Reversible membrane interaction of BAD requires two C-terminal lipid binding domains in conjunction with 14-3-3 protein binding

BAD is a Bcl-2 homology domain 3 (BH3)-only proapoptotic member of the Bcl-2 protein family that is regulated by phosphorylation in response to survival factors. Binding of BAD to mitochondria is thought to be exclusively mediated by its BH3 domain. We show here that BAD binds to lipids with high affinities, predominantly to negatively charged phospholipids, such as phosphatidylserine, phosphatidic acid, and cardiolipin, as well as to cholesterol-rich liposomes. Two lipid binding domains (LBD1 and LBD2) with different binding preferences were identified, both located in the C-terminal part of the BAD protein. BAD facilitates membrane translocation of Bcl-XL in a process that requires LBD2. Integrity of LBD1 and LBD2 is also required for proapoptotic activity in vivo. Phosphorylation of BAD does not affect membrane binding but renders BAD susceptible to membrane extraction by 14-3-3 proteins. BAD can be removed efficiently by 14-3-3zeta, -eta, -tau and lesxs efficiently by other 14-3-3 isoforms. The assembled BAD.14-3-3 complex exhibited high affinity for cholesterol-rich liposomes but low affinity for mitochondrial membranes. We conclude that BAD is a membrane-associated protein that has the hallmarks of a receptor rather than a ligand. Lipid binding is essential for the proapoptotic function of BAD in vivo. The data support a model in which BAD shuttles in a phosphorylation-dependent manner between mitochondria and other membranes and where 14-3-3 is a key regulator of this relocation. The dynamic interaction of BAD with membranes is tied to activation and membrane translocation of Bcl-XL.     

14-3-3 proteins and survival kinases cooperate to inactivate BAD by BH3 domain phosphorylation

The Bcl-2 homology 3 (BH3) domain of prodeath Bcl-2 family members mediates their interaction with prosurvival Bcl-2 family members and promotes apoptosis. We report that survival factors trigger the phosphorylation of the proapoptotic Bcl-2 family member BAD at a site (Ser-155) within the BAD BH3 domain. When BAD is bound to prosurvival Bcl-2 family members, BAD Ser-155 phosphorylation requires the prior phosphorylation of Ser-136, which recruits 14-3-3 proteins that then function to increase the accessibility of Ser-155 to survival-promoting kinases. Ser-155 phosphorylation disrupts the binding of BAD to prosurvival Bcl-2 proteins and thereby promotes cell survival. These findings define a mechanism by which survival signals inactivate a proapoptotic Bcl-2 family member, and suggest a role for 14-3-3 proteins as cofactors that regulate sequential protein phosphorylation events.     

Mdm38 is a 14-3-3-like receptor and associates with the protein synthesis machinery at the inner mitochondrial membrane

Mitochondrial ribosomes synthesize core subunits of the inner membrane respiratory chain complexes. In mitochondria, translation is regulated by mRNA‐specific activator proteins and occurs on membrane‐associated ribosomes. Mdm38/Letm1 is a conserved membrane receptor for mitochondrial ribosomes and specifically involved in respiratory chain biogenesis. In addition, Mdm38 and its higher eukaryotic homolog Letm1, function as K⁺/H⁺ or Ca²⁺/H⁺ antiporters in the inner membrane. Here, we identify the conserved ribosome‐binding domain (RBD) of Mdm38 and determine the crystal structure at 2.1 Å resolution. Surprisingly, Mdm38^RBD displays a 14‐3‐3‐like fold despite any similarity to 14‐3‐3‐proteins at the primary sequence level and thus represents the first 14‐3‐3‐like protein in mitochondria. The 14‐3‐3‐like domain is critical for respiratory chain assembly through regulation of Cox1 and Cytb translation. We show that this function can be spatially separated from the ion transport activity of the membrane integrated portion of Mdm38. On the basis of the phenotypes observed for mdm38Δ as compared to Mdm38 lacking the RBD, we suggest a model that combining ion transport and translational regulation into one molecule allows for direct coupling of ion flux across the inner membrane, and serves as a signal for the translation of mitochondrial membrane proteins via its direct association with the protein synthesis machinery.     

Phosphorylation and subsequent interaction with 14-3-3 proteins regulate plastid glutamine synthetase in Medicago truncatula

In this report we demonstrate that plastid glutamine synthetase of Medicago truncatula (MtGS2) is regulated by phosphorylation and 14-3-3 interaction. To investigate regulatory aspects of GS2 phosphorylation, we have produced non-phosphorylated GS2 proteins by expressing the plant cDNA in E. coli and performed in vitro phosphorylation assays. The recombinant isoenzyme was phosphorylated by calcium dependent kinase(s) present in leaves, roots and nodules. Using an (His)₆-tagged 14-3-3 protein column affinity purification method, we demonstrate that phosphorylated GS2 interacts with 14-3-3 proteins and that this interaction leads to selective proteolysis of the plastid located isoform, resulting in inactivation of the isoenzyme. By site directed mutagenesis we were able to identify a GS2 phosphorylation site (Ser97) crucial for the interaction with 14-3-3s. Phosphorylation of this target residue can be functionally mimicked by replacing Ser97 by Asp, indicating that the introduction of a negative charge contributes to the interaction with 14-3-3 proteins and subsequent specific proteolysis. Furthermore, we document that plant extracts contain protease activity that cleaves the GS2 protein only when it is bound to 14-3-3 proteins following either phosphorylation or mimicking of phosphorylation by Ser97Asp.     

Seizure-like activity leads to the release of BAD from 14-3-3 protein and cell death in hippocampal neurons in vitro

Seizure-induced neuronal death may involve engagement of the BCL-2 family of apoptosis-regulating proteins. In the present study we examined the activation of proapoptotic BAD in cultured hippocampal neurons following seizures induced by removal of chronic glutamatergic transmission blockade. Kynurenic acid withdrawal elicited an increase in seizure-like electrical activity, which was inhibited by blockers of AMPA (CNQX) and NMDA (MK801 and AP5) receptor function. However, only NMDA receptor antagonists inhibited calcium entry as assessed by fura-2, and cell death of hippocampal neurons. Seizures increased proteolysis of caspase-3 and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) of cells. Seizure-like activity induced dephosphorylation of BAD and the disruption of its constitutive interaction with 14-3-3 proteins. In turn, BAD dimerized with antiapoptotic BCL-Xl after seizures. However, the absence of neuroprotective effects of pathway intervention suggests that BAD may perform a reinforcement rather than instigator role in cell death following seizures in vitro.     

14-3-3 Protects against stress-induced apoptosis

Expression of human Bax, a cardinal regulator of mitochondrial membrane permeabilization, causes death in yeast. We screened a human cDNA library for suppressors of Bax-mediated yeast death and identified human 14-3-3β/α, a protein whose paralogs have numerous chaperone-like functions. Here, we show that, yeast cells expressing human 14-3-3β/α are able to complement deletion of the endogenous yeast 14-3-3 and confer resistance to a variety of different stresses including cadmium and cycloheximide. The expression of 14-3-3β/α also conferred resistance to death induced by the target of rapamycin inhibitor rapamycin and by starvation for the amino acid leucine, conditions that induce autophagy. Cell death in response to these autophagic stimuli was also observed in the macroautophagic-deficient atg1Δ and atg7Δ mutants. Furthermore, 14-3-3β/α retained its ability to protect against the autophagic stimuli in these autophagic-deficient mutants arguing against so called ‘autophagic death''. In line, analysis of cell death markers including the accumulation of reactive oxygen species, membrane integrity and cell surface exposure of phosphatidylserine indicated that 14-3-3β/α serves as a specific inhibitor of apoptosis. Finally, we demonstrate functional conservation of these phenotypes using the yeast homolog of 14-3-3: Bmh1. In sum, cell death in response to multiple stresses can be counteracted by 14-3-3 proteins.     

Tropisetron restores normal expression of BAD,SIRT1, SIRT3, and SIRT7 in the rat pressure overload-induced cardiac hypertrophy model

Tropisetron exerts a protective effect against cardiac complications, particularly cardiac hypertrophy. Oxidative stress and apoptosis are the main contributors to the pathogenesis of cardiac hypertrophy. Sirtuins, a family of histone deacetylases, are connected to cellular oxidative stress signaling and antioxidant defense. Sirtuins are also linked to apoptosis which is an important mechanism in the progression of cardiac hypertrophy to heart failure. Literature also suggests that tropisetron impedes apoptosis, partly mediated through an antioxidant mechanism. Therefore, we examined if tropisetron fights cardiac hypertrophy by adjusting sirtuin family proteins (Sirts) and components of mitochondrial death pathway, Bcl-associated X (BAX), Bcl-2-associated death promoter (BAD). Male Sprague–Dawley rats got divided into four groups, including control (Ctl), tropisetron (Trop), cardiac hypertrophy (Hyp), and hypertrophic rats under tropisetron treatment (Hyp + Trop). Pathological cardiac hypertrophy was induced by surgical abdominal aortic constriction (AAC). The increased expression of brain natriuretic peptide (BNP) in the Hyp group confirms the cardiac hypertrophy establishment. The mRNA levels of SIRT1, SIRT3, SIRT7, and BAD also upregulated in the hypertrophic group (p < 0.001). Postoperational administration of tropisetron for 3 weeks lowered the increased expression of BNP (p < 0.05) and BAD (p < 0.001), though the reduction of BAX expression was statistically insignificant (p > 0.05). Tropisetron treatment also restored the normal level of SIRT1/3/7 genes expression in the Hyp + Trop group (p < 0.05). Present findings suggest that tropisetron can suppress cardiomyocyte hypertrophy progression to heart failure by counteracting BNP, SIRT1, SIRT3, Sirt7, and BAD overexpression-mediated apoptosis in a rat model of cardiac hypertrophy.     

The proapoptotic BH3-only protein BAD transduces cell death signals independently of its interaction with Bcl-2

The BH3-only protein BAD binds to Bcl-2 family proteins through its BH3 domain. Recent studies suggest that BAD binds to both Bcl-2 and Bcl-X(L), however mediates its pro-apoptotic functions through inhibition of Bcl-X(L), but not Bcl-2. In this paper we addressed this issue using a BAD mutant within the BH3 domain, by substitution of Asp 119 with Gly (BAD(D119G)), which selectively abrogates an ability to interact with Bcl-2. Confocal microscopy revealed that mutation of BAD at D119 does not affect BAD targeting to the mitochondrial membrane in serum-starved COS-7 cells. However, co-precipitation assays indicated that, whereas wild-type BAD (BADwt) directly interacts with Bcl-2 and Bcl-X(L), BAD(D119G) interacts only with Bcl-X(L). Nevertheless both BADwt and BAD(D119G) could introduce apoptosis and diminish the anti-apoptotic effect of Bcl-2 and Bcl-X(L) in a similar manner in a co-transfection assay. These data thus suggest that Asp119 is a crucial site within the BH3 domain of BAD for interaction of BAD with Bcl-2, but is dispensable for the interaction of BAD with Bcl-X(L), for its targeting to mitochondria, and most importantly, for its pro-apoptotic functions. Thus, we confirm that neutralization of Bcl-2 function is marginal for BAD-mediated apoptosis.     

Involvement of 14-3-3 proteins in the osmotic regulation of H+-ATPase in plant plasma membranes

 Taking the binding of fusicoccin to plasma membranes as an indicator of complex formation between the 14-3-3 dimer and H⁺-ATPase, we assessed the effect of osmotic stress on the interaction of these proteins in suspension-cultured cells of sugar beet (Beta vulgaris L.). An increase in osmolarity of the cell incubation medium, accompanied by a decrease in turgor, was found to activate the H⁺ efflux 5-fold. The same increment was observed in the number of high-affinity fusicoccin-binding sites in isolated plasma membranes; the 14-3-3 content in the membranes increased 2- to 3-fold, while the H⁺-ATPase activity changed only slightly. The data obtained indicate that osmotic regulation of H⁺-ATPase in the plant plasma membrane is achieved via modulation of the coupling between H⁺ transport and ATP hydrolysis, and that such regulation involves 14-3-3 proteins. Received: 10 February 2000 / Accepted: 31 March 2000     

Light-dependent phosphorylation of THRUMIN1 regulates its association with actin filaments and 14-3-3 proteins

Light-dependent chloroplast movements in leaf cells contribute to the optimization of photosynthesis. Low-light conditions induce chloroplast accumulation along periclinal cell surfaces, providing greater access to available light, whereas high light induces movement of chloroplasts to anticlinal cell surfaces, providing photodamage protection and allowing more light to reach underlying cell layers. The THRUMIN1 protein is required for normal chloroplast movements in Arabidopsis (Arabidopsis thaliana) and has been shown to localize at the plasma membrane and to undergo rapid light-dependent interactions with actin filaments through the N-terminal intrinsically disordered region (IDR). A predicted WASP-Homology 2 domain was found in the IDR but mutations in this domain did not disrupt localization of THRUMIN1:YFP to actin filaments. A series of other protein truncations and site-directed mutations of known and putative phosphorylation sites indicated that a phosphomimetic mutation (serine to aspartic acid) at position 170 disrupted localization of THRUMIN1 to actin filaments. However, the phosphomimetic mutant rescued the thrumin1-2 mutant phenotype for chloroplast movement and raises questions about the role of THRUMIN1’s interaction with actin. Mutation of serine 146 to aspartic acid also resulted in cytoplasmic localization of THRUMIN1:YFP in Nicotiana benthamiana. Mutations to a group of putative zinc-binding cysteine clusters implicate the C-terminus of THRUMIN1 in chloroplast movement. Phosphorylation-dependent association of THRUMIN1 with 14-3-3 KAPPA and OMEGA were also identified. Together, these studies provide insights into the mechanistic role of THRUMIN1 in light-dependent chloroplast movements.

Site-directed mutagenesis of THRUMIN1 revealed sites critical to its blue-light-dependent localization to actin filaments, to 14-3-3 proteins, and for its regulation of chloroplast movement.     

Regulation of H+Pumping by Plant Plasmalemma under Osmotic Stress: The Role of 14-3-3 Proteins

All higher plants have high-specific sites for binding fusicoccin (FCBS), a metabolite of the fungus Fusicoccum amygdaliDel. These sites are localized on the plasmalemma and produced by the association of the dimers of 14-3-3 proteins with the C-terminal autoinhibitory domain of H⁺-ATPase. Considering the fusicoccin binding to the plasmalemma as an index characterizing the formation of this complex, we studied the influence of osmotic stress on the interaction between 14-3-3 proteins and H⁺-ATPase in the suspension-cultured sugar beet cells and protoplasts obtained from them. An increase in the osmolarity of the extracellular medium up to 0.3 Osm was shown to enhance proton efflux from the cells by several times. The number of FCBS in isolated plasma membranes increased in parallel, whereas 14-3-3 proteins accumulated in this membrane to a lesser degree. The amount of H⁺-ATPase molecules did not change, and the ATP-hydrolase activity changed insignificantly. The data obtained indicate that osmotic stress affects H⁺-ATPase pumping in the plasmalemma through its influence on the coupling between H⁺-transport and ATP hydrolysis; 14-3-3 proteins are involved in this coupling. The interaction between the plasmalemma and the cell wall is suggested to be very important in this process.     

SIRT2 is a negative regulator of anoxia-reoxygenation tolerance via regulation of 14-3-3 zeta and BAD in H9c2 cells

Knockdown or inhibition of SIRT2 enhances biological stress-tolerance. We extend this phenotype showing that SIRT2 knockdown reduces anoxia-reoxygenation injury in H9c2 cells. Gene array analysis following SIRT2 siRNA knockdown identifies 14-3-3 zeta as the most robustly induced gene. SIRT2 knockdown evokes induction of this chaperone, facilitating cytosolic sequestration of BAD with a corresponding reduction in mitochondrial BAD localization. Concurrent siRNA against SIRT2 and 14-3-3 zeta abolishes the SIRT2-depleted cytoprotective phenotype. SIRT2 functions to moderate cellular stress-tolerance, in part, by modulating the levels of 14-3-3 zeta with the concordant control of BAD subcellular localization.     

Facilitation of glutamate,but not GABA,release in Familial Alzheimer's APP mutant Knock‐in rats with increased β‐cleavage of APP

Amyloid precursor protein (APP) modulates glutamate release via cytoplasmic and intravesicular interactions with the synaptic vesicle release machinery. The intravesicular domain, called ISVAID, contains the BACE1 cleavage site of APP. We have tested the functional significance of BACE1 processing of APP using App‐Swedish (App^s) knock‐in rats, which carry an App mutation that causes familial Alzheimer's disease (FAD) in humans. We show that in App^s rats, β‐cleavage of APP is favored over α‐cleavage. App^s rats show facilitated glutamate, but not GABA, release. Our data support the notion that APP tunes glutamate release, and that BACE1 cleavage of the ISVAID segment of APP facilitates this function. We define this phenomenon as BACE1 on APP‐dependent glutamate release (BAD‐Glu). Unsurprisingly, App^s rats show no evidence of AD‐related pathology at 15 days and 3 months of age, indicating that alterations in BAD‐Glu are not caused by pathological lesions. The evidence that a pathogenic APP mutation causes an early enhancement of BAD‐Glu suggests that alterations of BACE1 processing of APP in glutamatergic synaptic vesicles could contribute to dementia.     

Motif-Guided Identification of a Glycoside Hydrolase Family 1 α-L-Arabinofuranosidase in Bifidobacterium adolescentis

Members of glycoside hydrolase family 1 (GH1) cleave glycosidic linkages with a variety of physiological roles. Here we report a unique GH1 member encoded in the genome of Bifidobacterium adolescentis ATCC 15703. This enzyme, BAD0156, was identified from over 2,000 GH1 sequences accumulated in a database by a genome mining approach based on a motif sequence. A recombinant BAD0156 protein was characterized to confirm that this enzyme alone specifically hydrolyzes p-nitrophenyl-α-L-arabinofuranoside among the 24 p-nitrophenyl-glycosides examined. Among natural glycosides, α-1,5-linked arabino-oligosaccharides served as substrates, but arabinan, debranched arabinan, arabinoxylan, and arabinogalactan did not. A time course analysis of arabino-oligosaccharide hydrolysis indicated that BAD0156 is an exo-acting enzyme. These results suggest that BAD0156 is an α-L-arabinofuranosidase. This is the first report of a GH1 enzyme that acts specifically on arabinosides, providing information on GH1 substrate specificity.     

ZmMPK6, a Novel Maize MAP Kinase that Interacts with 14-3-3 Proteins

Although an increasing body of evidence indicates that plant MAP kinases are involved in a number of cellular processes, such as cell cycle regulation and cellular response to abiotic stresses, hormones and pathogen attack, very little is known about their biochemical properties and regulation mechanism. In this paper we report on the identification and characterization of a novel member of the MAP kinase family from maize, ZmMPK6. The amino acid sequence reveals a high degree of identity with group D plant MAP kinases. Recombinant ZmMPK6, expressed in Escherichia coli, is an active enzyme able to autophosphorylate. Remarkably, ZmMPK6 interacts in vitro with GF14-6, a maize 14-3-3 protein and the interaction is dependent on autophosphorylation. The interacting domain of ZmMPK6 is on the C-terminus and is comprised between amino acid 337 and amino acid 467. Our results represent the first evidence of an interaction between a plant MAP kinase and a 14-3-3 protein. Possible functional roles of this association in vivo are discussed.     

14-3-3 regulates the G2/M transition in the basidiomycete Ustilago maydis

14-3-3 proteins are a family of highly conserved polypeptides that function as small adaptors that facilitate a diverse array of cellular processes by binding phosphorylated target proteins. One of these processes is the regulation of the cell cycle. Here we characterized the role of Bmh1, a 14-3-3 protein, in the cell cycle regulation of the fungus Ustilago maydis. We found that this protein is essential in U. maydis and that it has roles during the G2/M transition in this organism. The function of 14-3-3 in U. maydis seems to mirror the proposed role for this protein during Schizosaccharomyces pombe cell cycle regulation. We provided evidence that in U. maydis 14-3-3 protein binds to the mitotic regulator Cdc25. Comparison of the roles of 14-3-3 during cell cycle regulation in other fungal system let us to discuss the connections between morphogenesis, cell cycle regulation and the evolutionary role of 14-3-3 proteins in fungi.     

Continuous preparation of (S)-3-hydroxy-3-phenylpropionate by asymmetric reduction of 3-oxo-3-phenylpropionic acid ethyl ester with <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> CGMCC No.2266 in a membrane reactor

In this study, (S)-3-hydroxy-3-phenylpropionate was prepared continuously by coupling microbial transformation and membrane separation. The effect of several factors on membrane flux, reactor capacity, and reaction conversion were investigated. A kinetic model of the continuous reduction process was also developed. The appropriate molecular weight cut-off of the ultrafiltration membrane was 30 kDa. The reactor capacity reached a maximum of 0.136/h at a biomass concentration and membrane flux of 86 g/L (dry weight/reaction volume) and 20 mL/h, respectively. The (S)-3-hydroxy-3-phenylpropionate yield was 3.68 mmol/L/day after continuous reduction over seven days. The enantiometric excess of (S)-3-hydroxy-3-phenylpropionate reached above 99.5%. The kinetic constants of continuous reduction were as follows: r _m = 3.00 × 10⁻³ mol/L/h, k _cat = 3.49 × 10⁻⁴ mol/L/h, k ₁ = 3.09 × 10⁻² mol/L, and k ₂ = 5.00 × 10⁻⁷ mol/L. The kinetic model was in good agreement with the experimental data obtained during continuous reduction. Compared with batch reduction, continuous reduction can significantly improve the catalytic efficiency of microbial cells and increase the reactor capacity.     

菠菜14-3-3蛋白基因的克隆及硝酸盐胁迫下的表达分析

利用RT-PCR和RACE技术,从菠菜中首次获得了14-3-3蛋白基因的全长cDNA序列(GenBank登录号JX952165),命名为So14-3-3.该基因全长1 166 bp,开放阅读框801 bp,编码266个氨基酸.序列比对发现So143 3蛋白与其他植物14-3-3蛋白氨基酸序列一致性高达77.6％～84.7％.半定量RT-PCR表明,随NO3-胁迫处理时间的延长和浓度的增加,菠菜根和叶中So14-3-3基因的表达增强.实验构建了pGEX4T-So14-3-3原核表达载体,并通过IPTG诱导后获得分子量约为56 kD的蛋白.进一步的蛋白质印迹检测结果表明,随着NO3处理时间的延长和浓度的增加,So14-3-3蛋白表达也增加.该实验结果为进一步研究So14 3-3蛋白功能提供了基本的实验基础.     

14-3-3 proteins: eukaryotic regulatory proteins with many functions

The enigmatically named 14-3-3 proteins have been the subject of considerable attention in recent years since they have been implicated in the regulation of diverse physiological processes, in eukaryotes ranging from slime moulds to higher plants. In plants they have roles in the regulation of the plasma membrane H⁺-ATPase and nitrate reductase, among others. Regulation of target proteins is achieved through binding of 14-3-3 to short, often phosphorylated motifs in the target, resulting either in its activation (e.g. H⁺-ATPase), inactivation (e.g. nitrate reductase) or translocation (although this function of 14-3-3 proteins has yet to be demonstrated in plants). The native 14-3-3 proteins are homo- or heterodimers and, as each monomer has a binding site, a dimer can potentially bind two targets, promoting their association. Alternatively, target proteins may have more than one 14-3-3-binding site. In this mini review, we present a synthesis of recent results from plant 14-3-3 research and, with reference to known 14-3-3-binding motifs, suggest further subjects for research.