The tumor suppressor DAP kinase is a target of RSK-mediated survival signaling

The viability of vertebrate cells depends on a complex signaling interplay between survival factors and cell-death effectors. Subtle changes in the equilibrium between these regulators can result in abnormal cell proliferation or cell death, leading to various pathological manifestations. Death-associated protein kinase (DAPK) is a multidomain calcium/calmodulin (CaM)-dependent Ser/Thr protein kinase with an important role in apoptosis regulation and tumor suppression. The molecular signaling mechanisms regulating this kinase, however, remain unclear. Here, we show that DAPK is phosphorylated upon activation of the Ras-extracellular signal-regulated kinase (ERK) pathway. This correlates with the suppression of the apoptotic activity of DAPK. We demonstrate that DAPK is a novel target of p90 ribosomal S6 kinases (RSK) 1 and 2, downstream effectors of ERK1/2. Using mass spectrometry, we identified Ser-289 as a novel phosphorylation site in DAPK, which is regulated by RSK. Mutation of Ser-289 to alanine results in a DAPK mutant with enhanced apoptotic activity, whereas the phosphomimetic mutation (Ser289Glu) attenuates its apoptotic activity. Our results suggest that RSK-mediated phosphorylation of DAPK is a unique mechanism for suppressing the proapoptotic function of this death kinase in healthy cells as well as Ras/Raf-transformed cells.     

Peptide combinatorial libraries identify TSC2 as a death-associated protein kinase (DAPK) death domain-binding protein and reveal a stimulatory role for DAPK in mTORC1 signaling

Death-associated protein kinase (DAPK) is a multidomain enzyme that plays a central role in autophagic and apoptotic signaling, although the protein-protein interactions regulating DAPK functions are not well defined. Peptide aptamer libraries were used to identify the tumor suppressor protein tuberin (TSC2) as a novel DAPK death domain-binding protein, and we evaluated whether DAPK is a positive or negative effector of the TSC2-regulated mammalian target of rapamycin (mTORC1) signaling pathway. Binding studies using death domain miniproteins in vitro and deletion analysis in vivo determined that the death domain of DAPK is the major site for the interaction with TSC2. Recombinant DAPK phosphorylates TSC2 in vitro, and DAPK kinase activity is stimulated by growth factor signaling. Transfection of DAPK promotes phosphorylation of TSC2 in vivo, whereas short interfering RNA-mediated attenuation of DAPK reduces growth factor-stimulated phosphorylation of TSC2. DAPK-dependent phosphorylation leads to TSC1-TSC2 complex dissociation, and consequently manipulation of DAPK by transfection or short interfering RNA demonstrated that DAPK is a positive regulator of mTORC1 in response to growth factor activation. Epistatic studies suggest that DAPK functions downstream from the RAS-MEK-ERK and phosphatidylinositol 3-kinase-AKT growth factor signaling pathways. DAPK(+/-) mouse embryo fibroblasts have attenuated mTORC1 signaling compared with DAPK+/+ counterparts, and overexpression of DAPK in DAPK(+/-) MEFs stimulates mTORC1 activity. These data uncover a novel interaction between DAPK and TSC2 proteins that has revealed a positive link between growth factor stimulation of DAPK and mTORC1 signaling that may ultimately affect autophagy, cell survival, or apoptosis.     

DAP‐kinase‐mediated phosphorylation on the BH3 domain of beclin 1 promotes dissociation of beclin 1 from Bcl‐XL and induction of autophagy

Autophagy, an evolutionarily conserved process, has functions both in cytoprotective and programmed cell death mechanisms. Beclin 1, an essential autophagic protein, was recently identified as a BH3‐domain‐only protein that binds to Bcl‐2 anti‐apoptotic family members. The dissociation of beclin 1 from its Bcl‐2 inhibitors is essential for its autophagic activity, and therefore should be tightly controlled. Here, we show that death‐associated protein kinase (DAPK) regulates this process. The activated form of DAPK triggers autophagy in a beclin‐1‐dependent manner. DAPK phosphorylates beclin 1 on Thr 119 located at a crucial position within its BH3 domain, and thus promotes the dissociation of beclin 1 from Bcl‐X_L and the induction of autophagy. These results reveal a substrate for DAPK that acts as one of the core proteins of the autophagic machinery, and they provide a new phosphorylation‐based mechanism that reduces the interaction of beclin 1 with its inhibitors to activate the autophagic machinery.     

The tumor suppressor DAPK inhibits cell motility by blocking the integrin-mediated polarity pathway

Death-associated protein kinase (DAPK) is a calmodulin-regulated serine/threonine kinase and possesses apoptotic and tumor-suppressive functions. However, it is unclear whether DAPK elicits apoptosis-independent activity to suppress tumor progression. We show that DAPK inhibits random migration by reducing directional persistence and directed migration by blocking cell polarization. These effects are mainly mediated by an inhibitory role of DAPK in talin head domain association with integrin, thereby suppressing the integrin–Cdc42 polarity pathway. We present evidence indicating that the antimigratory effect of DAPK represents a mechanism through which DAPK suppresses tumors. First, DAPK can block migration and invasion in certain tumor cells that are resistant to DAPK-induced apoptosis. Second, using an adenocarcinoma cell line and its highly invasive derivative, we demonstrate DAPK level as a determining factor in tumor invasiveness. Collectively, our study identifies a novel function of DAPK in regulating cell polarity during migration, which may act together with its apoptotic function to suppress tumor progression.     

The Cullin 3 substrate adaptor KLHL20 mediates DAPK ubiquitination to control interferon responses

Death‐associated protein kinase (DAPK) was identified as a mediator of interferon (IFN)‐induced cell death. How IFN controls DAPK activation remains largely unknown. Here, we identify the BTB–Kelch protein KLHL20 as a negative regulator of DAPK. KLHL20 binds DAPK and Cullin 3 (Cul3) via its Kelch‐repeat domain and BTB domain, respectively. The KLHL20–Cul3–ROC1 E3 ligase complex promotes DAPK polyubiquitination, thereby inducing the proteasomal degradation of DAPK. Accordingly, depletion of KLHL20 diminishes DAPK ubiquitination and degradation. The KLHL20‐mediated DAPK ubiquitination is suppressed in cells receiving IFN‐α or IFN‐γ, which induces an enrichment/sequestration of KLHL20 in the PML nuclear bodies, thereby separating KLHL20 from DAPK. Consequently, IFN triggers the stabilization of DAPK. This mechanism of DAPK stabilization is crucial for determining IFN responsiveness of tumor cells and contributes to IFN‐induced autophagy. This study identifies KLHL20–Cul3–ROC1 as an E3 ligase for DAPK ubiquitination and reveals a regulatory mechanism of DAPK, through blocking its accessibility to this E3 ligase, in IFN‐induced apoptotic and autophagic death. Our findings may be relevant to the problem of IFN resistance in cancer therapy.     

Identification of a new form of death-associated protein kinase that promotes cell survival

In this study, two alternatively spliced forms of the mouse death-associated protein kinase (DAPK) have been identified and their roles in apoptosis examined. The mouse DAPK-alpha sequence is 95% identical to the previously described human DAPK, and it has a kinase domain and calmodulin-binding region closely related to the 130-150 kDa myosin light chain kinases. A 12-residue extension of the carboxyl terminus of DAPK-beta distinguishes it from the human and mouse DAPK-alpha. DAPK phosphorylates at least one substrate in vitro and in vivo, the myosin II regulatory light chain. This phosphorylation occurs preferentially at Ser-19 and is stimulated by calcium and calmodulin. The mRNA encoding DAPK is widely distributed and detected in mouse embryos and most adult tissues, although the expression of the encoded 160-kDa DAPK protein is more restricted. Overexpression of DAPK-alpha, the mouse homolog of human DAPK has a negligible effect on tumor necrosis factor (TNF)-induced apoptosis. Overexpression of DAPK-beta has a strong cytoprotective effect on TNF-treated cells. Biochemical analysis of TNF-treated cell lines expressing mouse DAPK-beta suggests that the cytoprotective effect of DAPK is mediated through both intrinsic and extrinsic apoptotic signaling pathways and results in the inhibition of cytochrome c release from the mitochondria as well as inhibition of caspase-3 and caspase-9 activity. These results suggest that the mouse DAPK-beta is a negative regulator of TNF-induced apoptosis.     

Role of DAPK in neuronal cell death

Neuronal cell death happens as a result of the normal physiological process that occurs during development, or as part of the pathological process that occurs during disease. Death-associated protein kinase (DAPK) is an intracellular protein that mediates cell death by its serine/threonine kinase activity, and transmits apoptotic cell death signals in various cells, including neurons. DAPK is elevated in injured neurons in acute models of injury such as ischemia and seizure. The absence of DAPK has been shown to protect neurons from a wide variety of acute toxic insults. Moreover, DAPK also regulates neuronal cell death during central nervous system development. Neurons are initially overproduced in the developing nervous system, following which approximately one-half of the original cell population dies. This “naturally-occurring” or “programmed” cell death is essential for the construction of the developing nervous system. In this review, we focus on the role of DAPK in neuronal cell death after neuronal injury. The participation of DAPK in developmental neuronal death is also explained.     

Neogenin regulates neuronal survival through DAP kinase

The repulsive guidance molecule (RGM) is a membrane-bound protein that has diverse functions in the developing central nervous system. Identification of neogenin as a receptor for RGM provided evidence of its cell death-inducing activity in the absence of RGM. Here, we show that the serine/threonine kinase death-associated protein kinase (DAPK) is involved in the signal transduction of neogenin. Neogenin interacts with DAPK and reduces DAPK autophosphorylation on Ser308 in vitro. Neogenin-induced cell death is abolished in the presence of RGM or by blocking DAPK. Although neogenin overexpression or RGM downregulation in the chick neural tube in vivo induces apoptosis, coexpression of the dominant-negative mutant or small-interference RNA of DAPK attenuates this proapoptotic activity. Thus, RGM/neogenin regulates cell fate by controlling the DAPK activity.     

DAPK activates MARK1/2 to regulate microtubule assembly, neuronal differentiation, and tau toxicity

Death-associated protein kinase (DAPK) is a key player in several modes of neuronal death/injury and has been implicated in the late-onset Alzheimer's disease (AD). DAPK promotes cell death partly through its effect on regulating actin cytoskeletons. In this study, we report that DAPK inhibits microtubule (MT) assembly by activating MARK/PAR-1 family kinases MARK1/2, which destabilize MT by phosphorylating tau and related MAP2/4. DAPK death domain, but not catalytic activity, is responsible for this activation by binding to MARK1/2 spacer region, thereby disrupting an intramolecular interaction that inhibits MARK1/2. Accordingly, DAPK(-/-) mice brain displays a reduction of tau phosphorylation and DAPK enhances the effect of MARK2 on regulating polarized neurite outgrowth. Using a well-characterized Drosophila model of tauopathy, we show that DAPK exerts an effect in part through MARK Drosophila ortholog PAR-1 to induce rough eye and loss of photoreceptor neurons. Furthermore, DAPK enhances tau toxicity through a PAR-1 phosphorylation-dependent mechanism. Together, our study reveals a novel mechanism of MARK activation, uncovers DAPK functions in modulating MT assembly and neuronal differentiation, and provides a molecular link of DAPK to tau phosphorylation, an event associated with AD pathology.     

Evaluating DAPK as a therapeutic target

Death associated protein kinase 1 (DAPK) is an important serine/theoreine kinase involved in various cellular processes such as apoptosis, autophagy and inflammation. DAPK expression and activity are misregulated in multiple diseases including cancer, neuronal death, stoke, et al. Methylation of the DAPK gene is common in many types of cancer and can lead to loss of DAPK expression. In this review, we summarize the pathological status and functional roles of DAPK in disease and compare the published reagents that can manipulate the expression or activity of DAPK. The pleiotropic functions of DAPK make it an intriguing target and the barriers and opportunities for targeting DAPK for future clinical application are discussed.     

Hsp27 silencing coordinately inhibits proliferation and promotes Fas-induced apoptosis by regulating the PEA-15 molecular switch

Heat shock protein 27 (Hsp27) is emerging as a promising therapeutic target for treatment of various cancers. Although the role of Hsp27 in protection from stress-induced intrinsic cell death has been relatively well studied, its role in Fas (death domain containing member of the tumor necrosis factor receptor superfamily)-induced apoptosis and cell proliferation remains underappreciated. Here, we show that Hsp27 silencing induces dual coordinated effects, resulting in inhibition of cell proliferation and sensitization of cells to Fas-induced apoptosis through regulation of PEA-15 (15-kDa phospho-enriched protein in astrocytes). We demonstrate that Hsp27 silencing suppresses proliferation by causing PEA-15 to bind and sequester extracellular signal-regulated kinase (ERK), resulting in reduced translocation of ERK to the nucleus. Concurrently, Hsp27 silencing promotes Fas-induced apoptosis by inducing PEA-15 to release Fas-associating protein with a novel death domain (FADD), thus allowing FADD to participate in death receptor signaling. Conversely, Hsp27 overexpression promotes cell proliferation and suppresses Fas-induced apoptosis. Furthermore, we show that Hsp27 regulation of PEA-15 activity occurs in an Akt-dependent manner. Significantly, Hsp27 silencing in a panel of phosphatase and tensin homolog on chromosome 10 (PTEN) wild-type or null cell lines, and in LNCaP cells that inducibly express PTEN, resulted in selective growth inhibition of PTEN-deficient cancer cells. These data identify a dual coordinated role of Hsp27 in cell proliferation and Fas-induced apoptosis via Akt and PEA-15, and indicate that improved clinical responses to Hsp27-targeted therapy may be achieved by stratifying patient populations based on tumor PTEN expression.     

An aminopyridazine-based inhibitor of a pro-apoptotic protein kinase attenuates hypoxia-ischemia induced acute brain injury

Death associated protein kinase (DAPK) is a calcium and calmodulin regulated enzyme that functions early in eukaryotic programmed cell death, or apoptosis. To validate DAPK as a potential drug discovery target for acute brain injury, the first small molecule DAPK inhibitor was synthesized and tested in vivo. A single injection of the aminopyridazine-based inhibitor administered 6 h after injury attenuated brain tissue or neuronal biomarker loss measured, respectively, 1 week and 3 days later. Because aminopyridazine is a privileged structure in neuropharmacology, we determined the high-resolution crystal structure of a binary complex between the kinase domain and a molecular fragment of the DAPK inhibitor. The co-crystal structure describes a structural basis for interaction and provides a firm foundation for structure-assisted design of lead compounds with appropriate molecular properties for future drug development.     

A protein kinase associated with apoptosis and tumor suppression: structure, activity, and discovery of peptide substrates

Death-associated protein kinase (DAPK) has been implicated in apoptosis and tumor suppression, depending on cellular conditions, and associated with mechanisms of disease. However, DAPK has not been characterized as an enzyme due to the lack of protein or peptide substrates. Therefore, we determined the structure of DAPK catalytic domain, used a homology model of docked peptide substrate, and synthesized positional scanning substrate libraries in order to discover peptide substrates with K(m) values in the desired 10 microm range and to obtain knowledge about the preferences of DAPK for phosphorylation site sequences. Mutagenesis of DAPK catalytic domain at amino acids conserved among protein kinases or unique to DAPK provided a link between structure and activity. An enzyme assay for DAPK was developed and used to measure activity in adult brain and monitor protein purification based on the physical and chemical properties of the open reading frame of the DAPK cDNA. The results allow insight into substrate preferences and regulation of DAPK, provide a foundation for proteomic investigations and inhibitor discovery, and demonstrate the utility of the experimental approach, which can be extended potentially to kinase open reading frames identified by genome sequencing projects or functional genetics screens and lacking a known substrate.     

p38 kinase-dependent and -independent Inhibition of protein kinase C zeta and -alpha regulates nitric oxide-induced apoptosis and dedifferentiation of articular chondrocytes

In articular chondrocytes, nitric oxide (NO) production triggers dedifferentiation and apoptotic cell death that is regulated by the converse functions of two mitogen-activated protein kinase subtypes, extracellular signal-regulated kinase (ERK) and p38 kinase. Since protein kinase C (PKC) transduces signals that influence differentiation, survival, and apoptosis of various cell types, we investigated the roles and underlying molecular mechanisms of action of PKC isoforms in NO-induced dedifferentiation and apoptosis of articular chondrocytes. We report here that among the expressed isoforms, activities of PKCalpha and -zeta were reduced during NO-induced dedifferentiation and apoptosis. Inhibition of PKCalpha activity was independent of NO-induced activation of ERK or p38 kinase and occurred due to blockage of expression. On the other hand, PKCzeta activity was inhibited as a result of NO-induced p38 kinase activation and was observed prior to proteolytic cleavage by a caspase-mediated process to generate enzymatically inactive fragments. Inhibition of PKCalpha or -zeta activities potentiated NO-induced apoptosis, whereas ectopic expression of these isoforms significantly reduced the number of apoptotic cells and blocked dedifferentiation. Ectopic expression of PKCalpha or -zeta did not affect p38 kinase or ERK but inhibited the p53 accumulation and caspase-3 activation that are required for NO-induced apoptosis of chondrocytes. Therefore, our results collectively indicate that p38 kinase-independent and -dependent inhibition of PKCalpha and -zeta, respectively, regulates NO-induced apoptosis and dedifferentiation of articular chondrocytes.     

Tuberous sclerosis-2 (TSC2) regulates the stability of death-associated protein kinase-1 (DAPK) through a lysosome-dependent degradation pathway

We previously identified a novel interaction between tuberous sclerosis-2 (TSC2) and death-associated protein kinase-1 (DAPK), the consequence being that DAPK catalyses the inactivating phosphorylation of TSC2 to stimulate mammalian target of rapamycin complex 1 (mTORC1) activity. We now report that TSC2 binding to DAPK promotes the degradation of DAPK. We show that DAPK protein levels, but not gene expression, inversely correlate with TSC2 expression. Furthermore, altering mTORC1 activity does not affect DAPK levels, excluding indirect effects of TSC2 on DAPK protein levels through changes in mTORC1 translational control. We provide evidence that the C-terminus regulates TSC2 stability and is required for TSC2 to reduce DAPK protein levels. Importantly, using a GTPase-activating protein-dead missense mutation of TSC2, we demonstrate that the effect of TSC2 on DAPK is independent of GTPase-activating protein activity. TSC2 binds to the death domain of DAPK and we show that this interaction is required for TSC2 to reduce DAPK protein levels and half-life. Finally, we show that DAPK is regulated by the lysosome pathway and that lysosome inhibition blocks TSC2-mediated degradation of DAPK. Our study therefore establishes important functions of TSC2 and the lysosomal-degradation pathway in the control of DAPK stability, which taken together with our previous findings, reveal a regulatory loop between DAPK and TSC2 whose balance can either promote: (a) TSC2 inactivation resulting in mTORC1 stimulation, or (b) DAPK degradation via TSC2 signalling under steady-state conditions. The fine balance between DAPK and TSC2 in this regulatory loop may have subtle but important effects on mTORC1 steady-state function.     

VEGF prevents apoptosis of human microvascular endothelial cells via opposing effects on MAPK/ERK and SAPK/JNK signaling

Vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen, promotes endothelial cell survival and angiogenesis. We recently showed that VEGF can support the growth of human dermal microvascular endothelial cells (HDMEC) and human umbilical vein endothelial cells in serum-free medium. Reasoning that VEGF might be modulating apoptotic signal transduction pathways, we examined mechanisms involved in the anti-apoptotic effect of VEGF on starvation- and ceramide-induced apoptosis in HDMEC. We observed that VEGF ameliorated the time-dependent increase in apoptosis, as demonstrated by morphologic observations, TUNEL assay, and DNA fragmentation. On the other hand, basic fibroblast growth factor only partially prevented apoptosis in serum-starved HDMEC; platelet-derived growth factor-BB was completely ineffective. VEGF activated the phosphorylation of extracellular signal regulated kinase (ERK)1 (p44 mitogen-activated protein kinase; MAPK) and ERK2 (p42 MAPK) in a time- and concentration-dependent manner. Both the VEGF-induced activation and its anti-apoptotic effect were prevented by the specific MAPK/ERK inhibitor PD98059. The presence of VEGF also inhibited the sustained activation of stress-activated protein kinase/c-jun-NH2-kinase (SAPK/JNK) caused by serum starvation and ceramide treatment. Activation of the MAPK pathway together with inhibition of SAPK/JNK activity by VEGF appears to be a key event in determining whether an endothelial cell survives or undergoes programmed cell death.     

DAPK3 inhibits gastric cancer progression via activation of ULK1-dependent autophagy

Dysregulation of the balance between cell proliferation and cell death is a central feature of malignances. Death-associated protein kinase 3 (DAPK3) regulates programmed cell death including apoptosis and autophagy. Our previous study showed that DAPK3 downregulation was detected in more than half of gastric cancers (GCs), which was related to tumor invasion, metastasis, and poor prognosis. However, the precise molecular mechanism underlying DAPK3-mediated tumor suppression remains unclear. Here, we showed that the tumor suppressive function of DAPK3 was dependent on autophagy process. Mass spectrometry, in vitro kinase assay, and immunoprecipitation revealed that DAPK3 increased ULK1 activity by direct ULK1 phosphorylation at Ser556. ULK1 phosphorylation by DAPK3 facilitates the ULK1 complex formation, the VPS34 complex activation, and autophagy induction upon starvation. The kinase activity of DAPK3 and ULK1 Ser556 phosphorylation were required for DAPK3-modulated tumor suppression. The coordinate expression of DAPK3 with ULK1 Ser556 phosphorylation was confirmed in clinical GC samples, and this co-expression was correlated with favorable survival outcomes in patients. Collectively, these findings indicate that the tumor-suppressor roles of DAPK3 in GC are associated with autophagy and that DAPK3 is a novel autophagy regulator, which can directly phosphorylate ULK1 and activate ULK1. Thus, DAPK3 might be a promising prognostic autophagy-associated marker.Subject terms: Tumour-suppressor proteins, Macroautophagy     

Antisense depletion of death-associated protein kinase promotes apoptosis

Death-associated protein kinases (DAPK) are serine/threonine protein kinases that have an important role in regulating cell death. In this study two antisense approaches were employed to down-regulate expression of the endogenous DAPK-alpha and DAPK-beta proteins. Transient expression of an antisense DAPK cDNA or antisense morpholino oligonucleotides in HeLa, 3T3, or primary human vascular smooth muscle cells demonstrate that decreased DAPK expression promotes a spontaneous, caspase-mediated apoptosis as evidenced by increased activities of caspases-3 and -9. Clonal HeLa cell lines with attenuated levels of DAPK expression, obtained following selection in the presence of antisense DAPK cDNA, are more sensitive to tumor necrosis factor-induced caspase-mediated apoptosis, and their sensitivity is inversely related to DAPK expression. In contrast, HeLa cells with reduced DAPK expression are moderately resistant to cell death induced by interferon-gamma. This finding is consistent with previous studies showing that DAPK has a role in promoting caspase-independent cell death. Together, these studies demonstrate that the cellular activities of DAPK are critical for antagonizing caspase-dependent apoptosis to promote cell survival under normal cell growth conditions.     

DAPK catalytic activity in the hippocampus increases during the recovery phase in an animal model of brain hypoxic-ischemic injury

Death-associated protein kinase (DAPK) is a pro-apoptotic, calmodulin (CaM)-regulated protein kinase whose mRNA levels increase following cerebral ischemia. However, the relationship between DAPK catalytic activity and cerebral ischemia is not known. This knowledge is critical as DAPK function is dependent on the catalytic activity of its kinase domain. Consequently, we examined DAPK catalytic activity in a rat model of neonatal cerebral hypoxia-ischemia (HI). An increase in DAPK specific activity was found in homogenates of the hippocampus from the injured right hemisphere, compared to the uninjured left hemisphere, 7 days after injury. The results raised the possibility that an upregulation of DAPK activity might be associated with the recovery phase of HI, during which neuronal repair and differentiation are initiated. Therefore, we examined the change of DAPK in an experimentally tractable cell culture model of neuronal differentiation. We found that DAPK catalytic activity and protein levels increase after nerve growth factor (NGF)-induced differentiation of rat PC12 cells. These results suggest that DAPK may have a previously unappreciated role in neuronal development or recovery from injury, and that potential future therapies targeting DAPK should consider a restricted time window.     

Ceramide pathways modulate ethanol-induced cell death in astrocytes

We showed previously that alcohol exposure during in vivo brain development induced astroglial damage and caused cell death. Because ceramide modulates a number of biochemical and cellular responses to stress, including apoptosis, we now investigate whether ethanol-induced cell death in astrocytes is mediated by ceramide signalling pathways triggering apoptosis. Here we show that both ethanol and ceramide are able to induce apoptotic death in cultured astrocytes, in a dose-dependent manner, and that C2-ceramide addition potentiates the apoptotic effects of ethanol. Cell death induced by ethanol is associated with stimulation of neutral and acidic sphingomyelinase (SMase) and ceramide generation, as well as with activation of stress-related kinases, c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (p38) and extracellular signal-regulated kinase (ERK) pathways. We also provide evidence for the participation of JNK and p38 in ethanol-induced cell death, because pharmacological inhibitors of these kinases largely prevent the apoptosis induced by ethanol or by ethanol and C2-ceramide. Furthermore, we show that ethanol-induced ERK activation triggers the stimulation of cyclo-oxygenase-2 (COX-2) and the release of prostaglandin E2, and that blockade of the mitogen-activated protein kinase kinase (MEK)/ERK pathway by PD98059 abolishes the up-regulation of COX-2 induced by ethanol plus ceramide, and decreases the ethanol-induced apoptosis. These results strongly suggest that ethanol is able to stimulate the SMase-ceramide pathway, leading to the activation of signalling pathways implicated in cell death. These findings provide an insight into the mechanisms involved in ethanol-induced astroglial cell death during brain development.