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Charles E. Chalfant - One of the best experts on this subject based on the ideXlab platform.
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The Coordination of Prostaglandin E 2 Production by Sphingosine-1-phosphate and Ceramide-1-phosphate
2020Co-Authors: Benjamin J Pettus, Yusuf A Hannun, Lina M. Obeid, Charles E. Chalfant, Kazuyuki Kitatani, Tarek A Taha, Toshihiko Kawamori, Jacek Bielawski, Ralph Johnson, H VeteranAbstract:ABSTRACT The ability of pro-inflammatory cytokines such as interleukin-1 (IL-1) to induce the major inflammatory mediator prostaglandin (PG) E 2 depends on the activation of two rate-limiting enzymes, phospholipase A 2 (PLA 2 ) and cyclooxygenase 2 (COX-2). PLA 2 acts to generate arachidonic acid, which serves as the precursor substrate for COX-2 in the metabolic pathway leading to PGE 2 production. However, less is known about the mechanisms that coordinate the regulation of these two enzymes. We have provided prior evidence that sphingosine Kinase 1 and its bioactive lipid product sphingosine-1-phosphate (S1P) mediate the effects of cytokines on COX-2 induction, whereas Ceramide Kinase and its distinct product, Ceramide-1-phosphate (C1P), are required for the activation and translocation of cPLA 2 (FASE
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Ceramide Kinase is required for a normal eicosanoid response and the subsequent orderly migration of fibroblasts
Journal of Lipid Research, 2014Co-Authors: Dayanjan S. Wijesinghe, Jennifer A. Mietla, Charles E. Chalfant, Matthew Brentnall, Alexis L Hoeferlin, Robert F Diegelmann, Lawrence H BoiseAbstract: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.
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nonvesicular trafficking of Ceramide 1 phosphate by a lipid transfer protein that regulates eicosanoid production
Biophysical Journal, 2014Co-Authors: Dhirendra K. Simanshu, Dayanjan S. Wijesinghe, Julian G. Molotkovsky, Xiuhong Zhai, Lucy Malinina, Shrawan K. Mishra, Edward H. Hinchcliffe, Ravi Kanth Kamlekar, Xianqiong Zou, Charles E. ChalfantAbstract:Phosphorylated sphingolipids such as Ceramide-1-phosphate (C1P) and sphingosine-1-phosphate (S1P) have emerged as key regulators of cell growth, survival, migration, and inflammation. C1P produced by Ceramide Kinase is an activator of group IVA cytosolic phospholipase A2α (cPLA2α), the rate-limiting releaser of arachidonic acid used for pro-inflammatory eicosanoid production. To modulate eicosanoid action and avoid the damaging effects of chronic inflammation, cells require efficient targeting, trafficking, and presentation of C1P to specific cellular sites. Vesicular trafficking is likely but nonvesicular mechanisms for C1P sensing, transfer, and presentation remain unexplored. We have identified a ubiquitously-expressed lipid transfer protein (CPTP) that can specifically transfer C1P between membranes. Crystal structures establish C1P binding via a novel surface-localized, phosphate headgroup recognition center connected to an interior hydrophobic pocket that adaptively expands to ensheath differing-length lipid chains using a cleft-like gating mechanism. The two-layered, α helically-dominated 'sandwich' topology identifies CPTP as the prototype for a new GLTP-fold subfamily. CPTP resides in the cell cytosol but associates with the trans-Golgi/TGN, nucleus, and plasma membrane. RNAi-induced CPTP depletion elevates C1P steady-state levels and alters Golgi cisternae stack morphology. The resulting C1P decrease in plasma membranes and increase in the Golgi complex stimulate cPLA2α release of arachidonic acid, triggering pro-inflammatory eicosanoid generation. [Support: NCI CA121493 (DJP & REB), NIGMS GM45928 (REB), NIGMS GM072754 (EHH), NCI CA154314 (CEC), VA Merit Award (CEC), VA Research Career Scientist Award (CEC), VA Career Devel. Award (DSW), NRS-T32/NIGMS 008695 (DSW), Spanish Ministerio de Ciencia e Innovacion BFU2010-17711 (LM), Russian Fnd. for Basic Research #12-04-00168 (JGM), Abby Rockefeller Mauze Trust (DJP), Maloris Fnd. (DJP), and Hormel Fnd. (REB); Equal contributors to the work: DKS, RKK, & DSW; Corresponding authors: REB, EHH, CEC, DJP]
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non vesicular trafficking by a Ceramide 1 phosphate transfer protein regulates eicosanoids
Nature, 2013Co-Authors: Dhirendra K. Simanshu, Dayanjan S. Wijesinghe, Charles E. Chalfant, Julian G. Molotkovsky, Xiuhong Zhai, Lucy Malinina, Shrawan K. Mishra, Edward H. Hinchcliffe, Ravi Kanth KamlekarAbstract:Phosphorylated sphingolipids Ceramide-1-phosphate (C1P) and sphingosine-1-phosphate (S1P) have emerged as key regulators of cell growth, survival, migration and inflammation. C1P produced by Ceramide Kinase is an activator of group IVA cytosolic phospholipase A2α (cPLA2α), the rate-limiting releaser of arachidonic acid used for pro-inflammatory eicosanoid production, which contributes to disease pathogenesis in asthma or airway hyper-responsiveness, cancer, atherosclerosis and thrombosis. To modulate eicosanoid action and avoid the damaging effects of chronic inflammation, cells require efficient targeting, trafficking and presentation of C1P to specific cellular sites. Vesicular trafficking is likely but non-vesicular mechanisms for C1P sensing, transfer and presentation remain unexplored. Moreover, the molecular basis for selective recognition and binding among signalling lipids with phosphate headgroups, namely C1P, phosphatidic acid or their lyso-derivatives, remains unclear. Here, a ubiquitously expressed lipid transfer protein, human GLTPD1, named here CPTP, is shown to specifically transfer C1P between membranes. Crystal structures establish C1P binding through a novel surface-localized, phosphate headgroup recognition centre connected to an interior hydrophobic pocket that adaptively expands to ensheath differing-length lipid chains using a cleft-like gating mechanism. The two-layer, α-helically-dominated 'sandwich' topology identifies CPTP as the prototype for a new glycolipid transfer protein fold subfamily. CPTP resides in the cell cytosol but associates with the trans-Golgi network, nucleus and plasma membrane. RNA interference-induced CPTP depletion elevates C1P steady-state levels and alters Golgi cisternae stack morphology. The resulting C1P decrease in plasma membranes and increase in the Golgi complex stimulates cPLA2α release of arachidonic acid, triggering pro-inflammatory eicosanoid generation.
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Characterization of eicosanoid synthesis in a genetic ablation model of Ceramide Kinase.
Journal of lipid research, 2013Co-Authors: Jennifer A. Mietla, Dayanjan S. Wijesinghe, L. Alexis Hoeferlin, Michael D. Shultz, Ramesh Natarajan, Alpha A. Fowler, Charles E. ChalfantAbstract: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.
Frederic Bornancin - One of the best experts on this subject based on the ideXlab platform.
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the Ceramide Kinase inhibitor nvp 231 inhibits breast and lung cancer cell proliferation by inducing m phase arrest and subsequent cell death
British Journal of Pharmacology, 2014Co-Authors: Oleksandr Pastukhov, Stephanie Schwalm, Uwe Zangemeisterwittke, Doriano Fabbro, Frederic Bornancin, Lukasz Japtok, Burkhard Kleuser, Josef Pfeilschifter, Andrea HuwilerAbstract:Background and Purpose Ceramide Kinase (CerK) catalyzes the generation of Ceramide-1-phosphate which may regulate various cellular functions, including inflammatory reactions and cell growth. Here, we studied the effect of a recently developed CerK inhibitor, NVP-231, on cancer cell proliferation and viability and investigated the role of cell cycle regulators implicated in these responses. Experimental Approach The breast and lung cancer cell lines MCF-7 and NCI-H358 were treated with increasing concentrations of NVP-231 and DNA synthesis, colony formation and cell death were determined. Flow cytometry was performed to analyse cell cycle distribution of cells and Western blot analysis was used to detect changes in cell cycle regulator expression and activation. Key Results In both cell lines, NVP-231 concentration-dependently reduced cell viability, DNA synthesis and colony formation. Moreover it induced apoptosis, as measured by increased DNA fragmentation and caspase-3 and caspase-9 cleavage. Cell cycle analysis revealed that NVP-231 decreased the number of cells in S phase and induced M phase arrest with an increased mitotic index, as determined by increased histone H3 phosphorylation. The effect on the cell cycle was even more pronounced when NVP-231 treatment was combined with staurosporine. Finally, overexpression of CerK protected, whereas down-regulation of CerK with siRNA sensitized, cells for staurosporine-induced apoptosis. Conclusions and Implications Our data demonstrate for the first time a crucial role for CerK in the M phase control in cancer cells and suggest its targeted inhibition, using drugs such as NVP-231, in combination with conventional pro-apoptotic chemotherapy.
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Ceramide Kinase the first decade
Cellular Signalling, 2011Co-Authors: Frederic BornancinAbstract:Abstract It has been some 20 years since the initial discovery of Ceramide 1-phosphate (C1P) and nearly a decade since Ceramide Kinase (CERK) was cloned. Many studies have shown that C1P is important for membrane biology and for the regulation of membrane-bound proteins, and the CERK enzyme has appeared to be tightly regulated in order to control both Ceramide levels and production of C1P. Furthermore, C1P made by CERK has emerged as a genuine signalling entity. However, it represents only part of the C1P pool that is available in the cell, therefore suggesting that alternative unknown C1P-producing mechanisms may also play a role. Recent technological developments for measuring complex sphingolipids in biological samples, together with the availability of Cerk-deficient animals as well as potent CERK inhibitors, have now provided new grounds for investigating C1P biology further. Here, we will review the current understanding of CERK and C1P in terms of biochemistry and functional implications, with particular attention to C1P produced by CERK.
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ovalbumin induced plasma interleukin 4 levels are reduced in Ceramide Kinase deficient do11 10 rag1 mice
Lipids in Health and Disease, 2010Co-Authors: Satoru Niwa, Thomas Baumruker, Andreas Billich, Nicole Urtz, Frederic BornancinAbstract:Ceramide Kinase (CERK) produces the bioactive lipid Ceramide-1-phosphate (C1P) and is a key regulator of Ceramide and dihydroCeramide levels. It is likely that CERK and C1P play a role in inflammatory processes but the cells involved and the mechanisms used remain to be clarified. In particular, the impact of CERK on T-cell biology has not been studied so far. Here, we used Cerk-/- mice backcrossed with DO11.10/RAG1-/- mice to probe the effect of CERK ablation on T-cell activation. Levels of interleukin (IL)-2, IL-4, IL-5, IL-13, of tumor necrosis factor (TNF)-α, and of interferon (INF)-γ were recorded following ovalbumin challenge in vivo and using ovalbumin-treated splenocytes ex- vivo. Absence of CERK led to a significant decrease in the production of IL-4, thus suggesting that CERK may polarize T cells towards the TH2 cell subtype. However, the importance of CERK to TH2 cell biology will have to be investigated further because in a model of asthma, which is TH2-cell driven, Cerk-/- mice responded like wild-type animals.
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a secondary assay for Ceramide Kinase inhibitors based on cell growth inhibition by short chain Ceramides
Analytical Biochemistry, 2009Co-Authors: Christine Graf, Philipp Rovina, Frederic BornancinAbstract:We recently reported that ectopic expression of Ceramide Kinase (CerK) in various cell lines increases their sensitivity to cell death induced by the exogenous addition of short-chain (e.g., C2) Ceramides (Cer). Here we show that this higher sensitivity results from CerK catalytic activity and production of C2-Ceramide 1-phosphate (C2-C1P). If CerK activity is inhibited by the potent inhibitor NVP-231, C2-C1P is not produced and viability returns to control levels. The EC(50) of NVP-231 in this assay is in the low nanomolar range, consistent with the IC(50) determined in activity assays in vitro using purified CerK. NVP-995, a structurally related but inactive compound, does not protect against C2-Cer-induced cell death. This assay is robust and easy to implement and scale up, thereby providing a valuable secondary screen assay for CerK inhibitors.
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Targeting Ceramide Metabolism with a Potent and Specific Ceramide Kinase Inhibitor
Molecular Pharmacology, 2008Co-Authors: Christine Graf, Philipp Rovina, Thomas Baumruker, Andreas Billich, Martin Klumpp, Michael Habig, Berndt Oberhauser, Frederic BornancinAbstract:Ceramide Kinase (CerK) produces the bioactive lipid Ceramide-1-phosphate (C1P) and appears as a key enzyme for controlling Ceramide levels. In this study, we discovered and characterized adamantane-1-carboxylic acid (2-benzoylamino-benzothiazol-6-yl)amide (NVP-231), a potent, specific, and reversible CerK inhibitor that competitively inhibits binding of Ceramide to CerK. NVP-231 is active in the low nanomolar range on purified as well as cellular CerK and abrogates phosphorylation of Ceramide, resulting in decreased endogenous C1P levels. When combined with another Ceramide metabolizing inhibitor, such as tamoxifen, NVP-231 synergistically increased Ceramide levels and reduced cell growth. Therefore, NVP-231 represents a novel and promising compound for controlling Ceramide metabolism that may provide insight into CerK physiological function.
Dayanjan S. Wijesinghe - One of the best experts on this subject based on the ideXlab platform.
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Ceramide Kinase is required for a normal eicosanoid response and the subsequent orderly migration of fibroblasts
Journal of Lipid Research, 2014Co-Authors: Dayanjan S. Wijesinghe, Jennifer A. Mietla, Charles E. Chalfant, Matthew Brentnall, Alexis L Hoeferlin, Robert F Diegelmann, Lawrence H BoiseAbstract: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.
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nonvesicular trafficking of Ceramide 1 phosphate by a lipid transfer protein that regulates eicosanoid production
Biophysical Journal, 2014Co-Authors: Dhirendra K. Simanshu, Dayanjan S. Wijesinghe, Julian G. Molotkovsky, Xiuhong Zhai, Lucy Malinina, Shrawan K. Mishra, Edward H. Hinchcliffe, Ravi Kanth Kamlekar, Xianqiong Zou, Charles E. ChalfantAbstract:Phosphorylated sphingolipids such as Ceramide-1-phosphate (C1P) and sphingosine-1-phosphate (S1P) have emerged as key regulators of cell growth, survival, migration, and inflammation. C1P produced by Ceramide Kinase is an activator of group IVA cytosolic phospholipase A2α (cPLA2α), the rate-limiting releaser of arachidonic acid used for pro-inflammatory eicosanoid production. To modulate eicosanoid action and avoid the damaging effects of chronic inflammation, cells require efficient targeting, trafficking, and presentation of C1P to specific cellular sites. Vesicular trafficking is likely but nonvesicular mechanisms for C1P sensing, transfer, and presentation remain unexplored. We have identified a ubiquitously-expressed lipid transfer protein (CPTP) that can specifically transfer C1P between membranes. Crystal structures establish C1P binding via a novel surface-localized, phosphate headgroup recognition center connected to an interior hydrophobic pocket that adaptively expands to ensheath differing-length lipid chains using a cleft-like gating mechanism. The two-layered, α helically-dominated 'sandwich' topology identifies CPTP as the prototype for a new GLTP-fold subfamily. CPTP resides in the cell cytosol but associates with the trans-Golgi/TGN, nucleus, and plasma membrane. RNAi-induced CPTP depletion elevates C1P steady-state levels and alters Golgi cisternae stack morphology. The resulting C1P decrease in plasma membranes and increase in the Golgi complex stimulate cPLA2α release of arachidonic acid, triggering pro-inflammatory eicosanoid generation. [Support: NCI CA121493 (DJP & REB), NIGMS GM45928 (REB), NIGMS GM072754 (EHH), NCI CA154314 (CEC), VA Merit Award (CEC), VA Research Career Scientist Award (CEC), VA Career Devel. Award (DSW), NRS-T32/NIGMS 008695 (DSW), Spanish Ministerio de Ciencia e Innovacion BFU2010-17711 (LM), Russian Fnd. for Basic Research #12-04-00168 (JGM), Abby Rockefeller Mauze Trust (DJP), Maloris Fnd. (DJP), and Hormel Fnd. (REB); Equal contributors to the work: DKS, RKK, & DSW; Corresponding authors: REB, EHH, CEC, DJP]
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non vesicular trafficking by a Ceramide 1 phosphate transfer protein regulates eicosanoids
Nature, 2013Co-Authors: Dhirendra K. Simanshu, Dayanjan S. Wijesinghe, Charles E. Chalfant, Julian G. Molotkovsky, Xiuhong Zhai, Lucy Malinina, Shrawan K. Mishra, Edward H. Hinchcliffe, Ravi Kanth KamlekarAbstract:Phosphorylated sphingolipids Ceramide-1-phosphate (C1P) and sphingosine-1-phosphate (S1P) have emerged as key regulators of cell growth, survival, migration and inflammation. C1P produced by Ceramide Kinase is an activator of group IVA cytosolic phospholipase A2α (cPLA2α), the rate-limiting releaser of arachidonic acid used for pro-inflammatory eicosanoid production, which contributes to disease pathogenesis in asthma or airway hyper-responsiveness, cancer, atherosclerosis and thrombosis. To modulate eicosanoid action and avoid the damaging effects of chronic inflammation, cells require efficient targeting, trafficking and presentation of C1P to specific cellular sites. Vesicular trafficking is likely but non-vesicular mechanisms for C1P sensing, transfer and presentation remain unexplored. Moreover, the molecular basis for selective recognition and binding among signalling lipids with phosphate headgroups, namely C1P, phosphatidic acid or their lyso-derivatives, remains unclear. Here, a ubiquitously expressed lipid transfer protein, human GLTPD1, named here CPTP, is shown to specifically transfer C1P between membranes. Crystal structures establish C1P binding through a novel surface-localized, phosphate headgroup recognition centre connected to an interior hydrophobic pocket that adaptively expands to ensheath differing-length lipid chains using a cleft-like gating mechanism. The two-layer, α-helically-dominated 'sandwich' topology identifies CPTP as the prototype for a new glycolipid transfer protein fold subfamily. CPTP resides in the cell cytosol but associates with the trans-Golgi network, nucleus and plasma membrane. RNA interference-induced CPTP depletion elevates C1P steady-state levels and alters Golgi cisternae stack morphology. The resulting C1P decrease in plasma membranes and increase in the Golgi complex stimulates cPLA2α release of arachidonic acid, triggering pro-inflammatory eicosanoid generation.
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Characterization of eicosanoid synthesis in a genetic ablation model of Ceramide Kinase.
Journal of lipid research, 2013Co-Authors: Jennifer A. Mietla, Dayanjan S. Wijesinghe, L. Alexis Hoeferlin, Michael D. Shultz, Ramesh Natarajan, Alpha A. Fowler, Charles E. ChalfantAbstract: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.
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the role of Ceramide 1 phosphate in biological functions
Handbook of experimental pharmacology, 2013Co-Authors: Alexis L Hoeferlin, Dayanjan S. Wijesinghe, Charles E. ChalfantAbstract:In mammalian cells, cermide-1-phosphate (C1P) is produced via the ATP-dependent mechanism of converting Ceramide to C1P by the enzyme, Ceramide Kinase (CERK). CERK was first described as a calcium-stimulated lipid Kinase that co-purified with brain synaptic vesicles, and to date, CERK is the only identified mammalian enzyme known to produce C1P in cells. C1P has steadily emerged as a bioactive sphingolipid involved in cell proliferation, macrophage migration, and inflammatory events. The recent generation of the CERK knockout mouse and the development of CERK inhibitors have furthered our current understanding of CERK-derived C1P in regulating biological processes. In this chapter, the history of C1P as well as the biological functions attributed to C1P are reviewed.
Yasuyuki Igarashi - One of the best experts on this subject based on the ideXlab platform.
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Ceramide Kinase deficiency improves diet induced obesity and insulin resistance
FEBS Letters, 2012Co-Authors: Susumu Mitsutake, Takafumi Kohama, Tomomi Date, Hazuki Yokota, Masako Sugiura, Yasuyuki IgarashiAbstract:Ceramide Kinase (CERK) is an enzyme that phosphorylates Ceramide to produce Ceramide 1-phosphate. Recently, evidence has emerged that CERK has a role in inflammatory signaling of immune cells. Since obesity is accompanied by chronic, low-grade inflammation, we examined whether CERK might be involved using CERK-null mice. We determined that CERK deficiency suppresses diet-induced increases in body weight, and improves glucose intolerance. Furthermore, we demonstrated that CERK deficiency attenuates MCP-1/CCR2 signaling in macrophages infiltrating the adipose tissue, resulting in the suppression of inflammation in adipocytes, which might otherwise lead to obesity and diabetes.
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the generation and behavioral analysis of Ceramide Kinase null mice indicating a function in cerebellar purkinje cells
Biochemical and Biophysical Research Communications, 2007Co-Authors: Susumu Mitsutake, Mariko Kato, Takafumi Kohama, Masako Sugiura, Urara Yokose, Ichiro Matsuoka, Kazunori Fujimoto, Yasuke Ando, Yasuyuki IgarashiAbstract:The discovery of Ceramide Kinase (CerK), which phosphorylates Ceramide (Cer) to Ceramide 1-phisphate (C1P), established a new pathway for Cer metabolism. Among mouse tissues, brain contains the highest CerK activity. In this study, we found that CerK is highly expressed in cerebellar Purkinje cells. Since Purkinje cells are important for motor-related behaviors, we generated CerK-null mice and performed behavioral analyses. The CerK-null mice were healthy, and displayed no histological abnormalities. The mice lost CerK activity completely, suggesting that CerK is the only enzyme that phosphorylate Cer. However, cellular C1P levels were not different between the CerK-null and wild-type mice, indicating the presence of other C1P-producing pathway. The general motor-coordination was not impaired in the CerK-null mice, but emotional behavior was slightly affected. Our findings suggest that CerK is not necessary for survival at an individual level, but might be involved in higher brain function related to emotion.
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identification of a nuclear localization signal in the retinitis pigmentosa mutated rp26 protein Ceramide Kinase like protein
Biochemical and Biophysical Research Communications, 2006Co-Authors: Yuichi Inagaki, Susumu Mitsutake, Yasuyuki IgarashiAbstract:Retinitis pigmentosa (RP) is a genetically heterogeneous disease characterized by degeneration of the retina. A mutation in a new Ceramide Kinase (CERK) homologous gene, named CERK-like protein (CERKL), was found to cause autosomal recessive retinitis pigmentosa (RP26). Here, we show a point mutation of one of two putative nuclear localization signal (NLS) sequences inhibited the nuclear localization of the protein. Furthermore, the tetra-GFP-tagged NLS, which cannot passively enter the nucleus, was observed not only in the nucleus but also in the nucleolus. Our results provide the first evidence of the active nuclear import of CERKL and suggest that the identified NLS might be responsible for nucleolar retention of the protein. As recent studies have shown other RP-related proteins are localized in the nucleus or the nucleolus, our identification of NLS in CERKL suggests that CERKL likely plays important roles for retinal functions in the nucleus and the nucleolus.
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the interaction between the pleckstrin homology domain of Ceramide Kinase and phosphatidylinositol 4 5 bisphosphate regulates the plasma membrane targeting and Ceramide 1 phosphate levels
Biochemical and Biophysical Research Communications, 2006Co-Authors: Tack-joong Kim, Susumu Mitsutake, Yasuyuki IgarashiAbstract:Ceramide Kinase (CERK) converts Ceramide to Ceramide-1-phosphate (C1P), which has recently emerged as a new bioactive molecule capable of regulating diverse cellular functions. The N-terminus of the CERK protein encompasses a sequence motif known as a pleckstrin homology (PH) domain. Although the PH domain was previously demonstrated to be an important domain for the subcellular localization of CERK, the precise properties of this domain remained unclear. In this study, we reveal that the PH domain of CERK exhibits high affinity for phosphatidylinositol 4,5-bisphosphate (PI(4,5)P{sub 2}), among other lipids. Furthermore, in COS7 cells, GFP-fused CERK translocated rapidly from the cytoplasm to the plasma membrane in response to hyper-osmotic stress, which is known to increase the intracellular PI(4,5)P{sub 2} levels, whereas a PH domain deletion mutant did not. Additionally, in [{sup 32}P]orthophosphate-labeled COS7 cells, the translocation of CERK to the plasma membrane induced a 2.8-fold increase in C1P levels. The study presented here provides insight into the crucial role of the CERK-PH domain in plasma membrane targeting, through its binding to PI(4,5)P{sub 2}, and subsequent induction of C1P production in the vicinity of the membrane.
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calmodulin is involved in the ca2 dependent activation of Ceramide Kinase as a calcium sensor
Journal of Biological Chemistry, 2005Co-Authors: Susumu Mitsutake, Yasuyuki IgarashiAbstract:We recently demonstrated that the activation of Ceramide Kinase (CERK) and the formation of its product, Ceramide 1-phosphate (C1P), are necessary for the degranulation pathway in mast cells and that the Kinase activity of this enzyme is completely dependent on the intracellular concentration of Ca2+ (Mitsutake, S., Kim, T.-J., Inagaki, Y., Kato, M., Yamashita, T., and Igarashi, Y. (2004) J. Biol. Chem. 279, 17570-17577). Despite the demonstrated importance of Ca2+ as a regulator of CERK activity, there are no apparent binding domains in the enzyme and the regulatory mechanism has not been well understood. In the present study, we found that calmodulin (CaM) is involved in the Ca2+-dependent activation of CERK. The CaM antagonist W-7 decreased both CERK activity and intracellular C1P formation. Additionally, exogenously added CaM enhanced CERK activity even at low concentrations of Ca2+. The CERK protein was co-immunoprecipitated with an anti-CaM antibody, indicating formation of intracellular CaM·CERK complexes. An in vitro CaM binding assay also demonstrated Ca2+-dependent binding of CaM to CERK. These results strongly suggest that CaM acts as a Ca2+ sensor for CERK. Furthermore, a CaM binding assay using various mutants of CERK revealed that the binding site of CERK is located within amino acids 422-435. This region appears to include a type 1-8-14B CaM binding motif and is predicted to form an amphipathic helical wheel, which is utilized in CaM recognition. The expression of a deletion mutant of CERK that contained the CaM binding domain but lost CERK activity inhibited the Ca2+-dependent C1P formation. These results suggest that this domain could saturate the CaM and hence block Ca2+-dependent activation of CERK. Finally, we reveal that in mast cell degranulation CERK acts downstream of CaM, similar to CaM-dependent protein Kinase II, which had been assumed to be the main target of CaM in mast cells.
Sarah Spiegel - One of the best experts on this subject based on the ideXlab platform.
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Ceramide Kinase regulates growth and survival of a549 human lung adenocarcinoma cells
FEBS Letters, 2007Co-Authors: Poulami Mitra, Michael Maceyka, Sheldon Milstien, Charles E. Chalfant, Nadia F. Lamour, Shawn G Payne, Sarah SpiegelAbstract:Ceramide-1-phosphate (C1P) is emerging as a new addition to the family of bioactive sphingolipid metabolites. At low concentrations, C1P enhanced survival of NIH 3T3 fibroblasts and A549 lung cancer cells, while at high concentrations, it reduced survival and induced apoptosis. Apoptosis correlated with degradation of C1P to pro-apoptotic Ceramide. To examine the role of endogenous C1P, expression of Ceramide Kinase, the enzyme that produces C1P, was downregulated, which reduced cellular proliferation, progression into S phase and enhanced apoptosis induced by serum starvation. Our results suggest that Ceramide Kinase determines the balance between pro-apoptotic Ceramide and anti-apoptotic C1P to regulate cell fate, reminiscent of its function in plants.
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sphingosine 1 phosphate and Ceramide 1 phosphate expanding roles in cell signaling
Journal of Cell Science, 2005Co-Authors: Charles E. Chalfant, Sarah SpiegelAbstract:The phosphorylated sphingolipid metabolites sphingosine 1-phosphate (S1P) and Ceramide 1-phosphate (C1P) have emerged as potent bioactive agents. Recent studies have begun to define new biological functions for these lipids. Generated by sphingosine Kinases and Ceramide Kinase, they control numerous aspects of cell physiology, including cell survival and mammalian inflammatory responses. Interestingly, S1P is involved in cyclooxygenase-2 induction and C1P is required for the activation and translocation of cPLA2. This suggests that these two sphingolipid metabolites may act in concert to regulate production of eicosanoids, important inflammatory mediators. Whereas S1P functions mainly via G-protein-coupled receptors, C1P appears to bind directly to targets such as cPLA2 and protein phosphatase 1/2A. S1P probably also has intracellular targets, and in plants it appears to directly regulate the G protein α subunit GPA1.
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Ceramide Kinase mediates cytokine and calcium ionophore induced arachidonic acid release
Journal of Biological Chemistry, 2003Co-Authors: Benjamin J Pettus, Alicia Bielawska, Patrick Roddy, Sarah Spiegel, Yusuf A Hannun, Charles E. ChalfantAbstract:Despite the importance of prostaglandins, little is known about the regulation of prostanoid synthesis proximal to the activation of cytosolic phospholipase A2, the initial rate-limiting step. In this study, Ceramide-1-phosphate (C-1-P) was shown to be a specific and potent inducer of arachidonic acid (AA) and prostanoid synthesis in cells. This study also demonstrates that two well established activators of AA release and prostanoid synthesis, the cytokine, interleukin-1β (IL-1β), and the calcium ionophore, A23187, induce an increase in C-1-P levels within the relevant time-frame of AA release. Furthermore, the enzyme responsible for the production of C-1-P in mammalian cells, Ceramide Kinase, was activated in response to IL-1β and A23187. RNA interference targeted to Ceramide Kinase specifically down-regulated Ceramide Kinase mRNA and activity with a concomitant decrease of AA release in response to IL-1β and A23187. Down-regulation of Ceramide Kinase had no effect on AA release induced by exogenous C-1-P. Collectively, these results indicate that Ceramide Kinase, via the formation of C-1-P, is an upstream modulator of phospholipase A2 activation. This study identifies previously unknown roles for Ceramide Kinase and its product, C-1-P, in AA release and production of eicosanoids and provides clues for potential new targets to block inflammatory responses.
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A specific Ceramide Kinase assay to measure cellular levels of Ceramide.
Analytical Biochemistry, 2003Co-Authors: Meryem Bektas, Puneet S. Jolly, Sheldon Milstien, Sarah SpiegelAbstract:Human Ceramide Kinase was recently cloned and characterized. Recombinant Ceramide Kinase is highly active and Ceramide is the only lipid that it phosphorylates, indicating that it should be useful for the measurement of Ceramide levels in biological samples by conversion to Ceramide-1-phosphate, in a manner analogous to that of the widely used Escherichia coli diacylglycerol Kinase method. Using recombinant Ceramide Kinase, we have now developed a rapid and specific enzymatic method to quantify mass levels of long-chain Ceramides in cellular lipid extracts. This new Ceramide Kinase assay is more specific than the commonly used diacylglycerol Kinase method because the ubiquitous lipid diacylglycerol, the preferred substrate for diacyglycerol Kinase which is usually present at higher concentrations than Ceramide and can interfere with Ceramide phosphorylation, is completely inactive with Ceramide Kinase. Moreover, this high specificity eliminates the need for analysis of the lipid product by thin-layer chromatography since Ceramide-1-phosphate is the only radiolabeled lipid in organic solvent extracts of Ceramide Kinase reactions.
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Ceramide Kinase a novel lipid Kinase molecular cloning and functional characterization
Journal of Biological Chemistry, 2002Co-Authors: Masako Sugiura, Sarah Spiegel, Keita Kono, Hong Liu, Tetsuya Shimizugawa, Hiroyuki Minekura, Takafumi KohamaAbstract:Abstract Ceramide-1-phosphate is a sphingolipid metabolite that has been implicated in membrane fusion of brain synaptic vesicles and neutrophil phagolysosome formation. Ceramide-1-phosphate can be produced by ATP-dependent Ceramide Kinase activity, although little is known of this enzyme because it has not yet been highly purified or cloned. Based on sequence homology to sphingosine Kinase type 1, we have now cloned a related lipid Kinase, human Ceramide Kinase (hCERK). hCERK encodes a protein of 537 amino acids that has a catalytic region with a high degree of similarity to the diacylglycerol Kinase catalytic domain. hCERK also has a putative N-myristoylation site on its NH2 terminus followed by a pleckstrin homology domain. Membrane but not cytosolic fractions from HEK293 cells transiently transfected with a hCERK expression vector readily phosphorylated Ceramide but not sphingosine or other sphingoid bases, diacylglycerol or phosphatidylinositol. This activity was clearly distinguished from those of bacterial or human diacylglycerol Kinases. With natural Ceramide as a substrate, the enzyme had a pH optimum of 6.0–7.5 and showed Michaelis-Menten kinetics, with K m values of 187 and 32 μm for Ceramide and ATP, respectively. Northern blot analysis revealed that hCERK mRNA expression was high in the brain, heart, skeletal muscle, kidney, and liver. A BLAST search analysis using the hCERK sequence revealed that putative Ceramide Kinases (CERKs) exist widely in diverse multicellular organisms including plants, nematodes, insects, and vertebrates. Phylogenetic analysis revealed that CERKs are a new class of lipid Kinases that are clearly distinct from sphingosine and diacylglycerol Kinases. Cloning of CERK should provide new molecular tools to investigate the physiological functions of Ceramide-1-phosphate.