Oligomerization of Chicken Acetylcholinesterase Does Not Require Intersubunit Disulfide Bonds

Abstract: Acetylcholinesterase (AChE) is secreted from muscle and nerve cells and associates as multimers through intermolecular covalent and noncovalent bonds. The amino acid sequence of the C-terminus is thought to play an important role in these interactions. We generated mutants in the C-terminus of the catalytic T-subunit of chicken AChE to determine the importance of this region to oligomerization and to the amphipathic character of the protein. Wild-type recombinant chicken AChE secreted from human embryonic kidney 293 cells was assembled into dimers and tetramers exclusively. Mutants lacking the C-terminal Cys⁷⁶⁴, the only cysteine involved in interchain disulfide bonds, showed lower but significant levels of the secreted dimeric and tetrameric forms. A truncated mutant, lacking the C-terminal 39 amino acids, exhibited a severe decrease in content of the multimeric forms, yet small amounts of the dimer were detectable. The amphipathic character was dependent on the state of oligomerization. When analyzed by sucrose gradients, the sedimentation of tetramers was not affected by detergent, but monomers and dimers sedimented more slowly in the presence of detergent. Most of the recombinant wild-type enzyme, shown to be dimeric and tetrameric by sedimentation analysis, was monomeric when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions, indicating that much of the secreted oligomeric AChE was not disulfide bonded. These data suggest that disulfide bonding of Cys⁷⁶⁴ is not required for the catalytic subunit of chicken AChE to form oligomers and that regions outside of the C-terminus contribute to the hydrophobic interactions that are important for stabilizing the oligomeric forms.     

Ethylene-Stimulated Nutations Do Not Require ETR1 Receptor Histidine Kinase Activity

Ethylene influences the growth and development of plants through the action of receptors that have homology to bacterial two-component receptors. In bacteria these receptors function via autophosphorylation of a His residue in the kinase domain followed by phosphotransfer to a conserved Asp residue in a response regulator protein. In Arabidopsis, two of the five receptor isoforms are capable of His kinase activity. However, the role of His kinase activity and phosphotransfer is unclear in ethylene signaling. A previous study showed that ethylene stimulates nutations of the hypocotyl in etiolated Arabidopsis seedlings that are dependent on the ETR1 receptor isoform. The ETR1 receptor is the only isoform in Arabidopsis that contains both a functional His kinase domain and a receiver domain for phosphotransfer. Therefore, we examined the role that ETR1 His kinase activity and phosphotransfer plays in ethylene-stimulated nutations.Key Words: ethylene, nutations, signal transduction, receptors, histidine kinase, phosphotransfer, two component signallingThe gaseous plant hormone ethylene has a role in a variety of physiological events in higher plants such as seed germination, abscission, senescence, fruit ripening, and growth regulation.¹ In etiolated Arabidopsis seedlings, ethylene causes reduced growth of the hypocotyl and root, increased radial expansion of the hypocotyl, and increased tightening of the apical hook.^2,3Previous studies have identified components in the ethylene signaling pathway and led to an inverse-agonist model for signal transduction.^4,5 According to this model, responses to ethylene are mediated by a family of five receptors (ETR1, ERS1, ETR2, EIN4, ERS2) in Arabidopsis that have homology to bacterial two-component receptors.^6–9 In bacterial systems, two-component receptors transduce signal via the autophosphorylation of a His residue in the kinase domain, followed by the transfer of phosphate to a conserved Asp residue in the receiver domain of a response regulator protein.¹⁰ The ethylene receptors of plants can be divided into two subfamilies based on sequence homology in the ethylene-binding domains.¹¹ ETR1 and ERS1 belong to subfamily I, contain all amino acid residues needed for His kinase activity,^6,12 and show His kinase activity in vitro.^13,14 ETR2, EIN4, and ERS2 belong to subfamily II, contain degenerate His kinase domains^7,9 and have Ser/Thr kinase activity in vitro.¹⁴ ERS1 shows both His and Ser/Thr kinase activities in vitro depending on the assay conditions used.¹⁴ While the kinase domain of ETR1 appears to be required for signaling,¹⁵ kinase activity is not.^15–17 It is unclear whether or not histidine kinase activity is involved in ethylene signaling, although, this activity might be involved in growth recovery after ethylene removal.¹⁷Recently, high-resolution, time-lapse imaging revealed that prolonged treatment with ethylene stimulates nutational bending of etiolated Arabidopsis hypocotyls.¹⁸ Nutations are oscillatory bending movements caused by localized differential growth¹⁹ that were originally termed “circumnutations”.²⁰ Nutations have been posited to be important for seedlings to penetrate through the soil²⁰ and thus could be critical for seedling survival. In support of this hypothesis, nutations of rice roots have been reported to increase soil penetration.²¹Mutational analysis revealed that many of the known ethylene signaling components including CTR1, EIN2, EIN3 and EIL1 are involved in signaling leading to ethylene-stimulated nutations.¹⁸ Surprisingly, loss-of-function mutations in ETR1 eliminated ethylene-stimulated nutations while combinatorial loss-of-function mutations in the other four receptor isoforms led to constitutive nutations in air.¹⁸ These results support a model where all the receptors are involved in ethylene-stimulated nutations but the ETR1 receptor is required for and has a contrasting role from the other receptor isoforms in this nutation phenotype. Since the ETR1 receptor is the only receptor isoform that contains both a functional His-kinase domain and a receiver domain,^6,13,14 the roles of His kinase activity and phosphorelay in the nutation phenotype were examined in the current study.Previous work showed that the nutation phenotype in etr1-7 loss-of-function mutants could be rescued with a wild-type, genomic ETR1 transgene.¹⁸ Etr1-7 mutants transformed with a kinase-inactivated genomic ETR1 transgene (gETR1 (G₂)) where the two conserved glycines in the G₂ box of the histidine kinase domain (G545, G547) were changed to alanines were examined to determine if ETR1 His kinase activity is required for ethylene-stimulated nutations. This construct lacks histidine autophosphorylation in vitro.²² Figure 1 shows that ethylene stimulates nutations in etr1-7 gETR1(G₂) seedlings. The period of these nutations was 4.7 ± 1.5 h which is similar to values obtained previously for wild-type seedlings (4.7 ± 1h) and somewhat longer than etr1-7 seedlings transformed with wild-type, genomic ETR1 (3.2 ± 0.6 h). However, the amplitude of these nutations (3.7 ± 1.0°) was approximately half that of nutations previously observed in wild-type seedlings (9.1 ± 6.0°) as well as etr1-7 seedlings transformed with wild-type, genomic ETR1 (8.2 ± 3.6°). This suggests that ETR1 histidine kinase activity is not required for ethylene-stimulated nutations but might have a role in modulating nutation amplitudes.Open in a separate windowFigure 1Ethylene stimulates nutations of etr1-7 seedlings transformed with a kinase-inactivated ETR1 transgene. The hypocotyl angles for four etr1-7 mutants transformed with a kinase-inactivated genomic ETR1 transgene (gETR1(G₂)) are shown. Transformants were obtained from Eric Schaller and have been described previously.²² In this and the following figure, etiolated Arabidopsis seedlings were imaged from the side at 15 min intervals while growing along a vertically orientated agar plate and the hypocotyl angle measured as described previously.¹⁸ Black and gray lines are used to help distinguish the movements of individual seedlings. All seedlings were grown in the presence of 5 µM AVG to block biosynthesis of ethylene by the seedlings. Seedlings were grown in air for 2 h prior to treatment with 10 µL L⁻¹ ethylene (Open in a separate window).To determine whether phosphotransfer through the receiver domain of ETR1 is required for the nutation phenotype, seedlings deficient in ethylene receptor isoforms containing a receiver domain (ETR1, ETR2, EIN4) were transformed with a mutant ETR1 transgene lacking the conserved Asp₆₅₉ required for phosphotransfer (getr1-[D]). Previous work showed that etr1-6 etr2-3 ein4-4 triple loss-of-function mutant seedlings failed to nutate and this nutation phenotype could be rescued when these mutants were transformed with wild-type, genomic ETR1 transgene.¹⁸ Similarly, transformation of the etr1-6 etr2-3 ein4-4 triple mutants with getr1-[D] rescued the nutation phenotype in most seedlings observed (Fig. 2). However, some seedlings (four of the eleven observed) failed to nutate. The reason for this variable rescue is unclear but could reflect differences in expression levels of the mutant transgene in individual plants. Alternatively, this variable rescue could reflect functional differences between the mutant and wild-type transgene suggesting a modulating role for phosphotransfer through the receiver domain of ETR1. Two independent lines were observed with similar results. Of those that did nutate, the period of nutations was 5.0 ± 1.2 h and the amplitude 7.6 ± 3.8° which is similar to values obtained previously for wild-type plants as well as plants transformed with a wild-type, genomic ETR1 transgene.¹⁸Open in a separate windowFigure 2Ethylene stimulates nutations of etr1-6 etr2-3 ein4-4 seedlings transformed with an ETR1 transgene mutated at Asp₆₅₉. The hypocotyl angles from seven etr1-6 etr2-3 ein4-4 triple mutants transformed with an ETR1 transgene mutated at Asp₆₅₉ (getr1[D]) are shown in two panels. One seedling in (A) (black) had no measurable nutations while one in (B) (black) had very small nutations.Conclusions from this and the previous study are that the ETR1 receptor has a unique role in ethylene-stimulated nutations. However, this role does not require either histidine kinase activity or phosphotransfer through the receiver domain of ETR1.     

N-Terminal Protein Acylation Confers Localization to Cholesterol, Sphingolipid-enriched Membranes But Not to Lipid Rafts/Caveolae

When variably fatty acylated N-terminal amino acid sequences were appended to a green fluorescent reporter protein (GFP), chimeric GFPs were localized to different membranes in a fatty acylation-dependent manner. To explore the mechanism of localization, the properties of acceptor membranes and their interaction with acylated chimeric GFPs were analyzed in COS-7 cells. Myristoylated GFPs containing a palmitoylated or polybasic region colocalized with cholesterol and ganglioside GM(1), but not with caveolin, at the plasma membrane and endosomes. A dipalmitoylated GFP chimera colocalized with cholesterol and GM(1) at the plasma membrane and with caveolin in the Golgi region. Acylated GFP chimeras did not cofractionate with low-density caveolin-rich lipid rafts prepared with Triton X-100 or detergent-free methods. All GFP chimeras, but not full-length p62(c-yes) and caveolin, were readily solubilized from membranes with various detergents. These data suggest that, although N-terminal acylation can bring GFP to cholesterol and sphingolipid-enriched membranes, protein-protein interactions are required to localize a given protein to detergent-resistant membranes or caveolin-rich membranes. In addition to restricting acceptor membrane localization, N-terminal fatty acylation could represent an efficient means to enrich the concentration of signaling proteins in the vicinity of detergent-resistant membranes and facilitate protein-protein interactions mediating transfer to a detergent-resistant lipid raft core.     

Bacteriophage MS2 Lysis Protein Does Not Require Coat Protein to Mediate Cell Lysis

We inserted all but the extreme 5' end of a DNA copy of the bacteriophage MS2 lysis gene downstream of a lac-induced promoter on a multicopy plasmid. Upon induction, cells harboring this plasmid began to lyse, showing that phage coat protein is not required for the lytic process itself.     

Sphingolipid Domains in the Plasma Membranes of Fibroblasts Are Not Enriched with Cholesterol

The plasma membranes of mammalian cells are widely expected to contain domains that are enriched with cholesterol and sphingolipids. In this work, we have used high-resolution secondary ion mass spectrometry to directly map the distributions of isotope-labeled cholesterol and sphingolipids in the plasma membranes of intact fibroblast cells. Although acute cholesterol depletion reduced sphingolipid domain abundance, cholesterol was evenly distributed throughout the plasma membrane and was not enriched within the sphingolipid domains. Thus, we rule out favorable cholesterol-sphingolipid interactions as dictating plasma membrane organization in fibroblast cells. Because the sphingolipid domains are disrupted by drugs that depolymerize the cells actin cytoskeleton, cholesterol must instead affect the sphingolipid organization via an indirect mechanism that involves the cytoskeleton.     

Smy1p, a Kinesin-related Protein That Does Not Require Microtubules

Abstract. We have previously reported that a defect in Myo2p, a myosin in budding yeast (Saccharomyces cerevisiae), can be partially corrected by overexpression of Smy1p, which is by sequence a kinesin-related protein (Lillie, S.H., and S.S. Brown. 1992. Nature. 356:358– 361). Such a functional link between putative actin- and microtubule-based motors is surprising, so here we have tested the prediction that Smy1p indeed acts as a microtubule-based motor. Unexpectedly, we found that abolition of microtubules by nocodazole does not interfere with the ability of Smy1p to correct the mutant Myo2p defect, nor does it interfere with the ability of Smy1p to localize properly. In addition, other perturbations of microtubules, such as treatment with benomyl or introduction of tubulin mutations, do not exacerbate the Myo2p defect. Furthermore, a mutation in SMY1 strongly predicted to destroy motor activity does not destroy Smy1p function. We have also observed a genetic interaction between SMY1 and two of the late SEC mutations, sec2 and sec4. This indicates that Smy1p can play a role even when Myo2p is wild type, and that Smy1p acts at a specific step of the late secretory pathway. We conclude that Smy1p does not act as a microtubule-based motor to localize properly or to compensate for defective Myo2p, but that it must instead act in some novel way.     

Cholesterol Esterase Bound to Intestinal Brush Border Membranes Does Not Accelerate Incorporation of Micellar Cholesterol into Absorptive Cells

We confirmed that cholesterol esterase accelerated the incorporation of unesterified cholesterol solubilized in bile salt micelles into differentiated Caco-2 cells under various experimental conditions. Rat pancreatic juice and bovine cholesterol esterase increased the incorporation of micellar cholesterol into rat intestinal brush border membranes. The incorporation of micellar cholesterol was not changed in the brush border membranes enriched in and depleted of cholesterol esterase. The results suggest that the accelerated incorporation of micellar cholesterol by cholesterol esterase into absorptive cells is not mediated by the enzyme bound to the brush border membranes.     

Oligomerization of DHHC Protein S-Acyltransferases

The formation of dimers or higher-order oligomers is a property of numerous integral membrane proteins, including ion channels, transporters, and receptors. In this study, we examined whether members of the DHHC-S-acyltransferase family oligomerize in intact cells and in vitro. DHHC-S-acyltransferases are integral membrane proteins that catalyze the addition of palmitate to cysteine residues on proteins at the cytoplasmic face of cell membranes. Bioluminescence resonance energy transfer (BRET) experiments revealed that DHHC2 or DHHC3 (Golgi-specific DHHC zinc finger protein (GODZ)) self-associate when expressed in HEK-293 cells. Homomultimer formation was confirmed by coimmunoprecipitation. Purified DHHC3 resolved predominately as a monomer and dimer on blue native polyacrylamide gels. In intact cells and in vitro, catalytically inactive DHHC proteins displayed a greater propensity to form dimers. BRET signals were higher for the catalytically inactive DHHC2 or DHHC3 than their wild-type counterparts. DHHC3 BRET in cell membranes was decreased by the addition of its lipid substrate palmitoyl-CoA, a treatment that results in autoacylation of the enzyme. Enzyme activity of a covalently linked DHHC3 dimer was less than that of the monomeric form, suggesting that enzyme activity may be modulated by the oligomerization status of the protein.     

Glucose Signaling in Yeast Is Partially Mimicked by Galactose and Does Not Require the Tps1 Protein

Glucose produces multiple effects inSaccharomyces cerevisiae,as it controls the expression of many genes and the activity of various enzymes. However, the elements involved in glucose signaling are not well characterized. In this work the capacity of galactose to bring about the same effects than glucose has been assessed. Galactose mimics glucose only partially; it is suggested that it does not interact with a “sensor” in the plasma membrane and that it produces a weaker intracellular signal than glucose. To examine whether trehalose-6P synthase (Tps1) is required to transduce the glucose signal, we have constructed atps1 hxk2/tps1 HXK2strain which, at difference of atps1strain, grows on glucose, and, at difference of atps1 hxk2strain, still possess the Hxk2 protein, possibly involved in glucose repression. From the response of this strain to glucose, we conclude that Tps1 does not play a prominent role in glucose signaling.     

Segregated Phases in Pulmonary Surfactant Membranes Do Not Show Coexistence of Lipid Populations with Differentiated Dynamic Properties

The composition of pulmonary surfactant membranes and films has evolved to support a complex lateral structure, including segregation of ordered/disordered phases maintained up to physiological temperatures. In this study, we have analyzed the temperature-dependent dynamic properties of native surfactant membranes and membranes reconstituted from two surfactant hydrophobic fractions (i.e., all the lipids plus the hydrophobic proteins SP-B and SP-C, or only the total lipid fraction). These preparations show micrometer-sized fluid ordered/disordered phase coexistence, associated with a broad endothermic transition ending close to 37°C. However, both types of membrane exhibit uniform lipid mobility when analyzed by electron paramagnetic resonance with different spin-labeled phospholipids. A similar feature is observed with pulse-field gradient NMR experiments on oriented membranes reconstituted from the two types of surfactant hydrophobic extract. These latter results suggest that lipid dynamics are similar in the coexisting fluid phases observed by fluorescence microscopy. Additionally, it is found that surfactant proteins significantly reduce the average intramolecular lipid mobility and translational diffusion of phospholipids in the membranes, and that removal of cholesterol has a profound impact on both the lateral structure and dynamics of surfactant lipid membranes. We believe that the particular lipid composition of surfactant imposes a highly dynamic framework on the membrane structure, as well as maintains a lateral organization that is poised at the edge of critical transitions occurring under physiological conditions.     

The Structural Basis of Cholesterol Accessibility in Membranes

Although the majority of free cellular cholesterol is present in the plasma membrane, cholesterol homeostasis is principally regulated through sterol-sensing proteins that reside in the cholesterol-poor endoplasmic reticulum (ER). In response to acute cholesterol loading or depletion, there is rapid equilibration between the ER and plasma membrane cholesterol pools, suggesting a biophysical model in which the availability of plasma membrane cholesterol for trafficking to internal membranes modulates ER membrane behavior. Previous studies have predominantly examined cholesterol availability in terms of binding to extramembrane acceptors, but have provided limited insight into the structural changes underlying cholesterol activation. In this study, we use both molecular dynamics simulations and experimental membrane systems to examine the behavior of cholesterol in membrane bilayers. We find that cholesterol depth within the bilayer provides a reasonable structural metric for cholesterol availability and that this is correlated with cholesterol-acceptor binding. Further, the distribution of cholesterol availability in our simulations is continuous rather than divided into distinct available and unavailable pools. This data provide support for a revised cholesterol activation model in which activation is driven not by saturation of membrane-cholesterol interactions but rather by bulk membrane remodeling that reduces membrane-cholesterol affinity.     

The Structural Basis of Cholesterol Accessibility in Membranes

Although the majority of free cellular cholesterol is present in the plasma membrane, cholesterol homeostasis is principally regulated through sterol-sensing proteins that reside in the cholesterol-poor endoplasmic reticulum (ER). In response to acute cholesterol loading or depletion, there is rapid equilibration between the ER and plasma membrane cholesterol pools, suggesting a biophysical model in which the availability of plasma membrane cholesterol for trafficking to internal membranes modulates ER membrane behavior. Previous studies have predominantly examined cholesterol availability in terms of binding to extramembrane acceptors, but have provided limited insight into the structural changes underlying cholesterol activation. In this study, we use both molecular dynamics simulations and experimental membrane systems to examine the behavior of cholesterol in membrane bilayers. We find that cholesterol depth within the bilayer provides a reasonable structural metric for cholesterol availability and that this is correlated with cholesterol-acceptor binding. Further, the distribution of cholesterol availability in our simulations is continuous rather than divided into distinct available and unavailable pools. This data provide support for a revised cholesterol activation model in which activation is driven not by saturation of membrane-cholesterol interactions but rather by bulk membrane remodeling that reduces membrane-cholesterol affinity.     

Cholesterol Induces Specific Spatial and Orientational Order in Cholesterol/Phospholipid Membranes

Background

In lipid bilayers, cholesterol facilitates the formation of the liquid-ordered phase and enables the formation of laterally ordered structures such as lipid rafts. While these domains have an important role in a variety of cellular processes, the precise atomic-level mechanisms responsible for cholesterol''s specific ordering and packing capability have remained unresolved.

Methodology/Principal Findings

Our atomic-scale molecular dynamics simulations reveal that this ordering and the associated packing effects in membranes largely result from cholesterol''s molecular structure, which differentiates cholesterol from other sterols. We find that cholesterol molecules prefer to be located in the second coordination shell, avoiding direct cholesterol-cholesterol contacts, and form a three-fold symmetric arrangement with proximal cholesterol molecules. At larger distances, the lateral three-fold organization is broken by thermal fluctuations. For other sterols having less structural asymmetry, the three-fold arrangement is considerably lost.

Conclusions/Significance

We conclude that cholesterol molecules act collectively in lipid membranes. This is the main reason why the liquid-ordered phase only emerges for Chol concentrations well above 10 mol% where the collective self-organization of Chol molecules emerges spontaneously. The collective ordering process requires specific molecular-scale features that explain why different sterols have very different membrane ordering properties: the three-fold symmetry in the Chol-Chol organization arises from the cholesterol off-plane methyl groups allowing the identification of raft-promoting sterols from those that do not promote rafts.     

Kaede for Detection of Protein Oligomerization

Photoconvertible fluorescent proteins such as Kaede are routinely used for tracking proteins, organelles, and whole cells. Kaede was the first identified photoconvertible fluorescent protein and has since become the most commonly used photoconvertible fluorescent protein in vertebrates. Kaede can be irreversibly converted from a green to a red fluo- rescent form upon UV/blue light irradiation and fluorescence of each form can be isolated separately by appropriate filter sets. Spectral properties of the Kaede forms allow F6rster resonance energy transfer （FRET） from the green form as donor to the red form as acceptor. As a sample containing oligomerized Kaede-containing proteins is exposed to UV or blue light, FRET first increases as green Kaede is converted to red and then decreases as the green donor becomes depleted. Thus, FRET information is potentially obtained from a number of independent measurements taken as photoconversion proceeds. We demonstrate here the application of this approach to detect homo-aggregation and conformational dynamics of plant pro- tein constructs. Structural alterations of 2-cys peroxiredoxin-Kaede were successfully detected depending on the redox state in living plant cells. Photoconversion was performed gradually and donor emission, acceptor emission, and FRET-derived sensitized acceptor emission were measured at each step of conversion. Since photoconvertible proteins have not been rou- tinely used in plants, two plasmids have been designed to facilitate plant applications. The plasmids allow either transient expression of Kaede-containing protein constructs in plant cells or Gateway cloning and stable transformation of plants.     

Identification of Two Distinct Populations of Protein Kinase C in Rat Brain Membranes

The regulatory enzyme protein kinase C (PKC) is proposed to be activated on its translocation from the cytosol to the membrane. However, a portion of the native activity is always associated with the membrane fraction. Using a noninvasive procedure to extract this endogenous activity from rat brain membranes, it has been possible to characterize the activity in a partially purified reconstituted system bearing resemblance to the in vivo system. Two subpopulations of membrane-associated PKC were identified and characterized at the level of activation, inhibition, and isozyme immunologic characteristics and chromatographic properties. One peak had properties similar to those of cytosolic PKC, whereas the second population, extracted as protein-lipid complexes, had considerable constitutive activity that could be stimulated further on addition of PKC activators. This latter activity was relatively resistant to staurosporine inhibition and phorbol ester treatment, but it phosphorylated the exogenous PKC substrates, histone 1 and the epidermal growth factor receptor peptide KTRLRR. The constitutive activity was totally dependent on its endogenous associated lipids coextracted by the solubilization procedure. The ratio between these two populations was ontogenetically regulated and modulated by phorbol ester treatment, suggesting that different PKC populations may serve unique functions in the rat brain regulated by the lipid environment. Analyses of the phospholipids extracted in these protein-lipid complexes showed differences in the major classes correlating to age. However, apart from a markedly lower cholesterol content in these complexes, no direct relationship between a specific lipid composition and the amount of constitutive PKC activity was evident.     

Generation of Digital Responses in Stress Sensors

Ultrasensitivity, hysteresis (a form of biochemical memory), and all-or-none (digital) responses are important signaling properties for the control of irreversible processes and are well characterized in the c-Jun N-terminal kinase (JNK) system using Xenopus oocytes. Our aim was to study these properties in the AMP-activated protein kinase (AMPK) signaling system under stress conditions that could engage a cell death program, and compare them to the JNK responses. After characterization of Xenopus AMPK, we show here that the response to antimycin (nonapoptotic) was slightly cooperative and graded (analog) in individual oocytes, whereas the response to sorbitol (which induced cytochrome c release and caspase activation) was ultrasensitive, digital in single cells, and without hysteresis, hallmarks of a monostable system. Moreover, initial graded responses of AMPK and JNK turned into digital during a critical period for the execution of the cell death program, although single cell analysis did not show complete correlation between AMPK or JNK activation and cytochrome c release. We propose a model where the life or death decision in the cell is made by integration of multiple digital signals from stress sensors.The energy level in a cell/organism is probably the most remarkable vital constant and must be tightly regulated or cell death programs will otherwise be engaged. The AMP-activated protein kinase (AMPK)² is an energy sensor (activated by a high AMP/ATP ratio) and a homeostatic regulator of cellular ATP levels, taking central stage in orchestrating cell metabolism (1).Stress sensors should have, to function efficiently, ultrasensitive properties to respond to small changes in the important parameters of cell survival. In addition, all-or-none (digital) responses, at a single cell level, and sustained activation when the stimulus has disappeared (also named hysteresis) may be important properties of stress sensors in regulating cell death (2).Mammalian AMPK is a heterotrimeric complex consisting of a catalytic α-subunit and regulatory β- and γ-subunits. AMPK is activated by AMP in two ways, both antagonized by high concentrations of ATP. Binding of AMP to the γ-subunit induces: 1) allosteric activation that can account for a 5-fold increase in the activity, and 2) phosphorylation of the α-subunit at Thr-172 (in human), which is essential for activity and causes a much marked activation (3). The combination of the two effects causes a >1000-fold increase in kinase activity (4). The protein kinases LKB1 and CaMKKα/β have been reported to phosphorylate the AMPK α-subunit at Thr-172 (1, 5). This signaling system is a clear example of multistep sensitivity, which arises when a signal molecule (AMP) affects more than one step in a cascade (allosteric activation and phosphorylation). In addition, the AMPK upstream kinases have a very low K_m for AMPK (6), a phenomenon referred to as zero-order ultrasensitivity (7). In practice, the ultrasensitivity of a system is reflected in a stimulus/response curve with a very steep upstroke. These properties of the AMPK signaling system may be very important to maintain energy levels in the cell within narrow limits and can be useful for filtering noise. Although there is one report supporting that AMPK is an ultrasensitive system (6), no further research has considered its significance in the control of cell death as well as other important signaling properties associated with ultrasensitivity, such as hysteresis and all-or-none responses.It is well known that ultrasensitive systems embedded in a positive feedback loop have the potential to exhibit bistable behavior, switching between discrete stable steady states without being able to rest in intermediate states (8, 9). The three hallmarks of a bistable system are: 1) strong ultrasensitivity, 2) digital response at the individual cell level, and 3) hysteresis. Examples of such systems are JNK and the mitogen-activated protein kinase (MAPK) cascades, implicated in oocyte maturation and maybe in apoptosis (2, 10). The hallmarks of a monostable system are the same, but without hysteresis.Because AMPK is an energy sensor, it seems reasonable that the AMPK cascade would transmit at the individual cell level graded (analog) information about the energy status of the cell. In fact, AMPK can be considered part of a negative feedback loop because AMPK activation (by a high AMP/ATP ratio) regulates multiple steps in metabolism to restore ATP levels in the cell, which in turn down-regulates AMPK activity. However, sustained activation of AMPK by some stimuli has been considered a pro-apoptotic signal (11, 12). Because cell death is an all-or-none irreversible process, it may be more appropriate for the AMPK cascade in this situation to exhibit a digital (all-or-none) response to trigger cell death.Here, we analyze the sensitivity, the grade of hysteresis, and the single cell response of the AMPK system under different stress conditions that could engage a cell death program. We performed experiments with Xenopus oocytes, a suitable model for discerning the character of a signaling response, as has been previously reported for JNK and MAPK cascades. Based on our results, we propose that commitment to cell death will occur after integration of multiple digital responses from stress protein kinases.