-apoptotic PI(3)K Akt/Bad pathway. It has been shown that the interaction of ceramide with PI(3)K is independent of its p110 catalytic subunit. Regulatory subunit (p85) knockouts are resistent to oxidative apoptosis in a PI(3)K-independent, p53-dependent fashion. Because ceramide modulates the PI(3)K response at extremely early times following stress, it is feasible that the effect of ceramide on p85 could modulate p85's affinity for p110 and thus make it available for binding other potential targets that could be proapoptotic (Zundel, 1998).

The phosphatidylinositol 3-kinase (PI3K)-signaling pathway has emerged as an important component of cytokine-mediated survival of hemopoietic cells. Recently, the protein kinase PKB/akt (referred to here as PKB) has been identified as a downstream target of PI3K that is necessary for survival. PKB has also been implicated in the phosphorylation of Bad, potentially linking the survival effects of cytokines with the Bcl-2 family. Granulocyte/macrophage colony-stimulating factor (GM-CSF) maintains survival in the absence of PI3K activity; when PKB activation is also completely blocked, GM-CSF is still able to stimulate phosphorylation of Bad. In contrast, Interleukin 3 (IL-3) requires PI3K for survival, and blocking PI3K partially inhibits Bad phosphorylation. IL-4, unique among the cytokines in that it lacks the ability to activate the p21ras-mitogen-activated protein kinase (MAPK) cascade, was found to activate PKB and promote cell survival, but it does not stimulate Bad phosphorylation. Finally, although these data suggest that the MAPK pathway is not required for inhibition of apoptosis, evidence is provided that phosphorylation of Bad may be occurring via a MAPK/ERK kinase (MEK)-dependent pathway. Together, these results demonstrate that although PI3K may contribute to phosphorylation of Bad in some instances, there is at least one other PI3K-independent pathway involved, possibly via activation of MEK. These data also suggest that although phosphorylation of Bad may be one means by which cytokines can inhibit apoptosis, it may be neither sufficient nor necessary for the survival effect (Scheid, 1998).

The ratio of proapoptotic versus antiapoptotic Bcl-2 members is a critical determinant that plays a significant role in altering susceptibility to apoptosis. Therefore, a reduction of antiapoptotic protein levels in response to proximal signal transduction events may switch on the apoptotic pathway. In endothelial cells, tumor necrosis factor alpha (TNF-alpha) induces dephosphorylation and subsequent ubiquitin-dependent degradation of the antiapoptotic protein Bcl-2. The roles of different putative phosphorylation sites to facilitate Bcl-2 degradation were investigation. Mutation of the consensus protein kinase B/Akt site or of potential protein kinase C or cyclic AMP-dependent protein kinase sites does not affect Bcl-2 stability. In contrast, inactivation of the three consensus mitogen-activated protein (MAP) kinase sites leads to a Bcl-2 protein that is ubiquitinated and subsequently degraded by the 26S proteasome. Inactivation of these sites within Bcl-2 revealed that dephosphorylation of Ser87 appears to play a major role. A Ser-to-Ala substitution at this position results in 50% degradation, whereas replacement of Thr74 with Ala leads to 25% degradation, as assessed by pulse-chase studies. It was further demonstrated that incubation with TNF-alpha induces dephosphorylation of Ser87 of Bcl-2 in intact cells. Furthermore, MAP kinase triggers phosphorylation of Bcl-2, whereas a reduction in Bcl-2 phosphorylation was observed in the presence of MAP kinase-specific phosphatases or the MAP kinase-specific inhibitor PD98059. Moreover, oxidative stress mediates TNF-alpha-stimulated proteolytic degradation of Bcl-2 by reducing MAP kinase activity. Taken together, these results demonstrate a direct protective role for Bcl-2 phosphorylation by MAP kinase against apoptotic challenges to endothelial cells and other cells (Breitschopf, 2000).

Bad is a critical regulatory component of the intrinsic cell death machinery that exerts its death-promoting effect upon heterodimerization with the antiapoptotic proteins Bcl-2 and Bcl-x(L). Growth factors promote cell survival through phosphorylation of Bad, resulting in its dissociation from Bcl-2 and Bcl-x(L) and its association with 14-3-3tau. Survival of interleukin 3 (IL-3)-dependent FL5.12 lymphoid progenitor cells is attenuated upon treatment with the Rho GTPase-inactivating toxin B from Clostridium difficile. p21-activated kinase 1 (PAK1) is activated by IL-3 in FL5.12 cells, and this activation is reduced by the phosphatidylinositol 3-kinase inhibitor LY294002. Overexpression of a constitutively active PAK mutant (PAK1-T423E) promoted cell survival of FL5.12 and NIH 3T3 cells, while overexpression of the autoinhibitory domain of PAK (amino acids 83 to 149) enhanced apoptosis. PAK phosphorylates Bad in vitro and in vivo on Ser112 and Ser136, resulting in a markedly reduced interaction between Bad and Bcl-2 or Bcl-x(L) and the increased association of Bad with 14-3-3tau. These findings indicate that PAK inhibits the proapoptotic effects of Bad by direct phosphorylation and that PAK may play an important role in cell survival pathways (Schurmann, 2000).

Phosphorylation of the Bcl-2 family protein Bad may represent an important bridge between survival signaling by growth factor receptors and the prevention of apoptosis. Bad phosphorylation was examined following cytokine stimulation, which revealed phosphorylation on a critical residue, serine 112, in a MEK-dependent manner. Furthermore, Bad phosphorylation also increases on several sites distinct from serine 112 but could not be detected on serine 136, previously thought to be a protein kinase B/Akt-targeted residue. Serine 112 phosphorylation is be absolutely required for dissociation of Bad from Bcl-x(L). These results demonstrate for the first time in mammalian cells the involvement of the Ras-MAPK pathway in the phosphorylation of Bad and the regulation of its function (Scheid, 1999).

Growth factors activate an array of cell survival signaling pathways. Mitogen-activated protein (MAP) kinases transduce signals emanating from their upstream activators: MAP kinase kinases (MEKs). The MEK-MAP kinase signaling cassette is a key regulatory pathway promoting cell survival. The downstream effectors of the mammalian MEK-MAP kinase cell survival signal have not been previously described. Identified here is a pro-survival role for the serine/threonine kinase S6 kinase p90 ribosomal S6 kinase Rsk1 ( (see Drosophila RSK)), a downstream target of the MEK-MAP kinase signaling pathway. In cells that are dependent on interleukin-3 (IL-3) for survival, pharmacological inhibition of MEKs antagonize the IL-3 survival signal. In the absence of IL-3, a kinase-dead Rsk1 mutant eliminates the survival effect afforded by activated MEK. Conversely, a novel constitutively active Rsk1 allele restores the MEK-MAP kinase survival signal. Experiments in vitro and in vivo have demonstrated that Rsk1 directly phosphorylates the pro-apoptotic protein Bad at the serine residues that, when phosphorylated, abrogate Bad's pro-apoptotic function. Constitutively active Rsk1 causes constitutive Bad phosphorylation and protection from Bad-modulated cell death. Kinase-inactive Rsk1 mutants antagonize Bad phosphorylation. Bad mutations that prevent phosphorylation by Rsk1 also inhibit Rsk1-mediated cell survival. These data support a model in which Rsk1 transduces the mammalian MEK-MAP kinase signal in part by phosphorylating Bad (Shimamura, 2000).

Insulin-like growth factor-I (IGF-I) is known to prevent apoptosis induced by diverse stimuli. The present study examined the effect of IGF-I on the promoter activity of bcl-2, a gene with antiapoptotic function. A luciferase reporter driven by the promoter region of bcl-2 from -1640 to -1287 base pairs upstream of the translation start site containing a cAMP-response element was used in transient transfection assays. Treatment of PC12 cells with IGF-I enhances the bcl-2 promoter activity by 2.3-fold, which is inhibited significantly (p < 0.01) by SB203580, an inhibitor of p38 mitogen-activated protein kinase (MAPK). Cotransfection of the bcl-2 promoter with MAPK kinase 6 and the beta isozyme of p38 MAPK results in 2-3-fold increase in the reporter activity. The dominant negative form of MAPKAP-K3, a downstream kinase activated by p38 MAPK, and the dominant negative form of cAMP-response element-binding protein, inhibited the reporter gene activation by IGF-I and p38beta MAPK significantly. IGF-I increases the activity of p38beta MAPK introduced into the cells by adenoviral infection. Thus, a novel signaling pathway (MAPK kinase 6/p38beta MAPK/MAPKAP-K3) has been characterized that defines a transcriptional mechanism for the induction of the antiapoptotic protein Bcl-2 by IGF-I through the nuclear transcription factor cAMP-response element-binding protein in PC12 cells (Pugazhenthi, 1999).

The familial Alzheimer's disease gene products, presenilin-1 and presenilin-2, have been reported to be functionally involved in amyloid precursor protein processing, notch receptor signaling, and programmed cell death or apoptosis. However, the molecular mechanisms by which presenilins regulate these processes remain unknown. With regard to the latter, a molecular link is described between presenilins and the apoptotic pathway. Bcl-X(L), an anti-apoptotic member of the Bcl-2 family has been shown to interact with the carboxyl-terminal fragments of PS1 and PS2 by the yeast two-hybrid system. In vivo interaction analysis reveals that both PS2 and its naturally occurring carboxyl-terminal products, PS2short and PS2Ccas, associated with Bcl-X(L), whereas the caspase-3-generated amino-terminal PS2Ncas fragment do not. This interaction has been corroborated by demonstrating that Bcl-X(L) and PS2 partially co-localized to sites of the vesicular transport system. Functional analysis revealed that presenilins can influence mitochondrial-dependent apoptotic activities, such as cytochrome c release and Bax-mediated apoptosis. Together, these data support a possible role of the Alzheimer's presenilins in modulating the anti-apoptotic effects of Bcl-X(L) (Passer, 1999).

The p75 neurotrophin receptor (p75NTR) has been shown to mediate neuronal death through an unknown pathway. p75NTR expression plasmids were microinjected into sensory neurons in the presence of growth factors and the effect of the expressed proteins on cell survival were assessed. Unlike other members of the TNFR family, p75NTR signals death through a unique caspase-dependent death pathway that does not involve the "death domain" and is differentially regulated by Bcl-2 family members: the anti-apoptotic molecule Bcl-2 both promotes, and is required for, p75NTR killing, whereas killing is inhibited by its homologue Bcl-xL. These results demonstrate that Bcl-2, through distinct molecular mechanisms, either promotes or inhibits neuronal death depending on the nature of the death stimulus (Coulson, 1999).

IL-7 functions as a trophic factor during T lymphocyte development by a mechanism that is partly based on the induction of Bcl-2, which protects cells from apoptosis. Here, a mechanism is reported by which cytokine withdrawal activates the prodeath protein Bax. On loss of IL-7 in a dependent cell line, Bax protein translocated from the cytosol to the mitochondria, where it integrates into the mitochondrial membrane. This translocation is attributable to a conformational change in the Bax protein itself. A rise in intracellular pH precedes mitochondrial translocation and triggers the change in Bax conformation. Intracellular pH in the IL-7-dependent cells rises steadily to peak over pH 7.8 by 6 hr after cytokine withdrawal, paralleling the time point of Bax translocation (a similar alkalinization and Bax translocation was also observed after IL-3 withdrawal from a dependent cell line). The conformation of Bax is directly altered by pH of 7.8 or higher and has been demonstrated by increased protease sensitivity, exposure of N terminus epitopes, and exposure of a hydrophobic domain in the C terminus. Eliminating charged amino acids at the C or N termini of Bax induces a conformational change similar to that induced by raising pH, implicating these residues in the pH effect. Therefore, by either cytokine withdrawal, experimental manipulation of pH, or site-directed mutagenesis, it has been shown that Bax protein changes conformation, exposing membrane-seeking domains, thereby inducing mitochondrial translocation and initiating the cascade of events leading to apoptotic death (Khaled, 1999).

Neutrophils are important effector cells in immunity to microorganisms, particularly bacteria. The process of neutrophil apoptosis is delayed in several inflammatory diseases, suggesting that this phenomenon may represent a general feature contributing to the development of neutrophilia, and, therefore, in many cases to host defense against infection. The delay of neutrophil apoptosis is associated with markedly reduced levels of Bax, a pro-apoptotic member of the Bcl-2 family. Such Bax-deficient cells are also observed upon stimulation of normal neutrophils with cytokines present at sites of neutrophilic inflammation, such as granulocyte and granulocyte-macrophage colony-stimulating factors, in vitro. Moreover, Bax-deficient neutrophils generated by using Bax antisense oligodeoxynucleotides demonstrated delay apoptosis, providing direct evidence for a role of Bax as a pro-apoptotic molecule in these cells. Interestingly, the Bax gene is reexpressed in Bax-deficient neutrophils under conditions of cytokine withdrawal. Thus, both granulocyte expansion and the resolution of inflammation appear to be regulated by the expression of the Bax gene in neutrophils (Dibbert, 1999).

Plakoglobin is a vertebrate cytoplasmic protein and a homolog of beta-catenin and Armadillo in Drosophila, with similar adhesive and signaling functions. These proteins interact with cadherins to mediate cell-cell adhesion and associate with transcription factors to induce changes in the expression of genes involved in cell fate determination and proliferation. Unlike the relatively well characterized role of beta-catenin in cell proliferation via activation of c-MYC and cyclin D1 gene expression, the signaling function of plakoglobin in regulation of cell growth is undefined. High levels of plakoglobin expression in plakoglobin-deficient human SCC9 cells leads to uncontrolled growth and foci formation. Concurrent with the change in growth characteristics is observed a pronounced inhibition of apoptosis. This correlates with an induction of expression of BCL-2, a prototypic member of apoptosis-regulating proteins. The BCL-2 expression coincides with decreased proteolytic processing and activation of caspase-3, an executor of programmed cell death. These data suggest that the growth regulatory function of plakoglobin is independent of its role in mediating cell-cell adhesion. These observations clearly implicate plakoglobin in pathways regulating cell growth and provide initial evidence of its role as a pivotal molecular link between pathways regulating cell adherence and cell death (Hakimelahi, 2000).

Nitric oxide is a chemical messenger implicated in neuronal damage associated with ischemia, neurodegenerative disease, and excitotoxicity. Excitotoxic injury leads to increased NO formation, as well as stimulation of the p38 mitogen-activated protein (MAP) kinase in neurons. In the present study, it was determined if NO-induced cell death in neurons is dependent on p38 MAP kinase activity. Sodium nitroprusside (SNP), a NO donor, elevates caspase activity and induces death in human SH-SY5Y neuroblastoma cells and primary cultures of cortical neurons. Concomitant treatment with SB203580, a p38 MAP kinase inhibitor, diminishes caspase induction and protects SH-SY5Y cells and primary cultures of cortical neurons from NO-induced cell death, whereas the caspase inhibitor zVAD-fmk does not provide significant protection. A role for p38 MAP kinase is further substantiated by the observation that SB203580 blocks translocation of the cell death activator, Bax, from the cytosol to the mitochondria after treatment with SNP. Moreover, expressing a constitutively active form of MKK3, a direct activator of p38 MAP kinase promotes Bax translocation and cell death in the absence of SNP. Bax-deficient cortical neurons are resistant to SNP, further demonstrating the necessity of Bax in this mode of cell death. These results demonstrate that p38 MAP kinase activity plays a critical role in NO-mediated cell death in neurons by stimulating Bax translocation to the mitochondria, thereby activating the cell death pathway (Ghatan, 2000).

Sympathetic neurons require nerve growth factor for survival and die by apoptosis in its absence. Key steps in the death pathway include c-Jun activation, mitochondrial cytochrome c release, and caspase activation. Neurons rescued from NGF withdrawal-induced apoptosis by expression of dominant-negative c-Jun do not release cytochrome c from their mitochondria. Furthermore, mRNA for BIM_EL, a proapoptotic BCL-2 family member, increases in level after NGF withdrawal and this is reduced by dominant-negative c-Jun. Overexpression of BI_MEL in neurons induces cytochrome c redistribution and apoptosis in the presence of NGF, and neurons injected with Bim antisense oligonucleotides or isolated from Bim-/- knockout mice die more slowly after NGF withdrawal (Whitfield, 2001).

BH3-only proapoptotic proteins of the Bcl-2 family such as Bad, Bid, Bim, or Bik transduce death stimuli from the cell surface to the central death machinery. Following apoptosis stimulation, these molecules translocate from the cytosol to mitochondria where they bind to membrane-based Bcl-2 family members. Bid plays an essential role in Fas-mediated apoptosis of the so-called type II cells. In type II cells, such as Jurkat cells or hepatocytes, death-inducing signaling complex (DISC) formation is strongly reduced compared to type I cells in which activation of large amounts of caspase 8 by the DISC enables direct activation of downstream caspases leading to irreversible cell damage. In type II cells, following cleavage by caspase 8, the C-terminal fragment of Bid translocates to mitochondria and triggers the release of apoptogenic factors, thereby inducing cell death. Bid is phosphorylated by casein kinase I (CKI) and casein kinase II (CKII). Inhibition of CKI and CKII accelerates Fas-mediated apoptosis and Bid cleavage, whereas hyperactivity of the kinases delays apoptosis. When phosphorylated, Bid is insensitive to caspase 8 cleavage in vitro. Moreover, a mutant of Bid that cannot be phosphorylated was found to be more toxic than wild-type Bid. Together, these data indicate that phosphorylation of Bid represents a new mechanism whereby cells control apoptosis (Desagher, 2001).

A mechanism that triggers neuronal apoptosis has been characterized. The cell cycle-regulated protein kinase Cdc2 is expressed in postmitotic granule neurons of the developing rat cerebellum and Cdc2 mediates apoptosis of cerebellar granule neurons upon the suppression of neuronal activity. Cdc2 catalyzes the phosphorylation of the BH3-only protein BAD at a distinct site, serine 128, and thereby induces BAD-mediated apoptosis in primary neurons by opposing growth factor inhibition of the apoptotic effect of BAD. The phosphorylation of BAD serine 128 inhibits the interaction of growth factor-induced serine 136-phosphorylated BAD with 14-3-3 proteins. These results suggest that a critical component of the cell cycle couples an apoptotic signal to the cell death machinery via a phosphorylation-dependent mechanism that may generally modulate protein-protein interactions (Konishi, 2002).

Growth factor suppression of apoptosis correlates with the phosphorylation and inactivation of multiple proapoptotic proteins, including the BCL-2 family member BAD. However, the physiological events required for growth factors to block cell death are not well characterized. To assess the contribution of BAD inactivation to cell survival, mice were made with point mutations in the BAD gene that abolish BAD phosphorylation at specific sites. BAD phosphorylation protects cells from the deleterious effects of apoptotic stimuli and attenuates death pathway signaling by raising the threshold at which mitochondria release cytochrome c to induce cell death. These findings establish a function for endogenous BAD phosphorylation, and elucidate a mechanism by which survival kinases block apoptosis in vivo (Datta, 2002).

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