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Cdk2 phosphorylation of Bcl-xL after stress converts it to a pro-apoptotic protein mimicking Bax/Bak.

ABSTRACT: Apoptosis is a regulated form of cell death that proceeds by defined biochemical pathways. Most apoptosis is controlled by interactions between pro-survival and pro-apoptotic Bcl-2 family proteins in which death is often the consequence of permeabilization of the mitochondrial outer membrane. Many drugs affect this equilibrium to favor apoptosis but this process is not completely understood. We show that the chemotherapeutic drug cisplatin initiates an apoptotic pathway by phosphorylation of a pro-survival Bcl-2 family member, Bcl-xL, by cyclin-dependent kinase 2. The phosphorylation occurred at a previously unreported site and its biologic significance was demonstrated by a phosphomimetic modification of Bcl-xL that was able to induce apoptosis without addition of cisplatin. The mechanism of cell death induction was similar to that initiated by pro-apoptotic Bcl-2 family proteins, that is, phosphorylated Bcl-xL translocated to the mitochondrial membrane, and formed pores in the membrane. This initiated cytochrome c release and caspase activation that resulted in cell death.

SUBMITTER: Megyesi J

PROVIDER: S-EPMC4877050 | biostudies-literature |

REPOSITORIES: biostudies-literature

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BH3-only proteins target BCL-xL/MCL-1, not BAX/BAK, to initiate apoptosis.

Project description:It has been widely accepted that mitochondria-dependent apoptosis initiates when select BH3-only proteins (BID, BIM, etc.) directly engage and allosterically activate effector proteins BAX/BAK. Here, through reconstitution of cells lacking all eight pro-apoptotic BH3-only proteins, we demonstrate that all BH3-only proteins primarily target the anti-apoptotic BCL-2 proteins BCL-xL/MCL-1, whose simultaneous suppression enables membrane-mediated spontaneous activation of BAX/BAK. BH3-only proteins' apoptotic activities correlate with affinities for BCL-xL/MCL-1 instead of abilities to directly activate BAX/BAK. Further, BID and BIM do not distinguish BAX from BAK or accelerate BAX/BAK activation following inactivation of BCL-xL/MCL-1. Remarkably, death ligand-induced apoptosis in cells lacking BH3-only proteins and MCL-1 is fully restored by BID mutants capable of neutralizing BCL-xL, but not direct activation of BAX/BAK. Taken together, our findings provide a "Membrane-mediated Permissive" model, in which the BH3-only proteins only indirectly activate BAX/BAK by neutralizing the anti-apoptotic BCL-2 proteins, and thus allowing BAX/BAK to undergo unimpeded, spontaneous activation in the mitochondrial outer membrane milieu, leading to apoptosis initiation.

| S-EPMC6888900 | biostudies-literature

Mcl-1 and Bcl-xL regulate Bak/Bax-dependent apoptosis of the megakaryocytic lineage at multistages.

Project description:Anti-apoptotic Bcl-2 family proteins, which inhibit the mitochondrial pathway of apoptosis, are involved in the survival of various hematopoietic lineages and are often dysregulated in hematopoietic malignancies. However, their involvement in the megakaryocytic lineage is not well understood. In the present paper, we describe the crucial anti-apoptotic role of Mcl-1 and Bcl-xL in this lineage at multistages. The megakaryocytic lineage-specific deletion of both, in sharp contrast to only one of them, caused apoptotic loss of mature megakaryocytes in the fetal liver and systemic hemorrhage, leading to embryonic lethality. ABT-737, a Bcl-xL/Bcl-2/Bcl-w inhibitor, only caused thrombocytopenia in adult wild-type mice, but further induced massive mature megakaryocyte apoptosis in the Mcl-1 knockout mice, leading to severe hemorrhagic anemia. All these phenotypes were fully restored if Bak and Bax, downstream apoptosis executioners, were also deficient. In-vitro study revealed that the Jak pathway maintained Mcl-1 and Bcl-xL expression levels, preventing megakaryoblastic cell apoptosis. Similarly, both were involved in reticulated platelet survival, whereas platelet survival was dependent on Bcl-xL due to rapid proteasomal degradation of Mcl-1. In conclusion, Mcl-1 and Bcl-xL regulate the survival of the megakaryocytic lineage, which is critically important for preventing lethal or severe hemorrhage in both developing and adult mice.

| S-EPMC3469054 | biostudies-literature

Bax mitochondrial relocation is linked to its phosphorylation and its interaction with Bcl-xL.

Project description:The heterologous expression of Bax, and other Bcl-2 family members, in the yeast Saccharomyces cerevisiae, has proved to be a valuable reporter system to investigate the molecular mechanisms underlying their interaction with mitochondria. By combining the co-expression of Bax and Bcl-xL mutants with analyzes of their localization and interaction in mitochondria and post-mitochondrial supernatants, we showed that the ability of Bax and Bcl-xL to interact is dependent both on Bax phosphorylation - mimicked by a substitution S184D - and by Bax and Bcl-xL localization. This, and previous data, provide the molecular basis for a model of dynamic equilibrium for Bax localization and activation, regulated both by phosphorylation and Bcl-xL.

| S-EPMC5348979 | biostudies-literature

Levels of pro-apoptotic regulator Bad and anti-apoptotic regulator Bcl-xL determine the type of the apoptotic logic gate.

Project description:BackgroundApoptosis is a tightly regulated process: cellular survive-or-die decisions cannot be accidental and must be unambiguous. Since the suicide program may be initiated in response to numerous stress stimuli, signals transmitted through a number of checkpoints have to be eventually integrated.ResultsIn order to analyze possible mechanisms of the integration of multiple pro-apoptotic signals, we constructed a simple model of the Bcl-2 family regulatory module. The module collects upstream signals and processes them into life-or-death decisions by employing interactions between proteins from three subgroups of the Bcl-2 family: pro-apoptotic multidomain effectors, pro-survival multidomain restrainers, and pro-apoptotic single domain BH3-only proteins. Although the model is based on ordinary differential equations (ODEs), it demonstrates that the Bcl-2 family module behaves akin to a Boolean logic gate of the type dependent on levels of BH3-only proteins (represented by Bad) and restrainers (represented by Bcl-xL). A low level of pro-apoptotic Bad or a high level of pro-survival Bcl-xL implies gate AND, which allows for the initiation of apoptosis only when two stress stimuli are simultaneously present: the rise of the p53 killer level and dephosphorylation of kinase Akt. In turn, a high level of Bad or a low level of Bcl-xL implies gate OR, for which any of these stimuli suffices for apoptosis.ConclusionsOur study sheds light on possible signal integration mechanisms in cells, and spans a bridge between modeling approaches based on ODEs and on Boolean logic. In the proposed scheme, logic gates switching results from the change of relative abundances of interacting proteins in response to signals and involves system bistability. Consequently, the regulatory system may process two analogous inputs into a digital survive-or-die decision.

| S-EPMC3750306 | biostudies-literature

Bcl-XL inhibits membrane permeabilization by competing with Bax.

Project description:Although Bcl-XL and Bax are structurally similar, activated Bax forms large oligomers that permeabilize the outer mitochondrial membrane, thereby committing cells to apoptosis, whereas Bcl-XL inhibits this process. Two different models of Bcl-XL function have been proposed. In one, Bcl-XL binds to an activator, thereby preventing Bax activation. In the other, Bcl-XL binds directly to activated Bax. It has been difficult to sort out which interaction is important in cells, as all three proteins are present simultaneously. We examined the mechanism of Bax activation by tBid and its inhibition by Bcl-XL using full-length recombinant proteins and measuring permeabilization of liposomes and mitochondria in vitro. Our results demonstrate that Bcl-XL and Bax are functionally similar. Neither protein bound to membranes alone. However, the addition of tBid recruited molar excesses of either protein to membranes, indicating that tBid activates both pro- and antiapoptotic members of the Bcl-2 family. Bcl-XL competes with Bax for the activation of soluble, monomeric Bax through interaction with membranes, tBid, or t-Bid-activated Bax, thereby inhibiting Bax binding to membranes, oligomerization, and membrane permeabilization. Experiments in which individual interactions were abolished by mutagenesis indicate that both Bcl-XL-tBid and Bcl-XL-Bax binding contribute to the antiapoptotic function of Bcl-XL. By out-competing Bax for the interactions leading to membrane permeabilization, Bcl-XL ties up both tBid and Bax in nonproductive interactions and inhibits Bax binding to membranes. We propose that because Bcl-XL does not oligomerize it functions like a dominant-negative Bax in the membrane permeabilization process.

| S-EPMC2422857 | biostudies-literature

Phosphorylation of Rad9 at serine 328 by cyclin A-Cdk2 triggers apoptosis via interfering Bcl-xL.

Project description:Cyclin A-Cdk2, a cell cycle regulated Ser/Thr kinase, plays important roles in a variety of apoptoticprocesses. However, the mechanism of cyclin A-Cdk2 regulated apoptosis remains unclear. Here, we demonstrated that Rad9, a member of the BH3-only subfamily of Bcl-2 proteins, could be phosphorylated by cyclin A-Cdk2 in vitro and in vivo. Cyclin A-Cdk2 catalyzed the phosphorylation of Rad9 at serine 328 in HeLa cells during apoptosis induced by etoposide, an inhibitor of topoisomeraseII. The phosphorylation of Rad9 resulted in its translocation from the nucleus to the mitochondria and its interaction with Bcl-xL. The forced activation of cyclin A-Cdk2 in these cells by the overexpression of cyclin A,triggered Rad9 phosphorylation at serine 328 and thereby promoted the interaction of Rad9 with Bcl-xL and the subsequent initiation of the apoptotic program. The pro-apoptotic effects regulated by the cyclin A-Cdk2 complex were significantly lower in cells transfected with Rad9S328A, an expression vector that encodes a Rad9 mutant that is resistant to cyclin A-Cdk2 phosphorylation. These findings suggest that cyclin A-Cdk2 regulates apoptosis through a mechanism that involves Rad9phosphorylation.

| S-EPMC3441668 | biostudies-other

Deregulation of apoptotic factors Bcl-xL and Bax confers apoptotic resistance to myeloid-derived suppressor cells and contributes to their persistence in cancer.

Project description:Myeloid-derived suppressor cells (MDSCs) are heterogeneous immature myeloid cells that accumulate in response to tumor progression. Compelling data from mouse models and human cancer patients showed that tumor-induced inflammatory mediators induce MDSC differentiation. However, the mechanisms underlying MDSC persistence is largely unknown. Here, we demonstrated that tumor-induced MDSCs exhibit significantly decreased spontaneous apoptosis as compared with myeloid cells with the same phenotypes from tumor-free mice. Consistent with the decreased apoptosis, cell surface Fas receptor decreased significantly in tumor-induced MDSCs. Screening for changes of key apoptosis mediators downstream the Fas receptor revealed that expression levels of IRF8 and Bax are diminished, whereas expression of Bcl-xL is increased in tumor-induced MDSCs. We further determined that IRF8 binds directly to Bax and Bcl-x promoter in primary myeloid cells in vivo, and IRF8-deficient MDSC-like cells also exhibit increased Bcl-xL and decreased Bax expression. Analysis of CD69 and CD25 levels revealed that cytotoxic T lymphocytes (CTLs) are partially activated in tumor-bearing hosts. Strikingly, FasL but not perforin and granzymes were selectively activated in CTLs in the tumor-bearing host. ABT-737 significantly increased the sensitivity of MDSCs to Fas-mediated apoptosis in vitro. More importantly, ABT-737 therapy increased MDSC spontaneous apoptosis and decreased MDSC accumulation in tumor-bearing mice. Our data thus determined that MDSCs use down-regulation of IRF8 to alter Bax and Bcl-xL expression to deregulate the Fas-mediated apoptosis pathway to evade elimination by host CTLs. Therefore, targeting Bcl-xL is potentially effective in suppression of MDSC persistence in cancer therapy.

| S-EPMC3696683 | biostudies-literature

cBid, Bax and Bcl-xL exhibit opposite membrane remodeling activities.

Project description:The proteins of the Bcl-2 family have a crucial role in mitochondrial outer membrane permeabilization during apoptosis and in the regulation of mitochondrial dynamics. Current models consider that Bax forms toroidal pores at mitochondria that are responsible for the release of cytochrome c, whereas Bcl-xL inhibits pore formation. However, how Bcl-2 proteins regulate mitochondrial fission and fusion remains poorly understood. By using a systematic analysis at the single vesicle level, we found that cBid, Bax and Bcl-xL are able to remodel membranes in different ways. cBid and Bax induced a reduction in vesicle size likely related to membrane tethering, budding and fission, besides membrane permeabilization. Moreover, they are preferentially located at highly curved membranes. In contrast, Bcl-xL not only counterbalanced pore formation but also membrane budding and fission. Our findings support a mechanism of action by which cBid and Bax induce or stabilize highly curved membranes including non-lamellar structures. This molecular activity reduces the energy for membrane remodeling, which is a necessary step in toroidal pore formation, as well as membrane fission and fusion, and provides a common mechanism that links the two main functions of Bcl-2 proteins.

| S-EPMC4849160 | biostudies-literature

BCL-xL/BCL2L1 is a critical anti-apoptotic protein that suppresses BAK to promote pancreatic specification from human pluripotent stem cells

Project description:Purpose: From qPCR and western blot analyses, we observed that BCL-xL was upregulated from D7 onwards during differentiation of human pluripotent stem cells towards pancreatic progenitors using a combination of growth factors

2020-05-01 | GSE136064 | GEO

Key role for Bak activation and Bak-Bax interaction in the apoptotic response to vinblastine.

Project description:Microtubule inhibitors such as vinblastine cause mitotic arrest and subsequent apoptosis through the intrinsic mitochondrial pathway. However, although Bcl-2 family proteins have been implicated as distal mediators, their precise role is largely unknown. In this study, we investigated the role of Bak in vinblastine-induced apoptosis. Bak was mainly monomeric in untreated KB-3 cells, and multimers corresponding to dimer, trimer, and higher oligomers were observed after vinblastine treatment. The oligomeric Bak species were strongly diminished in cells stably overexpressing Bcl-xL. Immunoprecipitation with a conformation-dependent Bak antibody revealed that vinblastine induced Bak activation. Reciprocal immunoprecipitations indicated that vinblastine induced the interaction of active Bak with active Bax. Furthermore, Bcl-xL overexpression prevented Bak and Bax interaction and strongly inhibited apoptosis, whereas Bcl-2 overexpression did not prevent Bak-Bax interaction and only weakly inhibited apoptosis. The relative contributions of Bak and Bax were investigated using fibroblasts deficient in one or both of these proteins; double knockouts were highly resistant compared with single knockouts, with vinblastine sensitivities in the order of Bak(+)/Bax(+) > Bak(+)/Bax(-) > Bak(-)/Bax(+) > Bak(-)/Bax(-). These results highlight Bak as a key mediator of vinblastine-induced apoptosis and show for the first time activation and oligomerization of Bak by an antimitotic agent. In addition, our results suggest that the interaction of the activated forms of Bak and Bax represents a key distal step in the apoptotic response to this important chemotherapeutic drug.

| S-EPMC3276366 | biostudies-literature

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