N-acetylphytosphingosine-induced apoptosis of Jurkat cells is mediated by the conformational change in Bak

N-acetylphytosphingosine (NAPS), a sphingolipid derivative, is one of the well-known signal molecules that mediates various cellular functions, including cell growth, differentiation, and apoptosis. In this study, we demonstrated that NAPS induces apoptosis of Jurkat cells by activating Bak, but not Bax, which are both members of a proapoptotic subfamily of the Bcl-2 proteins. NAPS activated caspase-8 in a FADD-independent manner, but the lack of caspase-8 did not suppress the activation of caspase-3 and -9 and cell death, indicating that caspase-8 activation does not play an important role in NAPS-induced cell death. The overexpression of Bcl-x_L, an anti-apoptotic protein, completely inhibited the activation of the caspases and apoptosis, assuming that NAPS-induced apoptosis was initiated by the mitochondria. The expression levels of pro- and anti-apoptotic Bcl-2 family members were not changed by the NAPS treatment. However, Bad was translocated from the cytosol into the mitochondria, where it bound to Bcl-x_L, and Bak was dissociated from Bcl-x_L and conformationally changed. Taken together, these findings indicate that NAPS induced apoptosis of Jurkat cells in a mitochondria-dependent manner that was controlled by the translocation of Bad and the conformational change in Bak. These authors contributed equally to this paper     

Beyond Cell Death – Antiapoptotic Bcl-2 Proteins Regulate Migration and Invasion of Colorectal Cancer Cells In Vitro

Migration and invasion of malignant cells are prerequisites for cancer progression and metastasis. The Bcl-2 family of proteins consists of about 25 members and has been extensively studied in the context of apoptosis. Despite the fact that small molecules targeting Bcl-2 proteins have already entered clinical trials, very few studies investigated a role of antiapoptotic Bcl-2 proteins beside cell death in the context of metastasis. The aim of this study was to dissect a potential role of the antiapoptotic Bcl-2 proteins Mcl-1, Bcl-2 and Bcl-x_L on migration and invasion of colorectal cancer cells independent of their cell death control function. We used migration and invasion assays as well as three dimensional cell cultures to analyze colorectal cancer cell lines (HT29 and SW480) after siRNA mediated knockdown or overexpression of Mcl-1, Bcl-2 or Bcl-x_L. We observed neither spontaneous cell death induction nor impaired proliferation of cells lacking Mcl-1, Bcl-2 or Bcl-x_L. In contrast, knockdown of Mcl-1 led to increased proliferation. Strikingly, we demonstrate a profound impairment of both, migration and invasion, of colorectal cancer cells after Mcl-1, Bcl-2 or Bcl-x_L knockdown. This phenotype was completely revised in cells overexpressing Mcl-1, Bcl-2 or Bcl-x_L. The most pronounced effect among the investigated proteins was observed for Bcl-2. The data presented indicate a pivotal role of Mcl-1, Bcl-2 and Bcl-x_L for migration and invasion of colorectal cancer cells independent of their known antiapoptotic effects. Thus, our study illustrates novel antitumoral mechanisms of Bcl-2 protein targeting.     

E1B 19,000-Molecular-Weight Protein Interacts with and Inhibits CED-4-Dependent, FLICE-Mediated Apoptosis

Genetic studies of the nematode Caenorhabditis elegans (C. elegans) have identified several important components of the cell death pathway, most notably CED-3, CED-4, and CED-9. CED-4 directly interacts with the Bcl-2 homologue CED-9 (or the mammalian Bcl-2 family member Bcl-x_L) and the caspase CED-3 (or the mammalian caspases ICE and FLICE). This trimolecular complex of CED-4, CED-3, and CED-9 is functional in that CED-9 inhibits CED-4 from activating CED-3 and thereby inhibits apoptosis in heterologous systems. The E1B 19,000-molecular weight protein (E1B 19K) is a potent apoptosis inhibitor and the adenovirus homologue of Bcl-2-related apoptosis inhibitors. Since E1B 19K and Bcl-x_L have functional similarity, we determined if E1B 19K interacts with CED-4 and regulates CED-4-dependent caspase activation. Binding analysis indicated that E1B 19K interacts with CED-4 in a Saccharomyces cerevisiae two-hybrid assay, in vitro, and in mammalian cell lysates. The subcellular localization pattern of CED-4 was dramatically changed by E1B 19K, supporting the theory of a functional interaction between CED-4 and E1B 19K. Whereas expression of CED-4 alone could not induce cell death, coexpression of CED-4 and FLICE augmented cell death induction by FLICE, which was blocked by expression of E1B 19K. Even though E1B 19K did not prevent FLICE-induced apoptosis, it did inhibit CED-4-dependent, FLICE-mediated apoptosis, which suggested that CED-4 was required for E1B 19K to block FLICE activation. Thus, E1B 19K functions through interacting with CED-4, and presumably a mammalian homologue of CED-4, to inhibit caspase activation and apoptosis.     

Importance of Bcl-2-family proteins in murine hematopoietic progenitor and early B cells

Mitochondrial apoptosis regulates survival and development of hematopoietic cells. Prominent roles of some Bcl-2-family members in this regulation have been established, for instance for pro-apoptotic Bim and anti-apoptotic Mcl-1. Additional, mostly smaller roles are known for other Bcl-2-members but it has been extremely difficult to obtain a comprehensive picture of the regulation of mitochondrial apoptosis in hematopoietic cells by Bcl-2-family proteins. We here use a system of mouse ‘conditionally immortalized’ lymphoid-primed hematopoietic progenitor (LMPP) cells that can be differentiated in vitro to pro-B cells, to analyze the importance of these proteins in cell survival. We established cells deficient in Bim, Noxa, Bim/Noxa, Bim/Puma, Bim/Bmf, Bax, Bak or Bax/Bak and use specific inhibitors of Bcl-2, Bcl-X_L and Mcl-1 to assess their importance. In progenitor (LMPP) cells, we found an important role of Noxa, alone and together with Bim. Cell death induced by inhibition of Bcl-2 and Bcl-X_L entirely depended on Bim and could be implemented by Bax and by Bak. Inhibition of Mcl-1 caused apoptosis that was independent of Bim but strongly depended on Noxa and was completely prevented by the absence of Bax; small amounts of anti-apoptotic proteins were co-immunoprecipitated with Bim. During differentiation to pro-B cells, substantial changes in the expression of Bcl-2-family proteins were seen, and Bcl-2, Bcl-X_L and Mcl-1 were all partially in complexes with Bim. In differentiated cells, Noxa appeared to have lost all importance while the loss of Bim and Puma provided protection. The results strongly suggest that the main role of Bim in these hematopoietic cells is the neutralization of Mcl-1, identify a number of likely molecular events during the maintenance of survival and the induction of apoptosis in mouse hematopoietic progenitor cells, and provide data on the regulation of expression and importance of these proteins during differentiation along the B cell lineage.Subject terms: Apoptosis, Immune cell death     

The C-terminal helix of Bcl-xL mediates Bax retrotranslocation from the mitochondria

The proapoptotic Bcl-2 protein Bax can commit a cell to apoptosis by translocation from the cytosol to the mitochondria and permeabilization of the outer mitochondrial membrane. Prosurvival Bcl-2 family members, such as Bcl-x_L, control Bax activity. Bcl-x_L recognizes Bax after a conformational change in the N-terminal segment of Bax on the mitochondria and retrotranslocates it back into the cytoplasm, stabilizing the inactive form of Bax. Here we show that Bax retrotranslocation depends on the C-terminal helix of Bcl-x_L. Deletion or substitution of this segment reduces Bax retrotranslocation and correlates with the accumulation of GFP-tagged or endogenous Bax on the mitochondria of non-apoptotic cells. Unexpectedly, the substitution of the Bcl-x_L membrane anchor by the corresponding Bax segment reverses the Bax retrotranslocation activity of Bcl-x_L, but not that of Bcl-x_L shuttling. Bax retrotranslocation depends on interaction to the Bcl-x_L membrane anchor and interaction between the Bax BH3 domain and the Bcl-x_L hydrophobic cleft. Interference with either interaction increases mitochondrial levels of endogenous Bax. In healthy cells, mitochondrial Bax does not permeabilize the outer mitochondrial membrane, but increases cell death after apoptosis induction.     

Akt, a Target of Phosphatidylinositol 3-Kinase, Inhibits Apoptosis in a Differentiating Neuronal Cell Line

Phosphatidylinositol (PI) 3-kinase has been suggested to mediate cell survival. Consistent with this possibility, apoptosis of conditionally (simian virus 40 T^ts) immortalized rat hippocampal H19-7 neuronal cells was increased in response to wortmannin, an inhibitor of PI 3-kinase. Downstream effectors of PI 3-kinase include Rac1, protein kinase C, and the serine-threonine kinase Akt (protein kinase B). Here, we show that activation of Akt is one mechanism by which PI 3-kinase can mediate survival of H19-7 cells during serum deprivation or differentiation. While ectopic expression of wild-type Akt (c-Akt) does not significantly enhance survival in H19-7 cells, expression of activated forms of Akt (v-Akt or myristoylated Akt) results in enhanced survival which can be comparable to that conferred by Bcl-2. Conversely, expression of a dominant-negative mutant of Akt accelerates cell death upon serum deprivation or differentiation. Finally, the results indicate that Akt can transduce a survival signal for differentiating neuronal cells through a mechanism that is independent of induction of Bcl-2 or Bcl-x_L or inhibition of Jun kinase activity.     

DNA Damage–Induced Bcl-xL Deamidation Is Mediated by NHE-1 Antiport Regulated Intracellular pH

The pro-survival protein Bcl-x_L is critical for the resistance of tumour cells to DNA damage. We have previously demonstrated, using a mouse cancer model, that oncogenic tyrosine kinase inhibition of DNA damage–induced Bcl-x_L deamidation tightly correlates with T cell transformation in vivo, although the pathway to Bcl-x_L deamidation remains unknown and its functional consequences unclear. We show here that rBcl-x_L deamidation generates an iso-Asp⁵²/iso-Asp⁶⁶ species that is unable to sequester pro-apoptotic BH3-only proteins such as Bim and Puma. DNA damage in thymocytes results in increased expression of the NHE-1 Na/H antiport, an event both necessary and sufficient for subsequent intracellular alkalinisation, Bcl-x_L deamidation, and apoptosis. In murine thymocytes and tumour cells expressing an oncogenic tyrosine kinase, this DNA damage–induced cascade is blocked. Enforced intracellular alkalinisation mimics the effects of DNA damage in murine tumour cells and human B-lineage chronic lymphocytic leukaemia cells, thereby causing Bcl-x_L deamidation and increased apoptosis. Our results define a signalling pathway leading from DNA damage to up-regulation of the NHE-1 antiport, to intracellular alkalanisation to Bcl-x_L deamidation, to apoptosis, representing the first example, to our knowledge, of how deamidation of internal asparagine residues can be regulated in a protein in vivo. Our findings also suggest novel approaches to cancer therapy.     

Endogenous Bak inhibitors Mcl-1 and Bcl-xL: differential impact on TRAIL resistance in Bax-deficient carcinoma

Tumor necrosis factor (α)–related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent that preferentially kills tumor cells with limited cytotoxicity to nonmalignant cells. However, signaling from death receptors requires amplification via the mitochondrial apoptosis pathway (type II) in the majority of tumor cells. Thus, TRAIL-induced cell death entirely depends on the proapoptotic Bcl-2 family member Bax, which is often lost as a result of epigenetic inactivation or mutations. Consequently, Bax deficiency confers resistance against TRAIL-induced apoptosis. Despite expression of Bak, Bax-deficient cells are resistant to TRAIL-induced apoptosis. In this study, we show that the Bax dependency of TRAIL-induced apoptosis is determined by Mcl-1 but not Bcl-x_L. Both are antiapoptotic Bcl-2 family proteins that keep Bak in check. Nevertheless, knockdown of Mcl-1 but not Bcl-x_L overcame resistance to TRAIL, CD95/FasL and tumor necrosis factor (α) death receptor ligation in Bax-deficient cells, and enabled TRAIL to activate Bak, indicating that Mcl-1 rather than Bcl-x_L is a major target for sensitization of Bax-deficient tumors for death receptor–induced apoptosis via the Bak pathway.     

Direct Interaction of Bax and Bak Proteins with Bcl-2 Homology Domain 3 (BH3)-only Proteins in Living Cells Revealed by Fluorescence Complementation

The key event in the mitochondrial pathway of apoptosis is the activation of Bax and Bak by BH3-only proteins through a molecular mechanism that is still a matter of debate. Here we studied interactions among anti- and proapoptotic proteins of the Bcl-2 family in living cells by using bimolecular fluorescence complementation analysis. Our results indicate that the antiapoptotic proteins Mcl-1 and Bcl-x_L bind preferably to the BH3-only proteins Bim, PUMA, and Noxa but can also bind to Bak and Bax. We also found a direct interaction between Bim, PUMA, or Noxa with either Bax or Bak during apoptosis induction. In HeLa cells, interaction of Bim with Bax occurs in cytosol, and then Bim-Bax complexes translocate to mitochondria. Complexes of either PUMA or Noxa with Bax or Bak were always detected at mitochondria. Overexpression of Bcl-x_L or Mcl-1 delayed Bim/Bax translocation to mitochondria. These results reveal the ability of main BH3-only proteins to directly activate Bax and Bak in living cells and suggest that a complex network of interactions regulate the function of Bcl-2 family members during apoptosis.     

DNA damage-induced Bcl-xL deamidation is mediated by NHE-1 antiport regulated intracellular pH

The pro-survival protein Bcl-x_L is critical for the resistance of tumour cells to DNA damage. We have previously demonstrated, using a mouse cancer model, that oncogenic tyrosine kinase inhibition of DNA damage–induced Bcl-x_L deamidation tightly correlates with T cell transformation in vivo, although the pathway to Bcl-x_L deamidation remains unknown and its functional consequences unclear. We show here that rBcl-x_L deamidation generates an iso-Asp⁵²/iso-Asp⁶⁶ species that is unable to sequester pro-apoptotic BH3-only proteins such as Bim and Puma. DNA damage in thymocytes results in increased expression of the NHE-1 Na/H antiport, an event both necessary and sufficient for subsequent intracellular alkalinisation, Bcl-x_L deamidation, and apoptosis. In murine thymocytes and tumour cells expressing an oncogenic tyrosine kinase, this DNA damage–induced cascade is blocked. Enforced intracellular alkalinisation mimics the effects of DNA damage in murine tumour cells and human B-lineage chronic lymphocytic leukaemia cells, thereby causing Bcl-x_L deamidation and increased apoptosis. Our results define a signalling pathway leading from DNA damage to up-regulation of the NHE-1 antiport, to intracellular alkalanisation to Bcl-x_L deamidation, to apoptosis, representing the first example, to our knowledge, of how deamidation of internal asparagine residues can be regulated in a protein in vivo. Our findings also suggest novel approaches to cancer therapy.     

Differential Interactions Between Beclin 1 and Bcl-2 Family Members

Autophagy, a cellular degradation system, promotes both cell death and survival. The interaction between Bcl-2 family proteins and Beclin 1, a Bcl-2 interacting protein that promotes autophagy, can mediate crosstalk between autophagy and apoptosis. We investigated the interaction between anti-and pro-apoptotic Bcl-2 proteins with Beclin 1. Our results show that Beclin 1 directly interacts with Bcl-2, Bcl-x_L, Bcl-w and to a lesser extent with Mcl-1. Beclin 1 does not bind the pro-apoptotic Bcl-2 proteins. The interaction between Beclin 1 and the anti-apoptotic protein Bcl-x_L was inhibited by BH3-only proteins, but not by multi-domain proteins. Sequence alignment and structural modeling suggest that Beclin 1 contains a putative BH3-like domain which may interact with the hydrophobic grove of Bcl-x_L. Mutation of the Beclin 1 amino acids predicted to mediate this interaction inhibited the association of Beclin 1 with Bcl-x_L. Our results suggest that BH3 only proapoptotic Bcl-2 proteins may modulate the interactions between Bcl-x_L and Beclin 1.     

Loss of BH3-only Protein Bim Inhibits Apoptosis of Hemopoietic Cells in the Fetal Liver and Male Germ Cells but Not Neuronal Cells in Bcl-x�Cdeficient Mice

Members of the Bcl-2 family include pro- and antiapoptotic proteins that regulate programmed cell death of developing tissues and death in response to cellular damage. In developing mice, the antiapoptotic Bcl-x_L is necessary for survival of neural and hematopoietic cells, and consequently, bcl-x–deficient mice die around Day 13.5 of embryogenesis. Furthermore, adult bcl-x^+/− heterozygous male mice have reduced fertility because of testicular degeneration. Bax, a multi-BH (Bcl-2 homology) domain proapoptotic member of the Bcl-2 family, is regulated by Bcl-x_L and is required for the neuropathological abnormalities seen in bcl-x–deficient embryos. The BH3 domain only subgroup of the Bcl-2 family includes proapoptotic members that are essential for the initiation of apoptotic signaling. In this study, we investigated the role for Bim, a BH3 domain only protein, in the embryonic lethality and increased developmental cell death in bcl-x–deficient animals and the perturbed testicular function in bcl-x^+/− adults. Our studies show that bim deficiency attenuates hematopoietic cell death in the fetal liver of bcl-x–deficient animals, indicating that Bim contributes to programmed cell death in this cell population. In addition, we found that testicular degeneration of adult bcl-x^+/− males was rescued by concomitant Bim deficiency. However, concomitant Bim deficiency had no effect on the embryonic lethality and widespread nervous system abnormalities caused by bcl-x deficiency. Our work identifies Bim as an important regulator of bcl-x deficiency–induced cell death during hematopoiesis and testicular development. (J Histochem Cytochem 56:921–927, 2008)     

Neuroprotectin D1 Induces Dephosphorylation of Bcl-xL in a PP2A-dependent Manner during Oxidative Stress and Promotes Retinal Pigment Epithelial Cell Survival

Retinal pigment epithelial (RPE) cell integrity is critical for the survival of photoreceptor cells. Bcl-x_L is a major anti-apoptotic Bcl-2 protein required for RPE cell survival, and phosphorylation of Bcl-x_L at residue Ser-62 renders this protein pro-apoptotic. In this study, we identify serine/threonine protein phosphatase 2A (PP2A) as a key regulator of Bcl-x_L phosphorylation at residue Ser-62 in ARPE-19 cells, a spontaneously arising RPE cell line in which Bcl-x_L is highly expressed. We found that either PP2A inhibitor okadaic acid or depletion of catalytic subunit α of PP2A (PP2A/Cα) by small interfering RNA enhanced Bcl-x_L phosphorylation when activated with hydrogen peroxide and tumor necrosis factor α-induced oxidative stress. Disruption of PP2A/Cα exacerbated oxidative stress-induced apoptosis. PP2A/Cα colocalized and interacted with S62Bcl-x_L in cells stressed with H₂O₂/tumor necrosis factor α. By contrast, the omega-3 fatty acid docosahexaenoic acid derivative, neuroprotectin D1 (NPD1), a potent activator of survival signaling, down-regulated oxidative stress-induced phosphorylation of Bcl-x_L by increasing protein phosphatase activity. NPD1 also attenuated the oxidative stress-induced apoptosis by knockdown of PP2A/Cα and increased the association of PP2A/Cα with S62Bcl-x_L as well as total Bcl-x_L. NPD1 also enhanced the heterodimerization of Bcl-x_L with its counterpart, pro-apoptotic protein Bax. Thus, NPD1 modulates the activation of this Bcl-2 family protein by dephosphorylating in a PP2A-dependent manner, suggesting a coordinated, NPD1-mediated regulation of cell survival in response to oxidative stress.     

Discovery of a potent and selective Bcl-2 inhibitor using SAR by NMR

The Bcl-2 family of proteins plays a major role in the regulation of apoptosis, or programmed cell death. Overexpression of the anti-apoptotic members of this family (Bcl-2, Bcl-x_L, and Mcl-1) can render cancer cells resistant to chemotherapeutic agents and therefore these proteins are important targets for the development of new anti-cancer agents. Here we describe the discovery of a potent, highly selective, Bcl-2 inhibitor using SAR by NMR and structure-based drug design which could serve as a starting point for the development of a Bcl-2 selective anti-cancer agent. Such an agent would potentially overcome the Bcl-x_L mediated thrombocytopenia observed with ABT-263.     

Human IAP-Like Protein Regulates Programmed Cell Death Downstream of Bcl-xL and Cytochrome c

The gene encoding human IAP-like protein (hILP) is one of several mammalian genes with sequence homology to the baculovirus inhibitor-of-apoptosis protein (iap) genes. Here we show that hILP can block apoptosis induced by a variety of extracellular stimuli, including UV light, chemotoxic drugs, and activation of the tumor necrosis factor and Fas receptors. hILP also protected against cell death induced by members of the caspase family, cysteine proteases which are thought to be the principal effectors of apoptosis. hILP and Bcl-x_L were compared for their ability to affect several steps in the apoptotic pathway. Redistribution of cytochrome c from mitochondria, an early event in apoptosis, was not blocked by overexpression of hILP but was inhibited by Bcl-x_L. In contrast, hILP, but not Bcl-x_L, inhibited apoptosis induced by microinjection of cytochrome c. These data suggest that while Bcl-x_L may control mitochondrial integrity, hILP can function downstream of mitochondrial events to inhibit apoptosis.     

Syk Interacts with and Phosphorylates Nucleolin To Stabilize Bcl-xL mRNA and Promote Cell Survival

The Syk protein tyrosine kinase, a well-characterized regulator of immune cell function, plays an increasingly recognized role in tumorigenesis as a promoter of cell survival in both hematological and nonhematological malignancies. We show here that the expression of Syk in MCF7 or MDA-MB-231 breast cancer cells or in DG75 B-lymphoma cells protects cells from apoptosis induced by oxidative or genotoxic stress by stabilizing the mRNA for Bcl-x_L, an antiapoptotic protein. Syk binds robustly to nucleolin and phosphorylates it on tyrosine, enhancing its ability to bind the Bcl-x_L mRNA. Consequently, reducing the level of nucleolin by RNA interference attenuates the ability of Syk to protect cells from stress-induced cell death.     

Determinants of BH3 Binding Specificity for Mcl-1 versus Bcl-xL

Interactions among Bcl-2 family proteins are important for regulating apoptosis. Prosurvival members of the family interact with proapoptotic BH3 (Bcl-2-homology-3)-only members, inhibiting execution of cell death through the mitochondrial pathway. Structurally, this interaction is mediated by binding of the α-helical BH3 region of the proapoptotic proteins to a conserved hydrophobic groove on the prosurvival proteins. Native BH3-only proteins exhibit selectivity in binding prosurvival members, as do small molecules that block these interactions. Understanding the sequence and structural basis of interaction specificity in this family is important, as it may allow the prediction of new Bcl-2 family associations and/or the design of new classes of selective inhibitors to serve as reagents or therapeutics. In this work, we used two complementary techniques—yeast surface display screening from combinatorial peptide libraries and SPOT peptide array analysis—to elucidate specificity determinants for binding to Bcl-x_Lversus Mcl-1, two prominent prosurvival proteins. We screened a randomized library and identified BH3 peptides that bound to either Mcl-1 or Bcl-x_L selectively or to both with high affinity. The peptides competed with native ligands for binding into the conserved hydrophobic groove, as illustrated in detail by a crystal structure of a specific peptide bound to Mcl-1. Mcl-1-selective peptides from the screen were highly specific for binding Mcl-1 in preference to Bcl-x_L, Bcl-2, Bcl-w, and Bfl-1, whereas Bcl-x_L-selective peptides showed some cross-interaction with related proteins Bcl-2 and Bcl-w. Mutational analyses using SPOT arrays revealed the effects of 170 point mutations made in the background of a peptide derived from the BH3 region of Bim, and a simple predictive model constructed using these data explained much of the specificity observed in our Mcl-1 versus Bcl-x_L binders.