Dissociation of Bax from a Bcl-2/Bax heterodimer triggered by phosphorylation of serine 70 of Bcl-2.

Serine 70 in the loop region of Bcl-2 is specifically phosphorylated by paclitaxel-treatment in tumor cells and BHK cells expressing Bcl-2. The phosphorylation of serine 70 of Bcl-2 (pS70-Bcl-2) peaks 24 to 48 h after paclitaxel treatment and accelerates apoptosis. Phosphorylation is effectively inhibited in the presence of actinomycin D or cycloheximide, which restore cell viability to the same level as control cells not expressing Bcl-2. These results indicate that paclitaxel-induced kinase(s) and/or its activator(s) are synthesized de novo and play an important role in paclitaxel-induced apoptosis by phosphorylating Bcl-2. In binding assays using the phosphorylation-specific antibody against pS70-Bcl-2, the induction of serine 70 phosphorylation 70 results in a loss of the binding ability of Bcl-2 to Bax, a pro-apoptotic partner, and induces subsequent cell death. When the pS70-Bcl-2 antibody was added to human breast cancer tissue, serine 70 phosphorylation was also detected, even prior to treatment with anticancer agents. Further study of breast cancers revealed 83% of tumors with high pS70-Bcl-2 expression responded to paclitaxel or docetaxel treatment, whereas 57% of those with low expression not respond. These findings suggest that pS70-Bcl-2 might be a predictive factor for prognosis and sensitivity to paclitaxel treatment for breast cancer.     

Bcl-2 changes conformation to inhibit Bax oligomerization

Bcl-2 inhibits apoptosis by regulating the release of cytochrome c and other proteins from mitochondria. Oligomerization of Bax promotes cell death by permeabilizing the outer mitochondrial membrane. In transfected cells and isolated mitochondria, Bcl-2, but not the inactive point mutants Bcl-2-G145A and Bcl-2-V159D, undergoes a conformation change in the mitochondrial membrane in response to apoptotic agonists such as tBid and Bax. A mutant Bcl-2 with two cysteines introduced at positions predicted to result in a disulfide bond that would inhibit the mobility of alpha5-alpha6 helices (Bcl-2-S105C/E152C) was only active in a reducing environment. Thus, Bcl-2 must change the conformation to inhibit tBid-induced oligomerization of integral membrane Bax monomers and small oligomers. The conformationally changed Bcl-2 sequesters the integral membrane form of Bax. If Bax is in excess, apoptosis resumes as Bcl-2 is consumed by the conformational change and in complexes with Bax. Thus, Bcl-2 functions as an inhibitor of mitochondrial permeabilization by changing conformation in the mitochondrial membrane to bind membrane-inserted Bax monomers and prevent productive oligomerization of Bax.     

Methods of assaying Bcl-2 and Bax family proteins in yeast

Bcl-2 family proteins play an evolutionarily conserved role in regulating the life and death of the cell. Certain proapoptotic members of the Bcl-2 family, Bax and Bak, have intrinsic cytotoxic activities in that they not only induce or sensitize mammalian cells to undergo apoptosis but also display a lethal phenotype when ectopically expressed in two yeast species Saccharomyces cerevisiae and Schizosaccharomyces pombe. Furthermore, the antiapoptotic Bcl-2 and Bcl-XL proteins can protect yeast against Bax-mediated lethality, suggesting that the death-regulatory functions of these Bcl-2 family proteins are well preserved in yeast. These observations provide the opportunity to study the function of Bcl-2 family proteins in genetically tractable yeast and to apply classical yeast genetics and functional cloning approaches to the dissection of programmed cell death pathway regulated by Bcl-2 family proteins. We describe here methods used in our laboratory to express and to study the functions of Bcl-2 family proteins in both the budding yeast S. cerevisiae and the fission yeast S. pombe.     

Bcl-2 inhibits a Fas-induced conformational change in the Bax N terminus and Bax mitochondrial translocation

Members of the Bcl-2 family of proteins control the cellular commitment to apoptosis, although their role in Fas-induced apoptosis is ill-defined. In this report we demonstrate that activation of the Fas receptor present on a human breast epithelial cell line resulted in a conformational change in the N terminus of the pro-apoptotic protein Bax. This conformational change appeared to occur in the cytosol and precede Bax translocation to the mitochondria. Overexpression of the anti-apoptotic protein Bcl-2 inhibited both the conformational change of Bax as well as its relocalization to the mitochondria. Bcl-2 overexpression did not, however, inhibit Fas-induced cleavage of both procaspase-8 and the pro-apoptotic protein Bid, indicating that Bcl-2 functions downstream of these events. These results suggest that the mechanism by which Bcl-2 inhibits Bax mitochondrial translocation and subsequent amplification of the apoptotic cascade is not by providing a physical barrier to Bax, but rather by inhibiting an upstream event necessary for Bax conformational change.     

Phosphorylation of Bcl-2 and Bax Proteins during Hypoxia in Newborn Piglets

Studies indicate that phosphorylated Bcl-2 cannot form a heterodimer with Bax and thus may lose its antiapoptotic potential. The present study tests the hypothesis that graded hypoxia in cerebral tissue induces the phosphorylation of Bcl-2, thus altering the heterodimerization of Bcl-2 with Bax and subsequently leading to apoptosis. Anesthetized, ventilated newborn piglets were assigned to a normoxic and a graded hypoxic group. Cerebral cortical neuronal nuclei were isolated and immunoprecipitated; immune complexes were separated and reacted with Bcl-2 and Bax specific antibodies. The results show an increased level of serine/tyrosine phosphorylated Bcl-2 in nuclear membranes of hypoxic animals. The level of phosphorylated Bcl-2 protein increased linearly with decrease in tissue PCr. The level of phosphorylated Bax in the neuronal nuclear membranes was independent of cerebral tissue PCr. The data shows that during hypoxia, there is increased phosphorylation of Bcl-2, which may prevent its heterodimerization with Bax and lead to increased proapoptotic activity due to excess Bax in the hypoxic brain. Further increased phosphorylation of Bcl-2 may alter the Bcl-2/Bax-dependent antioxidant, lipid peroxidation and pore forming activity, as well as the regulation of intranuclear Ca²⁺ and caspase activation pathways. We speculate that increased phosphorylation of Bcl-2 in neuronal nuclear membranes is a potential mechanism of programmed cell death activation in the hypoxic brain.     

Bcl-2 prevents Bax oligomerization in the mitochondrial outer membrane

ATP depletion results in Bax translocation from cytosol to mitochondria and release of cytochrome c from mitochondria into cytosol in cultured kidney cells. Overexpression of Bcl-2 prevents cytochrome c release, without ameliorating ATP depletion or Bax translocation, with little or no association between Bcl-2 and Bax as demonstrated by immunoprecipitation (Saikumar, P., Dong, Z., Patel, Y., Hall, K., Hopfer, U., Weinberg, J. M., and Venkatachalam, M. A. (1998) Oncogene 17, 3401-3415). Now we show that translocated Bax forms homo-oligomeric structures, stabilized as chemical adducts by bifunctional cross-linkers in ATP-depleted wild type cells, but remains monomeric in Bcl-2-overexpressing cells. The protective effects of Bcl-2 did not require Bcl-2/Bax association, at least to a degree of proximity or affinity that was stable to conditions of immunoprecipitation or adduct formation by eight cross-linkers of diverse spacer lengths and chemical reactivities. On the other hand, nonionic detergents readily induced homodimers and heterodimers of Bax and Bcl-2. Moreover, associations between translocated Bax and the voltage-dependent anion channel protein or the adenine nucleotide translocator protein could not be demonstrated by immunoprecipitation of Bax, or by using bifunctional cross-linkers. Our data suggest that the in vivo actions of Bax are at least in part dependent on the formation of homo-oligomers without requiring associations with other molecules and that Bcl-2 cytoprotection involves mechanisms that prevent Bax oligomerization.     

Bcl-2 and Bax mammalian regulators of apoptosis are functional in Drosophila

Studies of apoptosis in C. elegans have allowed the identification of three genes, ced-3, ced-4 and ced-9. Their products constitute the components of an induction pathway of apoptosis conserved in the nematode and mammals. In Drosophila, homologues have been found for CED-3, CED-4 and CED-9. CED-9 belongs to the Bcl-2 family which includes negative (Bcl-2) and positive (Bax) regulators of apoptosis. The recently discovered Bcl-2 family member named Drob-1 acts as a positive regulator of cell death. To address whether a Bcl-2 anti-apoptotic pathway exists in the fly, we studied the effects of expressing the mammalian genes bcl-2 in Drosophila. In embryos, expression of bcl-2 inhibits developmental and X-ray-induced apoptosis. Expressing bcl-2 or the pro-apoptotic mammalian bax in the developing eye and wing alters these structures, bcl-2 increasing the number of cells, while bax reduces the number of cells. In addition, the functional interaction between Bcl-2 and Bax is conserved. These results indicate that factors necessary for the activity of bcl-2 and bax are present in Drosophila. Therefore, a Bcl-2 pathway for inhibition of cell death may exist in the fly.     

Alteration of the Bcl-2:Bax ratio in the placenta as pregnancy proceeds

The placenta is the primary site of nutrient and gas exchange between mother and foetus. During human placental development, proliferation, differentiation and apoptosis occur at different stages. In order to clarify some of the molecular mechanisms underlying these events, we investigated the pattern of expression of two members of the Bcl-2 family in human placenta samples and compared them to the level of apoptosis detected by the TUNEL method.In particular, we evaluated the expression of Bcl-2 and Bax and their ratio during the first and third trimester. We found that Bcl-2 was generally expressed at low levels during the entire gestational period. On the other hand, Bax was low during the first trimester but increased towards the end of gestation. In accordance with the change of ratio of these two molecules, the increase of apoptotic cells was observable in the third trimester. These data indicate that Bcl-2 and Bax are spatio-temporally regulated during placental development and that the different expression of the above mentioned genes is at least in part responsible for the delicate balance between cell proliferation and programmed cell death in the human placenta during pregnancy.     

Expression of Bcl-2 and Bax in mouse renal tubules during kidney development

Bcl-2 and Bax play an important role in apoptosis regulation, as well as in cell adhesion and migration during kidney morphogenesis, which is structurally and functionally related to mitochondria. In order to elucidate the role of Bcl-2 and Bax during kidney development, it is essential to establish the exact location of their expression in the kidney. The present study localized their expression during kidney development. Kidneys from embryonic (E) 16-, 17-, 18-day-old mouse fetuses, and postnatal (P) 1-, 3-, 5-, 7-, 14-, 21-day-old pups were embedded in Epon. Semi-thin serial sections from two E17 kidneys underwent computer assisted 3D tubule tracing. The tracing was combined with a newly developed immunohistochemical technique, which enables immunohistochemistry on glutaraldehyde fixated plastic embedded sections. Thereby, the microstructure could be described in detail, and the immunochemistry can be performed using exactly the same sections. The study showed that Bcl-2 and Bax were strongly expressed in mature proximal convoluted tubules at all time points, less strongly expressed in proximal straight tubules, and only weakly in immature proximal tubules and distal tubules. No expression was detected in ureteric bud and other earlier developing structures, such as comma bodies, S shaped bodies, glomeruli, etc. Tubules expressing Bcl-2 only were occasionally observed. The present study showed that, during kidney development, Bcl-2 and Bax are expressed differently in the proximal and distal tubules, although these two tubule segments are almost equally equipped with mitochondria. The functional significance of the different expression of Bcl-2 and Bax in proximal and distal tubules is unknown. However, the findings of the present study suggest that the mitochondrial function differs between mature proximal tubules and in the rest of the tubules. The function of Bcl-2 and Bax during tubulogenesis still needs to be investigated.     

雌激素对大鼠肾上腺皮质中Bcl-2和 Bax蛋白表达的影响

利用免疫组织化学SP法检测摘除卵巢及补充17β-雌二醇后大鼠肾上腺皮质各层Bcl-2和Bax蛋白表达的变化。结果表明,摘除卵巢后肾上腺皮质束状带和网状带Bcl-2和Bax表达都增加;球状带中Bax表达增加,而Bcl-2表达阴性。补充17β-雌二醇后,束状带和网状带中Bcl-2蛋白表达显著降低,Bax在束状带中表达极显著降低,而在网状带中没有阳性产物;在球状带中Bcl-2表达上升,而Bax表达下降。表明雌激素在肾上腺皮质各层发挥着复杂的作用。     

Bcl-2 and Bax proteins are present in interphase nuclei of mammalian cells

The Bcl-2 family of proteins comprises both cell death inhibiting and cell death promoting members, generally believed to be cytoplasmic and predominantly membrane-associated. Like Bcl-2, many Bcl-2-related proteins contain a C-terminal membrane insertion domain and much research is aimed at evaluating the functional role of their localization to the outer membranes of mitochondria, the endoplasmic reticulum, and perinuclear membranes. However, confocal fluorescence microscopy of human breast cancer cells and rat colon cancer cells immunostained with commercial antibodies raised against different epitopes of the anti-apoptotic Bcl-2 and the pro-apoptotic Bax protein revealed that these proteins are not only present in the cellular cytoplasm, but also within interphase nuclei. This was confirmed by Western blot analysis of isolated nuclei. In human cells, certain epitopes of Bcl-2, but not of Bax, were also found to be associated with mitotic chromatin. Anti-estrogen treatment of human breast cancer cells or transfection with antisense bcl-2 led to a reduction in both cytoplasmic and nuclear Bcl-2. Transfection of human bcl-2 and bax into rat cells resulted in cytoplasmic and nuclear Bcl-2 and Bax. This data seems in line with increasing evidence that the role of the Bcl-2 family of proteins should be extended to activities inside the nuclear compartment.     

Apoptotic factors (Bcl-2 and Bax) and diabetic retinopathy in type 2 diabetes

The expression of apoptotic factors Bcl-2 and Bax were studied in the conjunctiva of diabetic patients with and without retinopathy. All patients underwent a complete ophthalmic examination including ocular fundus and retinal fluorescein angiography. The indirect immunoperoxidase method was performed on 15 normal conjunctiva taken during cataract surgery (group 1), on 40 eyes of 40 patients with type 2 diabetes without diabetic retinopathy (group 2) and 13 eyes of 13 patients with diabetic retinopathy (group 3). In normal human conjunctiva, Bax showed positive expression in epithelial, vascular and stromal cells whereas Bcl-2 staining was negative. In the conjunctiva of diabetic patients without diabetic retinopathy, Bax was widely, and strongly, expressed in epithelial cells, vascular endothelial cells, fibroblasts and infiltrating cells such as macrophages. For patients with diabetic retinopathy, Bax was consistently strong to very strong. Bcl-2 protein expression became weak to negative for diabetic patients both with and without diabetic retinopathy. Immunoreactivity was not correlated between Bcl-2 and Bax in the conjunctiva of diabetic patients. Bax was always localized in tissues characterized by a high rate of apoptosis, whereas, Bcl-2 was absent. Our results suggest that diabetic human conjunctiva, with its inflammatory phenomena, is considered as a privileged target for programmed cell death.     

Enhanced heterodimerization of Bax by Bcl-2 mutants improves irradiated cell survival

B Cell Lymphoma-2 (Bcl-2) protein suppresses ionizing radiation-induced apoptosis in hemato-lymphoid system. To enhance the survival of irradiated cells, we have compared the effects and mechanism of Bcl-2 and its functional variants, D34A (caspase-3 resistant) and S70E (mimics phosphorylation on S70). Bcl-2 and its mutants were transfected into hematopoietic cell line and assessed for cell survival, clonogenicity and cell cycle perturbations upon exposure to ionizing radiation. The electrostatic potential of BH3 cleft of Bcl-2/mutants and their heterodimerization with Bcl-2 associated X protein (Bax) were computationally evaluated. Correspondingly, these results were verified by co-immunoprecipitation and western blotting. The mutants afford higher radioprotective effect than Bcl-2 in apoptotic and clonogenic assays at D₀ (radiation dose at which 37 % cell survival was observed). The computational and functional analysis indicates that mutants have higher propensity to neutralize Bax protein by heterodimerization and have increased caspase-9 suppression capability, which is responsible for enhanced survival. This study implies potential of Bcl-2 mutants or their chemical/peptide mimics to elicit radioprotective effect in cells exposed to radiation.     

Jak3 selectively regulates Bax and Bcl-2 expression to promote T-cell development

Jak3-deficient mice display vastly reduced numbers of lymphoid cells. Thymocytes and peripheral T cells from Jak3-deficient mice have a high apoptotic index, suggesting that Jak3 provides survival signals. Here we report that Jak3 regulates T lymphopoiesis at least in part through its selective regulation of Bax and Bcl-2. Jak3-deficient thymocytes express elevated levels of Bax and reduced levels of Bcl-2 relative to those in wild-type littermates. Notably, up-regulation of Bax in Jak3-deficient T cells is physiologically relevant, as Jak3 Bax double-null mice have marked increases in thymocyte and peripheral T-cell numbers. Rescue of T lymphopoiesis by Bax loss was selective, as mice deficient in Jak3 plus p53 or in Jak3 plus Fas remained lymphopenic. However, Bax loss failed to restore proper ratios of peripheral CD4/CD8 T cells, which are abnormally high in Jak3-null mice. Transplantation into Jak3-deficient mice of Jak3-null bone marrow transduced with a Bcl-2-expressing retrovirus also improved peripheral T-cell numbers and restored the ratio of peripheral CD4/CD8 T cells to wild-type levels. The data support the concepts that Jak kinases regulate cell survival through their selective and cell context-dependent regulation of pro- and antiapoptotic Bcl-2 family proteins and that Bax and Bcl-2 play distinct roles in T-cell development.     

Expressions and localizations of Bax/Bcl-2 proteins during metamorphosis of Pelophylax ridibundus

Bcl-2 and Bax proteins are expressed in cells of the tails of Pelophylax ridibundus larvae. We investigated the levels of these proteins in tails undergoing apoptosis. Apoptotic cells were observed in the epidermis, muscle and notochord of tails of different lengths. The apoptotic cells in epidermis exhibited the typical features of apoptosis. Amorphous masses and irregularities in striated muscle tissue undergoing apoptosis and apoptotic remnants in the notochord also were observed. In general, Bax staining in the epidermis, subepidermal fibroblast layer, muscle and notochord cells increased, while Bcl-2 staining decreased as the tail regressed. Our results suggest that during tail regression due to metamorphosis, Bcl-2 and Bax proteins play key roles in the apoptosis of tail epidermis, subepidermal fibroblast layer, muscle and notochord cells.     

Apoptosis modulatory activities of transiently expressed Bcl-2: Roles in cytochrome c release and Bax regulation

Bcl-2 and Bcl-X_L are pro-survival members of the Bcl-2 family. These proteins have been shown to antagonize the pro-apoptotic activity of Bax and promote cell survival through blocking Bax translocation from the cytosol to mitochondria and by preventing the release of cytochrome c. However, it has been recently reported that transiently expressed Bcl-2 unexpectedly leads to significant cell toxicity. To study this intriguing phenomenon, we have carried out further analyses into the properties of transiently expressed Bcl-2. We found that various isoforms of human and different species of Bcl-2 were equally capable of inducing apoptosis. In addition, we discovered that transient expression of Bcl-2, unlike its pro-survival homolog Bcl-X_L, can lead to the release of cytochrome c from mitochondria and that the resulting cell death can be inhibited by caspase and calpain inhibitors. Moreover, we have shown that unlike the pro-apoptotic protein Bid, the toxicity associated with the transient expression of Bcl-2 occurs independent of the activity of the endogenous Bax. Finally, we found that in spite of its intrinsic toxicity, transiently expressed Bcl-2 is fully capable of blocking the ectopically expressed Bax from localizing to mitochondria. Taken together, these studies demonstrate that transiently expressed Bcl-2 displays opposing functional properties.     

Inhibition of Bid-induced apoptosis by Bcl-2. tBid insertion,Bax translocation,and Bax/Bak oligomerization suppressed

Bcl-2 family proteins are important regulators of apoptosis. They can be pro-apoptotic (e.g. Bid, Bax, and Bak) or anti-apoptotic (e.g. Bcl-2 and Bcl-x(L)). The current study examined Bid-induced apoptosis and its inhibition by Bcl-2. Transfection of Bid led to apoptosis in HeLa cells. In these cells, Bid was processed into active forms of truncated Bid or tBid. Following processing, tBid translocated to the membrane-bound organellar fraction. Bcl-2 co-transfection inhibited Bid-induced apoptosis but did not prevent Bid processing or tBid translocation. On the other hand, Bcl-2 blocked the release of mitochondrial cytochrome c in Bid-transfected cells, suggesting actions at the mitochondrial level. Alkaline treatment stripped off tBid from the membrane-bound organellar fraction of Bid plus Bcl-2-co-transfected cells, but not from cells transfected with only Bid, suggesting inhibition of tBid insertion into mitochondrial membranes by Bcl-2. Bcl-2 also prevented Bid-induced Bax translocation from cytosol to the membrane-bound organellar fraction. Finally, Bcl-2 diminished Bid-induced oligomerization of Bax and Bak within the membrane-bound organellar fraction, shown by cross-linking experiments. In conclusion, Bcl-2 inhibited Bid-induced apoptosis at the mitochondrial level by blocking cytochrome c release, without suppressing Bid processing or activation. Critical steps blocked by Bcl-2 included tBid insertion, Bax translocation, and Bax/Bak oligomerization in the mitochondrial membranes.