Characterization of eicosanoid synthesis in a genetic ablation model of ceramide kinase

Multiple reports have demonstrated a role for ceramide kinase (CERK) in the production of eicosanoids. To examine the effects of the genetic ablation of CERK on eicosanoid synthesis, primary mouse embryonic fibroblasts (MEFs) and macrophages were isolated from CERK^−/− and CERK^+/+ mice, and the ceramide-1-phosphate (C1P) and eicosanoid profiles were investigated. Significant decreases were observed in multiple C1P subspecies in CERK−/− cells as compared to CERK^+/+ cells with overall 24% and 48% decreases in total C1P. In baseline experiments, the levels of multiple eicosanoids were significantly lower in the CERK^−/− cells compared with wild-type cells. Importantly, induction of eicosanoid synthesis by calcium ionophore was significantly reduced in the CERK^−/− MEFs. Our studies also demonstrate that the CERK^−/− mouse has adapted to loss of CERK in regards to airway hyper-responsiveness as compared with CERK siRNA treatment. Overall, we demonstrate that there are significant differences in eicosanoid levels in ex vivo CERK^−/− cells compared with wild-type counterparts, but the effect of the genetic ablation of CERK on eicosanoid synthesis and the serum levels of C1P was not apparent in vivo.     

Fatty acid transport protein 2 interacts with ceramide synthase 2 to promote ceramide synthesis

Dihydroceramide is a lipid molecule generated via the action of (dihydro)ceramide synthases (CerSs), which use two substrates, namely sphinganine and fatty acyl-CoAs. Sphinganine is generated via the sequential activity of two integral membrane proteins located in the endoplasmic reticulum. Less is known about the source of the fatty acyl-CoAs, although a number of cytosolic proteins in the pathways of acyl-CoA generation modulate ceramide synthesis via direct or indirect interaction with the CerSs. In this study, we demonstrate, by proteomic analysis of immunoprecipitated proteins, that fatty acid transporter protein 2 (FATP2) (also known as very long-chain acyl-CoA synthetase) directly interacts with CerS2 in mouse liver. Studies in cultured cells demonstrated that other members of the FATP family can also interact with CerS2, with the interaction dependent on both proteins being catalytically active. In addition, transfection of cells with FATP1, FATP2, or FATP4 increased ceramide levels although only FATP2 and 4 increased dihydroceramide levels, consistent with their known intracellular locations. Finally, we show that lipofermata, an FATP2 inhibitor which is believed to directly impact tumor cell growth via modulation of FATP2, decreased de novo dihydroceramide synthesis, suggesting that some of the proposed therapeutic effects of lipofermata may be mediated via (dihydro)ceramide rather than directly via acyl-CoA generation. In summary, our study reinforces the idea that manipulating the pathway of fatty acyl-CoA generation will impact a wide variety of down-stream lipids, not least the sphingolipids, which utilize two acyl-CoA moieties in the initial steps of their synthesis.     

Regulation of secretory transport by protein kinase D-mediated phosphorylation of the ceramide transfer protein

Protein kinase D (PKD) has been identified as a crucial regulator of secretory transport at the trans-Golgi network (TGN). Recruitment and activation of PKD at the TGN is mediated by the lipid diacylglycerol, a pool of which is generated by sphingomyelin synthase from ceramide and phosphatidylcholine. The nonvesicular transfer of ceramide from the endoplasmic reticulum to the Golgi complex is mediated by the lipid transfer protein CERT (ceramide transport). In this study, we identify CERT as a novel in vivo PKD substrate. Phosphorylation on serine 132 by PKD decreases the affinity of CERT toward its lipid target phosphatidylinositol 4-phosphate at Golgi membranes and reduces ceramide transfer activity, identifying PKD as a regulator of lipid homeostasis. We also show that CERT, in turn, is critical for PKD activation and PKD-dependent protein cargo transport to the plasma membrane. Thus, the interdependence of PKD and CERT is key to the maintenance of Golgi membrane integrity and secretory transport.     

Ceramide directly activates protein kinase C zeta to regulate a stress-activated protein kinase signaling complex

We have previously shown that interleukin 1 (IL-1)-receptor-generated ceramide induces growth arrest in smooth muscle pericytes by activating an upstream kinase in the stress-activated protein kinase (SAPK) cascade. We now report the mechanism by which ceramide activates the SAPK signaling pathway in human embryonic kidney cells (HEK-293). We demonstrate that ceramide activation of protein kinase C zeta (PKCzeta) mediates SAPK signal complex formation and subsequent growth suppression. Ceramide directly activates both immunoprecipitated and recombinant human PKCzeta in vitro. Additionally, ceramide activates SAPK activity, which is blocked with a dominant-negative mutant of PKCzeta. Co-immunoprecipitation studies reveal that ceramide induces the association of SAPK with PKCzeta, but not with PKCepsilon. In addition, ceramide treatment induces PKCzeta association with phosphorylated SEK and MEKK1, elements of the SAPK signaling complex. The biological role of ceramide to induce cell cycle arrest is mimicked by overexpression of a constitutively active PKCzeta. Together, these studies demonstrate that ceramide induces cell cycle arrest by enhancing the ability of PKCzeta to form a signaling complex with MEKK1, SEK, and SAPK.     

Regulation of insulin action by ceramide: dual mechanisms linking ceramide accumulation to the inhibition of Akt/protein kinase B

The sphingolipid ceramide negatively regulates insulin action by inhibiting Akt/protein kinase B (PKB), a serine/threonine kinase that is a central regulator of glucose uptake and anabolic metabolism. Despite considerable attention, the molecular mechanism accounting for this action of ceramide has remained both elusive and controversial. Herein we utilized deletion constructs encoding two different functional domains of Akt/PKB to identify which region of the enzyme conferred responsiveness to ceramide. Surprisingly the findings obtained with these separate domains reveal that ceramide blocks insulin stimulation of Akt/PKB by two independent mechanisms. First, using the isolated pleckstrin homology domain, we found that ceramide specifically blocks the translocation of Akt/PKB, but not its upstream activator phosphoinositide-dependent kinase-1, to the plasma membrane. Second, using a construct lacking this pleckstrin homology domain, which does not require translocation for activation, we found that ceramide stimulates the dephosphorylation of Akt/PKB by protein phosphatase 2A. Collectively these findings identify at least two independent mechanisms by which excessive ceramide accumulation in peripheral tissues could contribute to the development of insulin resistance. Moreover the results obtained provide a unifying theory to account for the numerous dissenting reports investigating the actions of ceramide toward Akt/PKB.     

Oenocytoid cell lysis to release prophenoloxidase is induced by eicosanoid via protein kinase C

Eicosanoids mediate insect cellular immune responses, which depend largely on phenoloxidase (PO) activity. In plasma, PO is activated by the proteolytic cleavage of proPO, which is stored in oenocytoids, a specific hemocyte type, of the beet armyworm, Spodoptera exigua. Eicosanoids induce an acute cell lysis of oenocytoids, which releases proPO into the plasma. We investigated an intracellular signal pathway following a functional interaction of eicosanoid(s) to a putative membrane receptor. U-73122 (a specific inhibitor of phospholipase C) inhibited oenocytoid lysis of S. exigua significantly after bacterial infection. We concluded that oenocytoid lysis required a certain level of calcium ion because EGTA (a calcium chelator) treatment inhibited cell lysis. Two protein kinase C (PKC) inhibitors (staurosporine and calphostin C) significantly inhibited the oenocytoid lysis. Oenocytoid lysis was likely induced by Na⁺ entry and subsequent osmotic shock because juvenile hormone analog, pyriproxyfen, which activates Na⁺-K⁺ ATPase and induces subsequent cell shrinkage, antagonized the effect of eicosaniod on cell lysis. Furthermore, ouabain (a specific Na⁺ pump inhibitor) significantly inhibited oenocytoid lysis. These results suggest that eicosanoid mediates oenocytoid lysis by activating the intracellular PKC pathway.     

The AMP-activated protein kinase prevents ceramide synthesis de novo and apoptosis in astrocytes

Fatty acids induce apoptosis in primary astrocytes by enhancing ceramide synthesis de novo. The possible role of the AMP-activated protein kinase (AMPK) in the control of apoptosis was studied in this model. Long-term stimulation of AMPK with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) prevented apoptosis. AICAR blunted fatty acid-mediated induction of serine palmitoyltransferase and ceramide synthesis de novo, without affecting fatty acid synthesis and oxidation. Prevention of ceramide accumulation by AICAR led to a concomitant blockade of the Raf-1/extracellular signal-regulated kinase cascade, which selectively mediates fatty acid-induced apoptosis. Data indicate that AMPK may protect cells from apoptosis induced by stress stimuli.     

Ceramide kinase is required for a normal eicosanoid response and the subsequent orderly migration of fibroblasts

In these studies, the role of ceramide-1-phosphate (C1P) in the wound-healing process was investigated. Specifically, fibroblasts isolated from mice with the known anabolic enzyme for C1P, ceramide kinase (CERK), ablated (CERK^−/− mice) and their wild-type littermates (CERK^+/+) were subjected to in vitro wound-healing assays. Simulation of mechanical trauma of a wound by scratching a monolayer of fibroblasts from CERK^+/+ mice demonstrated steadily increasing levels of arachidonic acid in a time-dependent manner in stark contrast to CERK^−/− fibroblasts. This observed difference was reflected in scratch-induced eicosanoid levels. Similar, but somewhat less intense, changes were observed in a more complex system utilizing skin biopsies obtained from CERK-null mice. Importantly, C1P levels increased during the early stages of human wound healing correlating with the transition from the inflammatory stage to the peak of the fibroplasia stage (e.g., proliferation and migration of fibroblasts). Finally, the loss of proper eicosanoid response translated into an abnormal migration pattern for the fibroblasts isolated from CERK^−/−. As the proper migration of fibroblasts is one of the necessary steps of wound healing, these studies demonstrate a novel requirement for the CERK-derived C1P in the proper healing response of wounds.     

Subcellular localization of ceramide kinase and ceramide kinase-like protein requires interplay of their Pleckstrin Homology domain-containing N-terminal regions together with C-terminal domains

Ceramide kinase (CERK) and the ceramide kinase-like protein (CERKL), two related members of the diacylglycerol kinase family, are ill-defined at the molecular level. In particular, what determines their distinctive subcellular localization is not well understood. Here we show that the Pleckstrin Homology (PH) domain of CERK, which is required for Golgi complex localization, can substitute for the N-terminal region of CERKL and allow for wild-type CERKL localization, which is typified by nucleolar accumulation. This demonstrates that determinants for localization of these two enzymes do not lie solely in their PH domain-containing N-terminal regions. Moreover, we present evidence for a previously unrecognized participation of CERK distal sequences in structural stability, localization and activity of the full-length protein. Progressive deletion of CERK and CERKL from the C-terminus revealed similar sequential organization in both proteins, with nuclear import signals in their N-terminal part, and nuclear export signals in their C-terminal part. Furthermore, mutagenesis of individual cysteine residues of a CERK-specific CXXXCXXC motif severely compromised both exportation of CERK from the nucleus and its association with the Golgi complex. Altogether, this work identifies conserved domains in CERK and CERKL as well as new determinants for their subcellular localization. It further suggests a nucleocytoplasmic shuttling mechanism for both proteins that may be defective in CERKL mutant proteins responsible for retinal degenerative diseases.     

Ceramide inhibits protein kinase B/Akt by promoting dephosphorylation of serine 473

The second messenger ceramide (N-alkylsphingosine) has been implicated in a host of cellular processes including growth arrest and apoptosis. Ceramide has been reported to have effects on both protein kinases and phosphatases and may constitute an important component of stress response in various tissues. We have examined in detail the relationship between ceramide signaling and the activation of an important signaling pathway, phosphatidylinositol (PI) 3-kinase and its downstream target, protein kinase B (PKB). PKB activation was observed following stimulation of cells with the cytokine granulocyte-macrophage colony-stimulating factor. Addition of cell-permeable ceramide analogs, C(2)- or C(6)-ceramide, caused a partial loss (50-60%) of PKB activation. This reduction was not a result of decreased PI(3,4,5)P(3) or PI(3,4)P(2) generation by PI 3-kinase. Two residues of PKB (threonine 308 and serine 473) require phosphorylation for maximal PKB activation. Serine 473 phosphorylation was consistently reduced by treatment with ceramide, whereas threonine 308 phosphorylation remained unaffected. In further experiments, ceramide appeared to accelerate serine 473 dephosphorylation, suggesting the activation of a phosphatase. Consistent with this, the reduction in serine 473 phosphorylation was inhibited by the phosphatase inhibitors okadaic acid and calyculin A. Surprisingly, threonine 308 phosphorylation was abolished in cells treated with these inhibitors, revealing a novel mechanism of regulation of threonine 308 phosphorylation. These results demonstrate that PI 3-kinase-dependent kinase 2-catalyzed phosphorylation of serine 473 is the principal target of a ceramide-activated phosphatase.     

Dendritic transport and localization of protein kinase Mzeta mRNA: implications for molecular memory consolidation

Protein kinase Mzeta (PKMzeta) is an atypical protein kinase C isoform that has been implicated in the protein synthesis-dependent maintenance of long term potentiation and memory storage in the brain. Synapse-associated kinases are uniquely positioned to promote enduring consolidation of structural and functional modifications at the synapse, provided that kinase mRNA is available on site for local input-specific translation. We now report that the mRNA encoding PKMzeta is rapidly transported and specifically localized to synaptodendritic neuronal domains. Transport of PKMzeta mRNA is specified by two cis-acting dendritic targeting elements (Mzeta DTEs). Mzeta DTE1, located at the interface of the 5'-untranslated region and the open reading frame, directs somato-dendritic export of the mRNA. Mzeta DTE2, in contrast, is located in the 3'-untranslated region and is required for delivery of the mRNA to distal dendritic segments. Colocalization with translational repressor BC1 RNA in hippocampal dendrites suggests that PKMzeta mRNA may be subject to translational control in local domains. Dendritic localization of PKMzeta mRNA provides a molecular basis for the functional integration of synaptic signal transduction and translational control pathways.     

Ceramide synthesis enhances transport of GPI-anchored proteins to the Golgi apparatus in yeast.

Inhibition of ceramide synthesis by a fungal metabolite, myriocin, leads to a rapid and specific reduction in the rate of transport of glycosylphosphatidylinositol (GPI)-anchored proteins to the Golgi apparatus without affecting transport of soluble or transmembrane proteins. Inhibition of ceramide biosynthesis also quickly blocks remodelling of GPI anchors to their ceramide-containing, mild base-resistant forms. These results suggest that the pool of ceramide is rapidly depleted from early points of the secretory pathway and that its presence at these locations enhances transport of GPI-anchored proteins specifically. A mutant that is resistant to myriocin reverses its effect on GPI-anchored protein transport without reversing its effects on ceramide synthesis and remodelling. Two hypotheses are proposed to explain the role of ceramide in the transport of GPI-anchored proteins.     

Regulation and traffic of ceramide 1-phosphate produced by ceramide kinase: comparative analysis to glucosylceramide and sphingomyelin

Ceramide 1-phosphate (C1P) has been characterized as a sphingolipid that participates in cell signaling. Although C1P synthesis is thought to occur via phosphorylation of ceramide by ceramide kinase (CerK), the processes that regulate C1P formation and fate remain largely unknown. In this study we analyzed bone marrow-derived macrophages (BMDM) from CerK-null mice (Cerk(-/-)) and found significant levels of C1P, suggesting that previously unrecognized pathways may also lead to C1P formation. After these experiments we used an overexpression system, BMDM from Cerk(-/-) mice, and short-chain fluorescent ceramides to trace CerK-dependent formation of C1P. Because the ceramide analogs can also be converted to glucosylceramide (GlcCer) and sphingomyelin (SM), they allowed us to directly compare all three metabolites. We found that C1P produced by CerK is turned over rapidly when serum is removed or upon calcium chelation, whereas GlcCer and SM are stable under these conditions. We further demonstrated that ceramide must be transported to the Golgi complex to be phosphorylated by CerK. Inhibition of the ceramide transfer protein slowed down SM formation without decreasing C1P, suggesting an alternate route of ceramide transport. Other experiments indicated that, like GlcCer and SM, C1P traffics along the secretory pathway to reach the plasma membrane. Furthermore, in BMDM C1P was secreted more readily than was GlcCer or SM. Altogether, our results indicate that CerK is essential to C1P formation via phosphorylation of Cer, providing the first insights into mechanisms underlying ceramide access to CerK and C1P trafficking as well as clarifying C1P as a signaling entity.     

Ceramide/long-chain base phosphate rheostat in Saccharomyces cerevisiae: regulation of ceramide synthesis by Elo3p and Cka2p

Sphingolipid precursors, namely, ceramide and long-chain base phosphates (LCBPs), are important growth regulators with often opposite effects on mammalian cells. A set of enzymes that regulate the levels of these precursors, referred to as a ceramide/LCBP rheostat, is conserved in all eukaryotes. In order to gain further insight into the function of the rheostat in Saccharomyces cerevisiae, we searched for mutants that are synthetically lethal with a deletion of the LCB3 gene encoding LCBP phosphatase. In addition to acquiring expected mutants lacking the LCBP lyase, the screen revealed elo3 (sur4) mutants that were defective in fatty acid elongation and cka2 mutants lacking the α′ subunit of the protein kinase CK2 (casein kinase). Both mutations affected the in vivo activity of the acyl coenzyme A (acyl-CoA)-dependent and fumonisin B₁-sensitive ceramide synthase (CS). The Elo3 protein is necessary for synthesis of C₂₆-CoA, which in wild-type yeast is a source of C₂₆ fatty acyls found in the ceramide moieties of all sphingolipids. In the in vitro assay, CS had a strong preference for acyl-CoAs containing longer acyl chains. This finding suggests that a block in the formation of C₂₆-CoA in yeast may cause a reduction in the conversion of LCBs into ceramides and lead to an overaccumulation of LCBPs that is lethal in strains lacking the Lcb3 phosphatase. In fact, elo3 mutants were found to accumulate high levels of LCBs and LCBPs. The cka2 mutants, on the other hand, exhibited only 25 to 30% of the in vitro CS activity found in wild-type membranes, indicating that the α′ subunit of CK2 kinase is necessary for full activation of CS. The cka2 mutants also accumulated high levels of LCBs and had elevated levels of LCBPs. In addition, both the elo3 and cka2 mutants showed increased sensitivity to the CS inhibitors australifungin and fumonisin B₁. Together, our data demonstrate that the levels of LCBPs in yeast are regulated by the rate of ceramide synthesis, which depends on CK2 kinase activity and is also strongly affected by the supply of C₂₆-CoA. This is the first evidence indicating the involvement of protein kinase in the regulation of de novo sphingolipid synthesis in any organism.     

Akt protein kinase inhibits non-apoptotic programmed cell death induced by ceramide.

A growing body of evidence now suggests that programmed cell death (PCD) occurs via non-apoptotic mechanisms as well as by apoptosis. In contrast to apoptosis, however, the molecular mechanisms involved in the regulation of non-apoptotic PCD remain only poorly understood. Here we show that ceramide induces a non-apoptotic PCD with a necrotic-like morphology in human glioma cells. Characteristically, the cell death was not accompanied by loss of the mitochondrial transmembrane potential, cytosolic release of cytochrome c from mitochondria, or the activation of the caspase cascade. Consistent with these characteristics, this ceramide-induced cell death was inhibited neither by the overexpression of Bcl-xL nor by the pan-caspase inhibitor zVAD-fmk. However, strikingly, the ceramide-induced non-apoptotic cell death was inhibited by the activation of the Akt/protein kinase B pathway through the expression of a constitutively active version of Akt. The results for the first time indicate that the Akt kinase, known to play an essential role in survival factor-mediated inhibition of apoptotic cell death, is also involved in the regulation of non-apoptotic PCD.     

Co-evolution of sphingomyelin and the ceramide transport protein CERT

Life creates many varieties of lipids. The choline-containing sphingophospholipid sphingomyelin (SM) exists ubiquitously or widely in vertebrates and lower animals, but is absent or rare in bacteria, fungi, protists, and plants. In the biosynthesis of SM, ceramide, which is synthesized in the endoplasmic reticulum, is transported to the Golgi region by the ceramide transport protein CERT, probably in a non-vesicular manner, and is then converted to SM by SM synthase, which catalyzes the reaction of phosphocholine transfer from phosphatidylcholine (PtdCho) to ceramide. Recent advances in genomics and lipidomics indicate that the phylogenetic occurrence of CERT and its orthologs is nearly parallel to that of SM. Based on the chemistry of lipids together with evolutionary aspects of SM and CERT, several concepts are here proposed. SM may serve as a chemically inert and robust, but non-covalently interactive lipid class at the outer leaflet of the plasma membrane. The functional domains and peptidic motifs of CERT are separated by exon units, suggesting an exon-shuffling mechanism for the generation of an ancestral CERT gene. CERT may have co-evolved with SM to bypass a competing metabolic reaction at the bifurcated point in the anabolism of ceramide. Human CERT is identical to the splicing variant of human Goodpasture antigen-binding protein (GPBP) annotated as an extracellular non-canonical serine/threonine protein kinase. The relationship between CERT and GPBP has also been discussed from an evolutionary aspect. Moreover, using an analogy of “compatible (or osmoprotective) solutes” that can accumulate to very high concentrations in the cytosol without denaturing proteins, choline phospholipids such as PtdCho and SM may act as compatible phospholipids in biomembranes. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.     

Involvement of protein kinase C-regulated ceramide generation in inostamycin-induced apoptosis

Activation of caspases is commonly involved in the apoptosis induced by various anticancer drugs. However, the upstream events leading to the activation of caspases seem to be specific to each anticancer drug. In the present study, we examined the possible involvement of protein kinase C (PKC) and ceramide generation in caspase-3(-like) protease activation induced by inostamycin, a phosphatidylinositol synthesis inhibitor. Treatment of cells with 12-O-tetradecanoyl phorbol-13-acetate (TPA), an activator of PKC, suppressed the release of cytochrome c from mitochondria and the activation of caspase-3(-like) proteases in inostamycin-treated cells, but not in other anticancer drug-treated cells. Inostamycin induced the elevation of intracellular ceramide levels, and fumonisin B1, an inhibitor of ceramide synthase, inhibited inostamycin-induced cytochrome c release, caspase-3(-like) protease activation, and apoptosis. Moreover, TPA also inhibited inostamycin-induced ceramide synthesis. Taken together, our results suggest that inostamycin-induced apoptosis is mediated by PKC-regulated ceramide generation, leading to the activation of a caspase cascade.     

Regulation of brain-type creatine kinase by AMP-activated protein kinase: Interaction,phosphorylation and ER localization

AMP-activated protein kinase (AMPK) and cytosolic brain-type creatine kinase (BCK) cooperate under energy stress to compensate for loss of adenosine triphosphate (ATP) by either stimulating ATP-generating and inhibiting ATP-consuming pathways, or by direct ATP regeneration from phosphocreatine, respectively. Here we report on AMPK-dependent phosphorylation of BCK from different species identified by in vitro screening for AMPK substrates in mouse brain. Mass spectrometry, protein sequencing, and site-directed mutagenesis identified Ser6 as a relevant residue with one site phosphorylated per BCK dimer. Yeast two-hybrid analysis revealed interaction of active AMPK specifically with non-phosphorylated BCK. Pharmacological activation of AMPK mimicking energy stress led to BCK phosphorylation in astrocytes and fibroblasts, as evidenced with a highly specific phospho-Ser6 antibody. BCK phosphorylation at Ser6 did not affect its enzymatic activity, but led to the appearance of the phosphorylated enzyme at the endoplasmic reticulum (ER), close to the ER calcium pump, a location known for muscle-type cytosolic creatine kinase (CK) to support Ca²⁺-pumping.