Engagement of phospholipid scramblase 1 in activated cells: implication for phosphatidylserine externalization and exocytosis

Phosphatidylserine (PS) in quiescent cells is predominantly confined to the inner leaflet of the plasma membrane. Externalization of PS is a marker of apoptosis, exocytosis, and some nonapoptotic activation events. It has been proposed that PS externalization is regulated by the activity of PLSCR1 (phospholipid scramblase 1), a Ca(2+)-dependent endofacial plasma membrane protein, which is tyrosine-phosphorylated in activated cells. It is, however, unclear how the phosphorylation of PLSCR1 is related to its membrane topography, PS externalization, and exocytosis. Using rat basophilic leukemia cells as a model, we show that nonapoptotic PS externalization induced through the high affinity IgE receptor (FcepsilonRI) or the glycosylphosphatidylinositol-anchored protein Thy-1 does not correlate with enhanced tyrosine phosphorylation of PLSCR1. In addition, PS externalization in FcepsilonRI- or Thy-1-activated cells is not associated with alterations of PLSCR1 fine topography as detected by electron microscopy on isolated plasma membrane sheets. In contrast, activation by calcium ionophore A23187 induces changes in the cellular distribution of PLSCR1. We also show for the first time that in pervanadate-activated cells, exocytosis occurs even in the absence of PS externalization. Finally, we document here that tyrosine-phosphorylated PLSCR1 is preferentially located in detergent-insoluble membranes, suggesting its involvement in the formation of membrane-bound signaling assemblies. The combined data indicate that changes in the topography of PLSCR1 and its tyrosine phosphorylation, PS externalization, and exocytosis are independent phenomena that could be distinguished by employing specific conditions of activation.     

Plasma membrane phospholipid scramblase 1 promotes EGF-dependent activation of c-Src through the epidermal growth factor receptor

Phospholipid scramblase (PLSCR1) is a multiply palmitoylated, calcium-binding endofacial membrane protein proposed to mediate transbilayer movement of plasma membrane phospholipids. PLSCR1 is a component of membrane lipid rafts and has been shown to both physically and functionally interact with activated epidermal growth factor (EGF) receptors and other raft-associated cell surface receptors. Cell stimulation by EGF results in Tyr phosphorylation of PLSCR1, its association with both Shc and EGF receptors, and rapid cycling of PLSCR1 between plasma membrane and endosomal compartments. We now report evidence that upon EGF stimulation, PLSCR1 is phosphorylated by c-Src, within the tandem repeat sequence 68VYNQPVYNQP77. The in vivo interaction between PLSCR1 and Shc requires the Src-mediated phosphorylation on tyrosines 69 and 74. In in vitro pull down studies, phosphorylated PLSCR1 was found to bind directly to Shc through the phosphotyrosine binding domain. Consistent with the potential role of PLSCR1 in growth factor signaling pathways, granulocyte precursors derived from mice deficient in PLSCR1 show impaired proliferation and maturation under cytokine stimulation. Using PLSCR1-/- embryonic fibroblasts and kidney epithelial cells, we now demonstrate that deletion of PLSCR1 from the plasma membrane reduces the activation of c-Src by EGF, implying that PLSCR1 normally facilitates receptor-dependent activation of this kinase. We propose that PLSCR1, through its interaction with Shc, promotes Src kinase activation through the EGF receptor.     

Aminophospholipid translocase TAT-1 promotes phosphatidylserine exposure during C. elegans apoptosis

Phospholipids are distributed asymmetrically across the plasma-membrane bilayer of eukaryotic cells: Phosphatidylserine (PS), phosphatidylethanolamine, and phosphoinositides are predominantly restricted to the inner leaflet, whereas phophatidylcholine and sphingolipids are enriched on the outer leaflet [1, 2]. Exposure of PS on the cell surface is a conserved feature of apoptosis and plays an important role in promoting the clearance of apoptotic cells by phagocytosis [3]. However, the molecular mechanism that drives PS exposure remains mysterious. To address this issue, we studied cell-surface changes during apoptosis in the nematode C. elegans. Here, we show that PS exposure can readily be detected on apoptotic C. elegans cells. We generated a transgenic strain expressing a GFP::Annexin V reporter to screen for genes required for this process. Although none of the known engulfment genes was required, RNAi knockdown of the putative aminophospholipid transporter gene tat-1 abrogated PS exposure on apoptotic cells. tat-1(RNAi) also reduced the efficiency of cell-corpse clearance, suggesting that PS exposure acts as an "eat-me" signal in worms. We propose that tat-1 homologs might also play an important role in PS exposure in mammals.     

ABC1 promotes engulfment of apoptotic cells and transbilayer redistribution of phosphatidylserine

ATP-binding-cassette transporter 1 (ABC1) has been implicated in processes related to membrane-lipid turnover. Here, using in vivo loss-of-function and in vitro gain-of-function models, we show that ABC1 promotes Ca2+-induced exposure of phosphatidylserine at the membrane, as determined by a prothrombinase assay, membrane microvesiculation and measurement of transbilayer redistribution of spin-labelled phospholipids. That ABC1 promotes engulfment of dead cells is shown by the impaired ability of ABC1-deficient macrophages to engulf apoptotic preys and by the acquisition of phagocytic behaviour by ABC1 transfectants. Release of membrane phospholipids and cholesterol to apo-AI, the protein core of the cholesterol-shuttling high-density lipoprotein (HDL) particle, is also ABC1-dependent. We propose that both the efficiency of apoptotic-cell engulfment and the efflux of cellular lipids depend on ABC1-induced perturbation of membrane phosphatidylserine turnover. Transient local exposure of anionic phospholipids in the outer membrane leaflet may be sufficient to alter the general properties of the membrane and thus influence discrete physiological functions.     

Apical phosphatidylserine externalization in auditory hair cells

In hair cells of the inner ear, phosphatidylserine (PS), detected with fluorescent annexin V labeling, was rapidly exposed on the external leaflet of apical plasma membranes upon dissection of the organ of Corti. PS externalization was unchanged by caspase inhibition, suggesting that externalization did not portend apoptosis or necrosis. Consistent with that conclusion, mitochondrial membrane potential and hair-cell nuclear structure remained normal during externalization. PS externalization was triggered by forskolin, which raises cAMP, and blocked by inhibitors of adenylyl cyclase. Blocking Na⁺ influx by inhibiting the mechanoelectrical transduction channels and P2X ATP channels also inhibited external PS externalization. Diminished PS externalization was also seen in cells exposed to LY 294002, which blocks membrane recycling in hair cells by inhibiting phosphatidylinositol 3-kinase. These results indicate that PS exposure on the external leaflet, presumably requiring vesicular transport, results from elevation of intracellular cAMP, which can be triggered by Na⁺ entry into hair cells.     

Apical phosphatidylserine externalization in auditory hair cells

In hair cells of the inner ear, phosphatidylserine (PS), detected with fluorescent annexin V labeling, was rapidly exposed on the external leaflet of apical plasma membranes upon dissection of the organ of Corti. PS externalization was unchanged by caspase inhibition, suggesting that externalization did not portend apoptosis or necrosis. Consistent with that conclusion, mitochondrial membrane potential and hair-cell nuclear structure remained normal during externalization. PS externalization was triggered by forskolin, which raises cAMP, and blocked by inhibitors of adenylyl cyclase. Blocking Na(+) influx by inhibiting the mechanoelectrical transduction channels and P2X ATP channels also inhibited external PS externalization. Diminished PS externalization was also seen in cells exposed to LY 294002, which blocks membrane recycling in hair cells by inhibiting phosphatidylinositol 3-kinase. These results indicate that PS exposure on the external leaflet, presumably requiring vesicular transport, results from elevation of intracellular cAMP, which can be triggered by Na(+) entry into hair cells.     

Stimulation of phosphatidylserine biosynthesis and facilitation of UV-induced apoptosis in Chinese hamster ovary cells overexpressing phospholipid scramblase 1

Members of the phospholipid scramblase (PLSCR) family play active roles in altering lipid asymmetry at the plasma membrane including phosphatidylserine (PtdSer) exposure on the cell surface. To determine whether PtdSer biosynthesis and externalization are altered by PLSCR activities during apoptosis, Chinese hamster ovary K1 cell lines stably overexpressing PLSCR1 and PLSCR2 were established. PLSCR1 was localized on the plasma membrane, whereas PLSCR2 was predominantly in the nucleus. Cells overexpressing PLSCR1 showed suppressed growth, altered cell morphology, and higher basal levels of cell death. Following UV irradiation, these cells showed earlier and enhanced PtdSer exposure, increased caspase-3 activation, apoptotic nuclear changes, and PARP cleavage indicative of apoptosis. UV irradiation in cells overexpressing PLSCR1 led to a 4-fold stimulation of PtdSer synthesis (accompanied by increased movement of newly made PtdSer into microvesicles) relative to untreated PLSCR1 cells, whereas PtdSer formation in UV-irradiated vector control cells increased only by 2-fold. No differences in these responses were observed between PLSCR2-expressing cells and vector controls. PtdSer synthesis and its transbilayer movement stimulated by PLSCR1 overexpression were blocked by a caspase inhibitor along with progression of apoptosis. Thus, our studies showed that overexpression of PLSCR1 in Chinese hamster ovary K1 cells stimulated caspase-dependent PtdSer externalization and synthesis, implying an up-regulation of PtdSer formation in response to enhanced outward movement of this phospholipid to the cell surface during apoptosis. PLSCR1 also appears to influence progression of UV-induced apoptosis and could be a point of regulation or intervention during programmed cell death.     

Lipid antioxidant, etoposide, inhibits phosphatidylserine externalization and macrophage clearance of apoptotic cells by preventing phosphatidylserine oxidation

Apoptosis is associated with the externalization of phosphatidylserine (PS) in the plasma membrane and subsequent recognition of PS by specific macrophage receptors. Selective oxidation of PS precedes its externalization/recognition and is essential for the PS-dependent engulfment of apoptotic cells. Because etoposide is a potent and selective lipid antioxidant that does not block thiol oxidation, we hypothesized that it may affect PS externalization/recognition without affecting other features of the apoptotic program. We demonstrate herein that etoposide induced apoptosis in HL-60 cells without the concomitant peroxidation of PS and other phospholipids. HL-60 cells also failed to externalize PS in response to etoposide treatment. In contrast, oxidant (H2O2)-induced apoptosis was accompanied by PS externalization and oxidation of different phospholipids, including PS. Etoposide potentiated H2O2-induced apoptosis but completely blocked H2O2-induced PS oxidation. Etoposide also inhibited PS externalization as well as phagocytosis of apoptotic cells by J774A.1 macrophages. Integration of exogenous PS or a mixture of PS with oxidized PS in etoposide-treated HL-60 cells reconstituted the recognition of these cells by macrophages. The current data demonstrate that lipid antioxidants, capable of preventing PS peroxidation, can block PS externalization and phagocytosis of apoptotic cells by macrophages and hence dissociate PS-dependent signaling from the final common pathway for apoptosis.     

Preferential externalization of newly synthesized phosphatidylserine in apoptotic U937 cells is dependent on caspase-mediated pathways

Externalization of phosphatidylserine (PtdSer) is a common feature of programmed cell death and plays an important role in the recognition and removal of apoptotic cells. In this study with U937 cells, PtdSer synthesis from [(3)H]serine was stimulated and newly synthesized PtdSer was transferred preferentially to cell-free medium vesicles (CFMV) from cells when apoptosis was induced with a topoisomerase I inhibitor, camptothecin (CAM). When CAM-induced apoptosis was blocked by a caspase inhibitor, z-VAD-fmk, stimulation of PtdSer synthesis and movement to CFMV were abolished. In contrast, changes in synthesis and transport of sphingomyelin (SM) or phosphatidylethanolamine (PtdEtn) were minor; total phosphatidylcholine (PtdCho) synthesis was below control levels. All phospholipids appeared in CFMV but PtdSer displayed a 6-fold increase relative to controls compared to 3-fold for SM, 2-fold for PtdCho and 1.8-fold for PtdEtn. Even greater effects on specificity of PtdSer synthesis, movement to CFMV and inhibition by z-VAD-fmk were observed in apoptotic cells induced by UV irradiation or tumor necrosis factor-alpha/cycloheximide treatment. Thus, PtdSer biosynthesis stimulated during apoptosis in U937 cells was specific for this phospholipid and was correlated with caspase-mediated exposure of PtdSer at the cell surface and preferential movement to vesicles during apoptosis.     

Appetizing rancidity of apoptotic cells for macrophages: oxidation,externalization, and recognition of phosphatidylserine

Programmed cell death (apoptosis) functions as a mechanism to eliminate unwanted or irreparably damaged cells ultimately leading to their orderly phagocytosis in the absence of calamitous inflammatory responses. Recent studies have demonstrated that the generation of free radical intermediates and subsequent oxidative stress are implicated as part of the apoptotic execution process. Oxidative stress may simply be an unavoidable yet trivial byproduct of the apoptotic machinery; alternatively, intermediates or products of oxidative stress may act as essential signals for the execution of the apoptotic program. This review is focused on the specific role of oxidative stress in apoptotic signaling, which is realized via phosphatidylserine-dependent pathways leading to recognition of apoptotic cells and their effective clearance. In particular, the mechanisms involved in selective phosphatidylserine oxidation in the plasma membrane during apoptosis and its association with disturbances of phospholipid asymmetry leading to phosphatidylserine externalization and recognition by macrophage receptors are at the center of our discussion. The putative importance of this oxidative phosphatidylserine signaling in lung physiology and disease are also discussed.     

Involvement of phosphatidylserine externalization in the down-regulation of c-myb expression in differentiating C2C12 cells

Abstract Analysis of c-myb gene down-regulation in differentiating C212 cells revealed that in proliferating cells, c-myb expression is high and ceases as the proliferation rate decreases. However, a low level of c-myb mRNA was detected in confluent non-proliferating differentiating cells for an extended period of time before it declined to an undetectable level. The time course of c-myb gene silencing in differentiating cells correlated with exposition of phosphatidylserine (PS) on the cell surface. Moreover, the interaction of exposed PS with exogenously added annexin V perturbed PS-mediated cell signaling and transiently up-regulated the declining c-myb expression. We, therefore, suggest that cell surface-exposed PS, which plays a role in the process of myotube formation, is also involved in the down-regulation of c-myb expression.     

DEPS-1 promotes P-granule assembly and RNA interference in C. elegans germ cells

P granules are germ-cell-specific cytoplasmic structures containing RNA and protein, and required for proper germ cell development in C. elegans. PGL-1 and GLH-1 were previously identified as critical components of P granules. We have identified a new P-granule-associated protein, DEPS-1, the loss of which disrupts P-granule structure and function. DEPS-1 is required for the proper localization of PGL-1 to P granules, the accumulation of glh-1 mRNA and protein, and germ cell proliferation and fertility at elevated temperatures. In addition, DEPS-1 is required for RNA interference (RNAi) of germline-expressed genes, possibly because DEPS-1 promotes the accumulation of RDE-4, a dsRNA-binding protein required for RNAi. A genome wide analysis of gene expression in deps-1 mutant germ lines identified additional targets of DEPS-1 regulation, many of which are also regulated by the RNAi factor RDE-3. Our studies suggest that DEPS-1 is a key component of the P-granule assembly pathway and that its roles include promoting accumulation of some mRNAs, such as glh-1 and rde-4, and reducing accumulation of other mRNAs, perhaps by collaborating with RDE-3 to generate endogenous short interfering RNAs (endo-siRNAs).     

Small GTPase CDC-42 promotes apoptotic cell corpse clearance in response to PAT-2 and CED-1 in C. elegans

The rapid clearance of dying cells is important for the well-being of multicellular organisms. In C. elegans, cell corpse removal is mainly mediated by three parallel engulfment signaling cascades. These pathways include two small GTPases, MIG-2/RhoG and CED-10/Rac1. Here we present the identification and characterization of CDC-42 as a third GTPase involved in the regulation of cell corpse clearance. Genetic analyses performed by both loss of cdc-42 function and cdc-42 overexpression place cdc-42 in parallel to the ced-2/5/12 signaling module, in parallel to or upstream of the ced-10 module, and downstream of the ced-1/6/7 module. CDC-42 accumulates in engulfing cells at membranes surrounding apoptotic corpses. The formation of such halos depends on the integrins PAT-2/PAT-3, UNC-112 and the GEF protein UIG-1, but not on the canonical ced-1/6/7 or ced-2/5/12 signaling modules. Together, our results suggest that the small GTPase CDC-42 regulates apoptotic cell engulfment possibly upstream of the canonical Rac GTPase CED-10, by polarizing the engulfing cell toward the apoptotic corpse in response to integrin signaling and ced-1/6/7 signaling in C. elegans.During development and in tissue homeostasis, multicellular organisms frequently use apoptosis to eliminate cells that are useless or potentially dangerous. Apoptotic cells are readily recognized, internalized and degraded by neighboring or specialized engulfing cells. Rapid clearance of unwanted cells avoids the release of harmful intracellular contents into the surroundings that can lead to inflammation and autoimmune disease.¹The nematode C. elegans serves as a simple yet powerful genetic model organism to study cell corpse clearance in vivo. Many genes involved in recognition, internalization or degradation of apoptotic corpses have been identified through forward and reverse genetic screens in the past two decades.² Loss of engulfment activity not only results in the persistence of cell corpses, but also leads to the survival of some cells destined to die,³ and – in some cases – leads to impaired cell migration.⁴Phenotypic, genetic and biochemical analyses of the early ‘classical'' ced (cell death abnormal) genes led to the identification of three partially redundant signaling cascades that cooperate to regulate cytoskeletal rearrangements and the migration of the engulfing cell around the apoptotic corpse.^{5, 6, 7, 8, 9} In the first pathway, the transmembrane protein CED-1/MEGF10 has been proposed to act as a cell corpse receptor¹⁰ that binds to exposed phosphatidylserine (PS), either directly or indirectly through the action of the bridging molecule TTR-52/TTR.^{11, 12} The lipid transporter homolog CED-7 also plays a role at this stage, at least in part by promoting the exposure of PS in the outer leaflet of the doomed cell.¹³ The adaptor protein CED-6/GULP transduces the signal(s) from CED-1 downstream to CED-10/Rac1 and DYN-1/Dynamin to drive cytoskeletal rearrangements and phagosome maturation.^{8, 14, 15, 16} In the second pathway, activation of CED-10 is promoted by the bipartite GEF (guanine exchange factor) complex composed of CED-12/Elmo–CED-5/Dock180.^{17, 18, 19, 20} This GEF complex in turn is regulated by CED-2/CrkII and the small GTPase MIG-2/RhoG. In the third pathway, the cytoskeletal regulator ABL-1/Abl suppresses corpse clearance by inhibiting ABI-1/Abl-interacting protein.²¹ Active GTP-loaded CED-10 promotes the extensive cytoskeletal rearrangements that are essential for proper cell corpse internalization.⁸ This process is negatively regulated by the GTPase-activating protein (GAP) SRGP-1/srGAP1 that facilitates GTP hydrolysis in CED-10.²²Here we present the identification and characterization of cdc-42 (cell division control protein-42) as an additional mediator of engulfment signaling regulated by SRGP-1 (Slit/Robo GTPase activating protein 1). Our epistatic analyses, performed with cdc-42(lf) mutants and cdc-42 overexpression experiments, suggest that cdc-42 acts downstream or in parallel to the ced-1/6/7 and in parallel to the ced-2/5/12 signaling cascades. Using a functional and rescuing GFP::CDC-42 protein, we show that CDC-42 is recruited to the cell membrane surrounding apoptotic corpses, and that this localization requires the integrin-α PAT-2 but not the canonical ced-1/6/7 or ced-2/5/12 cascades.Taken together, our results suggest that the small GTPase CDC-42 regulates apoptotic cell engulfment upstream of the canonical Rac GTPase CED-10, possibly by polarizing the engulfing cell toward the apoptotic corpse in response to integrin signaling. Our data confirm and significantly expand on recent results published by Hsieh et al.,²³ who independently identified CDC-42 as an engulfment regulator downstream of integrin-α PAT-2.     

The phospholipid scramblase PLSCR1 increases UV induced apoptosis primarily through the augmentation of the intrinsic apoptotic pathway and independent of direct phosphorylation by protein kinase C delta

Cell death by apoptosis can be caused by the DNA mutagen UV light whose exposure causes the direct activation of both the caspase 9 regulated cell damage intrinsic pathway and the caspase 8 regulated plasma membrane extrinsic pathway. We determined that increased activity of the plasma membrane phospholipid scramblase, PLSCR1, amplified UV mediated apoptosis primarily through the activation of the intrinsic apoptotic pathway. The caspase 8 inhibitor z-IETD-fmk was not as effective an inhibitor of PLSCR1 augmented UV induced apoptosis compared to treatment with caspase 3, caspase 9, or pan-caspase inhibitors. The inability of the caspase 8 inhibitor to decrease UV induced apoptosis was dependent on PLSCR1, as UV induced apoptosis was decreased by a similar amount in the control cells in the presence of inhibitors of caspase 8, caspase 9, caspase 3, or the pan-caspase inhibitor. PKC-delta directly phosphorylates human PLSCR1 resulting in increased PLSCR1 scramblase activity. PKC-delta can also be activated by caspase mediated cleavage resulting in the release of a constitutively active kinase domain. We observed that replacing the PKC-delta phosphorylation site of PLSCR1 with an alanine did not affect the ability of PLSCR1 to enhance UV induced apoptosis implying that PKC-delta does not directly phosphorylate PLSCR1 to increase plasma membrane scramblase activity during apoptosis. Cells transfected with a PLSCR1 mutant that contained an alanine substitution at its known PKC-delta phosphorylation site underwent UV induced apoptosis at a level similar to those transfected with wild type PLSCR1. The combined results indicate that UV exposure in cells possessing PLSCR1 increases apoptosis primarily by enhancement of the intrinsic apoptotic pathway, and also imply that the increased apoptosis observed upon exposure to UV light is not through direct phosphorylation of PLSCR1 by PKC-delta.     

TIEG1 induces apoptosis through mitochondrial apoptotic pathway and promotes apoptosis induced by homoharringtonine and velcade

Overexpression of TGFbeta inducible early gene (TIEG1) mimics TGFbeta action and induces apoptosis. In this study, we found that TIEG1 was significantly up-regulated during apoptosis induced by homoharringtonine or velcade. Overexpression of TIEG1 could induce apoptosis in K562 cells and promote apoptosis induced by HHT or velcade. TIEG1-induced apoptosis was shown to involve Bax and Bim up-regulation, Bcl-2 and Bcl-XL down-regulation, release of cytochrome c from mitochondria into the cytosol, activation of caspase 3 and disruption of the mitochondrial membrane potential (DeltaPsim). We concluded that TIEG1 is a key regulator which induces and promotes apoptosis through the mitochondrial apoptotic pathway.     

The phosphoinositide phosphatase MTM-1 regulates apoptotic cell corpse clearance through CED-5-CED-12 in C. elegans

Multicellular organisms use programmed cell death to eliminate unwanted or potentially harmful cells. Improper cell corpse removal can lead to autoimmune diseases. The development of interventional therapies that increase engulfment activity could represent an attractive approach to treat such diseases. Here, we describe mtm-1, the Caenorhabditis elegans homolog of human myotubularin 1, as a potential negative regulator of apoptotic cell corpse clearance. Loss of mtm-1 function leads to substantially reduced numbers of persistent cell corpses in engulfment mutants, which is a result of a restoration of engulfment function rather than of impaired or delayed programmed cell death. Epistatic analyses place mtm-1 upstream of the ternary GEF complex, which consists of ced-2, ced-5 and ced-12, and parallel to mig-2. Over-activation of engulfment results in the removal of viable cells that have been brought to the verge of death under limiting caspase activity. In addition, mtm-1 also promotes phagosome maturation in the hermaphrodite gonad, potentially through CED-1 receptor recycling. Finally, we show that the CED-12 PH domain can bind to PtdIns(3,5)P(2) (one target of MTM-1 phosphatase activity), suggesting that MTM-1 might regulate CED-12 recruitment to the plasma membrane.     

C. elegans EIF-3.K promotes programmed cell death through CED-3 caspase

Programmed cell death (apoptosis) is essential for the development and homeostasis of metazoans. The central step in the execution of programmed cell death is the activation of caspases. In C. elegans, the core cell death regulators EGL-1(a BH3 domain-containing protein), CED-9 (Bcl-2), and CED-4 (Apaf-1) act in an inhibitory cascade to activate the CED-3 caspase. Here we have identified an additional component eif-3.K (eukaryotic translation initiation factor 3 subunit k) that acts upstream of ced-3 to promote programmed cell death. The loss of eif-3.K reduced cell deaths in both somatic and germ cells, whereas the overexpression of eif-3.K resulted in a slight but significant increase in cell death. Using a cell-specific promoter, we show that eif-3.K promotes cell death in a cell-autonomous manner. In addition, the loss of eif-3.K significantly suppressed cell death-induced through the overexpression of ced-4, but not ced-3, indicating a distinct requirement for eif-3.K in apoptosis. Reciprocally, a loss of ced-3 suppressed cell death induced by the overexpression of eif-3.K. These results indicate that eif-3.K requires ced-3 to promote programmed cell death and that eif-3.K acts upstream of ced-3 to promote this process. The EIF-3.K protein is ubiquitously expressed in embryos and larvae and localizes to the cytoplasm. A structure-function analysis revealed that the 61 amino acid long WH domain of EIF-3.K, potentially involved in protein-DNA/RNA interactions, is both necessary and sufficient for the cell death-promoting activity of EIF-3.K. Because human eIF3k was able to partially substitute for C. elegans eif-3.K in the promotion of cell death, this WH domain-dependent EIF-3.K-mediated cell death process has potentially been conserved throughout evolution.