Hydrolyzes the sphingolipid ceramide into sphingosine and free fatty acid. Unsaturated long-chain ceramides are the best substrates, saturated long-chain ceramides and unsaturated very long-chain ceramides are good substrates, whereas saturated very long-chain ceramides and short-chain ceramides were poor substrates. The substrate preference is D-erythro-C(18:1)-, C(20:1)-, C(20:4)-ceramide > D-erythro-C(16:0)-, C(18:0), C(20:0)-ceramide > D-erythro-C(24:1)-ceramide > D-erythro-C(12:0)-ceramide, D-erythro-C(14:0)-ceramides > D-erythro-C(24:0)-ceramide > D-erythro-C(6:0)-ceramide. Inhibits the maturation of protein glycosylation in the Golgi complex, including that of integrin beta-1 (ITGB1) and of LAMP1, by increasing the levels of sphingosine. Inhibits cell adhesion by reducing the level of ITGB1 in the cell surface. May have a role in cell proliferation and apoptosis that seems to depend on the balance between sphingosine and sphingosine-1-phosphate.
Sphingosine-1-phosphate (S1P), a sphingolipid metabolite, promotes cell proliferation and survival whereas its precursor, sphingosine, has the opposite effects. However, much remains unknown about their regulation. Here we identify a novel human ceramidase (haCER2) that regulates the levels of both sphingosine and S1P by controlling the hydrolysis of ceramides. haCER2 is localized to the Golgi complex and is highly expressed in the placenta. High ectopic expression of haCER2 caused fragmentation of the Golgi complex and growth arrest in HeLa cells due to sphingosine accumulation. Low ectopic expression of haCER2 increased S1P without sphingosine accumulation, promoting cell proliferation in serum-free medium. This proliferative effect was suppressed by dimethylsphingosine, an inhibitor of the S1P formation, or by the RNA interference (RNAi) -mediated inhibition of S1P(1,) a G-protein-coupled receptor for S1P. The RNAi-mediated down-regulation of haCER2 enhanced the serum deprivation-induced growth arrest and apoptosis of HeLa cells, which was inhibited by addition of exogenous S1P. Serum deprivation up-regulated both haCER2 mRNA and activity in HeLa cells. haCER2 mRNA is also up-regulated in some tumors. Taken together, these results suggest that haCER2 is important for the generation of S1P and S1P-mediated cell proliferation and survival, but that its overexpression may cause cell growth arrest due to an accumulation of sphingosine.
The polypeptide core of the integrin beta1 subunit (beta1) is glycosylated sequentially in the endoplasmic reticulum and the Golgi complex to form beta1 precursor and mature beta1, respectively. The beta1 precursor to mature beta1 conversion, termed beta1 maturation, regulates the cell surface levels and function of beta1-containing integrins, beta1 integrins. Here we demonstrate that the human alkaline ceramidase 2 (ACER2), a Golgi enzyme, regulates beta1 maturation by controlling the generation of sphingosine. ACER2 overexpression inhibited beta1 maturation, thus leading to a decrease in the levels of mature beta1 in T-REx HeLa cells, whereas RNA interference-mediated knockdown of ACER2 enhanced beta1 maturation in MCF-7 cells. ACER2 overexpression decreased the cell surface levels of beta1 integrins, thus inhibiting cell adhesion to fibronectin or collagen, whereas ACER2 knockdown has the opposite effects. Treatment with all-trans retinoic acid (ATRA) increased both the expression of ACER2 and the generation of sphingosine in HeLa cells and inhibited beta1 maturation. ACER2 knockdown attenuated the inhibitory effects of ATRA on both beta1 maturation and cell adhesion. In contrast, treatment with phorbol myristate acetate (PMA), a protein kinase C activator, decreased the expression of ACER2 and sphingosine in T-REx HeLa cells, thus enhancing beta1 maturation. ACER2 overexpression inhibited the stimulatory effects of PMA on both beta1 maturation and cell adhesion. These results suggest that the ACER2/sphingosine pathway plays an important role in regulating beta1 maturation and cell adhesion mediated by beta1 integrins.
Human alkaline ceramidase 2 (ACER2) plays an important role in cellular responses by regulating the hydrolysis of ceramides in cells. Here we report its biochemical characterization, membrane topology, and activity regulation. Recombinant ACER2 was expressed in yeast mutant cells (Deltaypc1Deltaydc1) that lack endogenous ceramidase activity, and microsomes from ACER2-expressiong yeast cells were used to biochemically characterize ACER2. ACER2 catalyzed the hydrolysis of various ceramides and followed Michaelis-Menten kinetics. ACER2 required Ca(2+) for both its in vitro and cellular activities. ACER2 has 7 putative transmembrane domains, and its amino (N) and carboxyl (C) termini were found to be oriented in the lumen of the Golgi complex and cytosol, respectively. ACER2 mutant (ACER2DeltaN36) lacking the N-terminal tail (the first 36 amino acid residues) exhibited undetectable activity and was mislocalized to the endoplasmic reticulum, suggesting that the N-terminal tail is necessary for both ACER2 activity and Golgi localization. ACER2 mutant (ACER2DeltaN13) lacking the first 13 residues was also mislocalized to the endoplasmic reticulum although it retained ceramidase activity. Overexpression of ACER2, ACER2DeltaN13, but not ACER2DeltaN36 increased the release of sphingosine 1-phosphate from cells, suggesting that its mislocalization does not affect the ability of ACER2 to regulate sphingosine 1-phosphate secretion. However, overexpression of ACER2 but not ACER2DeltaN13 or ACER2DeltaN36 inhibited the glycosylation of integrin beta1 subunit and Lamp1, suggesting that its mistargeting abolishes the ability of ACER2 to regulation protein glycosylation. These data suggest that ACER2 has broad substrate specificity and requires Ca(2+) for its activity and that ACER2 has the cytosolic C terminus and luminal N terminus, which are essential for its activity, correct cellular localization, and regulation for protein glycosylation.
Human alkaline ceramidase 2 (ACER2) plays an important role in cellular responses by regulating the hydrolysis of ceramides in cells. Here we report its biochemical characterization, membrane topology, and activity regulation. Recombinant ACER2 was expressed in yeast mutant cells (Deltaypc1Deltaydc1) that lack endogenous ceramidase activity, and microsomes from ACER2-expressiong yeast cells were used to biochemically characterize ACER2. ACER2 catalyzed the hydrolysis of various ceramides and followed Michaelis-Menten kinetics. ACER2 required Ca(2+) for both its in vitro and cellular activities. ACER2 has 7 putative transmembrane domains, and its amino (N) and carboxyl (C) termini were found to be oriented in the lumen of the Golgi complex and cytosol, respectively. ACER2 mutant (ACER2DeltaN36) lacking the N-terminal tail (the first 36 amino acid residues) exhibited undetectable activity and was mislocalized to the endoplasmic reticulum, suggesting that the N-terminal tail is necessary for both ACER2 activity and Golgi localization. ACER2 mutant (ACER2DeltaN13) lacking the first 13 residues was also mislocalized to the endoplasmic reticulum although it retained ceramidase activity. Overexpression of ACER2, ACER2DeltaN13, but not ACER2DeltaN36 increased the release of sphingosine 1-phosphate from cells, suggesting that its mislocalization does not affect the ability of ACER2 to regulate sphingosine 1-phosphate secretion. However, overexpression of ACER2 but not ACER2DeltaN13 or ACER2DeltaN36 inhibited the glycosylation of integrin beta1 subunit and Lamp1, suggesting that its mistargeting abolishes the ability of ACER2 to regulation protein glycosylation. These data suggest that ACER2 has broad substrate specificity and requires Ca(2+) for its activity and that ACER2 has the cytosolic C terminus and luminal N terminus, which are essential for its activity, correct cellular localization, and regulation for protein glycosylation.
Sphingosine-1-phosphate (S1P), a sphingolipid metabolite, promotes cell proliferation and survival whereas its precursor, sphingosine, has the opposite effects. However, much remains unknown about their regulation. Here we identify a novel human ceramidase (haCER2) that regulates the levels of both sphingosine and S1P by controlling the hydrolysis of ceramides. haCER2 is localized to the Golgi complex and is highly expressed in the placenta. High ectopic expression of haCER2 caused fragmentation of the Golgi complex and growth arrest in HeLa cells due to sphingosine accumulation. Low ectopic expression of haCER2 increased S1P without sphingosine accumulation, promoting cell proliferation in serum-free medium. This proliferative effect was suppressed by dimethylsphingosine, an inhibitor of the S1P formation, or by the RNA interference (RNAi) -mediated inhibition of S1P(1,) a G-protein-coupled receptor for S1P. The RNAi-mediated down-regulation of haCER2 enhanced the serum deprivation-induced growth arrest and apoptosis of HeLa cells, which was inhibited by addition of exogenous S1P. Serum deprivation up-regulated both haCER2 mRNA and activity in HeLa cells. haCER2 mRNA is also up-regulated in some tumors. Taken together, these results suggest that haCER2 is important for the generation of S1P and S1P-mediated cell proliferation and survival, but that its overexpression may cause cell growth arrest due to an accumulation of sphingosine.
Increased generation of dihydrosphingosine (DHS), a bioactive sphingolipid, has been implicated in the cytotoxicity of the synthetic retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) in tumor cells. However, how 4-HPR increases DHS remains unclear. Here we demonstrate that 4-HPR increases the expression of ACER2, which catalyzes the hydrolysis of dihydroceramides to generate DHS, and that ACER2 up-regulation plays a key role in mediating the 4-HPR-induced generation of DHS as well as the cytotoxicity of 4-HPR in tumor cells. Treatment with 4-HPR induced the accumulation of dihydroceramides (DHCs) in tumor cells by inhibiting dihydroceramide desaturase (DES) activity, which catalyzes the conversion of DHCs to ceramides. Treatment with 4-HPR also increased ACER2 expression through a retinoic acid receptor-independent and caspase-dependent manner. Overexpression of ACER2 augmented the 4-HPR-induced generation of DHS as well as 4-HPR cytotoxicity, and 4-HPR-induced death in tumor cells, whereas knocking down ACER2 had the opposite effects. ACER2 overexpression, along with treatment with GT11, another DES inhibitor, markedly increased cellular DHS, leading to tumor cell death, whereas ACER2 overexpression or GT11 treatment alone failed to do so, suggesting that both ACER2 up-regulation and DES inhibition are necessary and sufficient to mediate 4-HPR-induced DHS accumulation, cytotoxicity, and death in tumor cells. Taken together, these results suggest that up-regulation of the ACER2/DHS pathway mediates the cytotoxicity of 4-HPR in tumor cells and that up-regulating or activating ACER2 may improve the anti-cancer activity of 4-HRR and other DHC-inducing agents.
Increased generation of dihydrosphingosine (DHS), a bioactive sphingolipid, has been implicated in the cytotoxicity of the synthetic retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) in tumor cells. However, how 4-HPR increases DHS remains unclear. Here we demonstrate that 4-HPR increases the expression of ACER2, which catalyzes the hydrolysis of dihydroceramides to generate DHS, and that ACER2 up-regulation plays a key role in mediating the 4-HPR-induced generation of DHS as well as the cytotoxicity of 4-HPR in tumor cells. Treatment with 4-HPR induced the accumulation of dihydroceramides (DHCs) in tumor cells by inhibiting dihydroceramide desaturase (DES) activity, which catalyzes the conversion of DHCs to ceramides. Treatment with 4-HPR also increased ACER2 expression through a retinoic acid receptor-independent and caspase-dependent manner. Overexpression of ACER2 augmented the 4-HPR-induced generation of DHS as well as 4-HPR cytotoxicity, and 4-HPR-induced death in tumor cells, whereas knocking down ACER2 had the opposite effects. ACER2 overexpression, along with treatment with GT11, another DES inhibitor, markedly increased cellular DHS, leading to tumor cell death, whereas ACER2 overexpression or GT11 treatment alone failed to do so, suggesting that both ACER2 up-regulation and DES inhibition are necessary and sufficient to mediate 4-HPR-induced DHS accumulation, cytotoxicity, and death in tumor cells. Taken together, these results suggest that up-regulation of the ACER2/DHS pathway mediates the cytotoxicity of 4-HPR in tumor cells and that up-regulating or activating ACER2 may improve the anti-cancer activity of 4-HRR and other DHC-inducing agents.
Any process that results in a change in state or activity of a cell (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of a drug stimulus. A drug is a substance used in the diagnosis, treatment or prevention of a disease.
Evidence
1:
Inferred from Expression PatternBHF-UCL
Increased generation of dihydrosphingosine (DHS), a bioactive sphingolipid, has been implicated in the cytotoxicity of the synthetic retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) in tumor cells. However, how 4-HPR increases DHS remains unclear. Here we demonstrate that 4-HPR increases the expression of ACER2, which catalyzes the hydrolysis of dihydroceramides to generate DHS, and that ACER2 up-regulation plays a key role in mediating the 4-HPR-induced generation of DHS as well as the cytotoxicity of 4-HPR in tumor cells. Treatment with 4-HPR induced the accumulation of dihydroceramides (DHCs) in tumor cells by inhibiting dihydroceramide desaturase (DES) activity, which catalyzes the conversion of DHCs to ceramides. Treatment with 4-HPR also increased ACER2 expression through a retinoic acid receptor-independent and caspase-dependent manner. Overexpression of ACER2 augmented the 4-HPR-induced generation of DHS as well as 4-HPR cytotoxicity, and 4-HPR-induced death in tumor cells, whereas knocking down ACER2 had the opposite effects. ACER2 overexpression, along with treatment with GT11, another DES inhibitor, markedly increased cellular DHS, leading to tumor cell death, whereas ACER2 overexpression or GT11 treatment alone failed to do so, suggesting that both ACER2 up-regulation and DES inhibition are necessary and sufficient to mediate 4-HPR-induced DHS accumulation, cytotoxicity, and death in tumor cells. Taken together, these results suggest that up-regulation of the ACER2/DHS pathway mediates the cytotoxicity of 4-HPR in tumor cells and that up-regulating or activating ACER2 may improve the anti-cancer activity of 4-HRR and other DHC-inducing agents.
The polypeptide core of the integrin beta1 subunit (beta1) is glycosylated sequentially in the endoplasmic reticulum and the Golgi complex to form beta1 precursor and mature beta1, respectively. The beta1 precursor to mature beta1 conversion, termed beta1 maturation, regulates the cell surface levels and function of beta1-containing integrins, beta1 integrins. Here we demonstrate that the human alkaline ceramidase 2 (ACER2), a Golgi enzyme, regulates beta1 maturation by controlling the generation of sphingosine. ACER2 overexpression inhibited beta1 maturation, thus leading to a decrease in the levels of mature beta1 in T-REx HeLa cells, whereas RNA interference-mediated knockdown of ACER2 enhanced beta1 maturation in MCF-7 cells. ACER2 overexpression decreased the cell surface levels of beta1 integrins, thus inhibiting cell adhesion to fibronectin or collagen, whereas ACER2 knockdown has the opposite effects. Treatment with all-trans retinoic acid (ATRA) increased both the expression of ACER2 and the generation of sphingosine in HeLa cells and inhibited beta1 maturation. ACER2 knockdown attenuated the inhibitory effects of ATRA on both beta1 maturation and cell adhesion. In contrast, treatment with phorbol myristate acetate (PMA), a protein kinase C activator, decreased the expression of ACER2 and sphingosine in T-REx HeLa cells, thus enhancing beta1 maturation. ACER2 overexpression inhibited the stimulatory effects of PMA on both beta1 maturation and cell adhesion. These results suggest that the ACER2/sphingosine pathway plays an important role in regulating beta1 maturation and cell adhesion mediated by beta1 integrins.
The polypeptide core of the integrin beta1 subunit (beta1) is glycosylated sequentially in the endoplasmic reticulum and the Golgi complex to form beta1 precursor and mature beta1, respectively. The beta1 precursor to mature beta1 conversion, termed beta1 maturation, regulates the cell surface levels and function of beta1-containing integrins, beta1 integrins. Here we demonstrate that the human alkaline ceramidase 2 (ACER2), a Golgi enzyme, regulates beta1 maturation by controlling the generation of sphingosine. ACER2 overexpression inhibited beta1 maturation, thus leading to a decrease in the levels of mature beta1 in T-REx HeLa cells, whereas RNA interference-mediated knockdown of ACER2 enhanced beta1 maturation in MCF-7 cells. ACER2 overexpression decreased the cell surface levels of beta1 integrins, thus inhibiting cell adhesion to fibronectin or collagen, whereas ACER2 knockdown has the opposite effects. Treatment with all-trans retinoic acid (ATRA) increased both the expression of ACER2 and the generation of sphingosine in HeLa cells and inhibited beta1 maturation. ACER2 knockdown attenuated the inhibitory effects of ATRA on both beta1 maturation and cell adhesion. In contrast, treatment with phorbol myristate acetate (PMA), a protein kinase C activator, decreased the expression of ACER2 and sphingosine in T-REx HeLa cells, thus enhancing beta1 maturation. ACER2 overexpression inhibited the stimulatory effects of PMA on both beta1 maturation and cell adhesion. These results suggest that the ACER2/sphingosine pathway plays an important role in regulating beta1 maturation and cell adhesion mediated by beta1 integrins.
Any process that decreases the rate, frequency, or extent of the addition of a carbohydrate or carbohydrate derivative unit to a protein amino acid in any compartment of the Golgi apparatus.
Evidence
1:
Inferred from Mutant PhenotypeUniProtKB
Human alkaline ceramidase 2 (ACER2) plays an important role in cellular responses by regulating the hydrolysis of ceramides in cells. Here we report its biochemical characterization, membrane topology, and activity regulation. Recombinant ACER2 was expressed in yeast mutant cells (Deltaypc1Deltaydc1) that lack endogenous ceramidase activity, and microsomes from ACER2-expressiong yeast cells were used to biochemically characterize ACER2. ACER2 catalyzed the hydrolysis of various ceramides and followed Michaelis-Menten kinetics. ACER2 required Ca(2+) for both its in vitro and cellular activities. ACER2 has 7 putative transmembrane domains, and its amino (N) and carboxyl (C) termini were found to be oriented in the lumen of the Golgi complex and cytosol, respectively. ACER2 mutant (ACER2DeltaN36) lacking the N-terminal tail (the first 36 amino acid residues) exhibited undetectable activity and was mislocalized to the endoplasmic reticulum, suggesting that the N-terminal tail is necessary for both ACER2 activity and Golgi localization. ACER2 mutant (ACER2DeltaN13) lacking the first 13 residues was also mislocalized to the endoplasmic reticulum although it retained ceramidase activity. Overexpression of ACER2, ACER2DeltaN13, but not ACER2DeltaN36 increased the release of sphingosine 1-phosphate from cells, suggesting that its mislocalization does not affect the ability of ACER2 to regulate sphingosine 1-phosphate secretion. However, overexpression of ACER2 but not ACER2DeltaN13 or ACER2DeltaN36 inhibited the glycosylation of integrin beta1 subunit and Lamp1, suggesting that its mistargeting abolishes the ability of ACER2 to regulation protein glycosylation. These data suggest that ACER2 has broad substrate specificity and requires Ca(2+) for its activity and that ACER2 has the cytosolic C terminus and luminal N terminus, which are essential for its activity, correct cellular localization, and regulation for protein glycosylation.
Any process that increases the rate or frequency of cell death. Cell death is the specific activation or halting of processes within a cell so that its vital functions markedly cease, rather than simply deteriorating gradually over time, which culminates in cell death.
Increased generation of dihydrosphingosine (DHS), a bioactive sphingolipid, has been implicated in the cytotoxicity of the synthetic retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) in tumor cells. However, how 4-HPR increases DHS remains unclear. Here we demonstrate that 4-HPR increases the expression of ACER2, which catalyzes the hydrolysis of dihydroceramides to generate DHS, and that ACER2 up-regulation plays a key role in mediating the 4-HPR-induced generation of DHS as well as the cytotoxicity of 4-HPR in tumor cells. Treatment with 4-HPR induced the accumulation of dihydroceramides (DHCs) in tumor cells by inhibiting dihydroceramide desaturase (DES) activity, which catalyzes the conversion of DHCs to ceramides. Treatment with 4-HPR also increased ACER2 expression through a retinoic acid receptor-independent and caspase-dependent manner. Overexpression of ACER2 augmented the 4-HPR-induced generation of DHS as well as 4-HPR cytotoxicity, and 4-HPR-induced death in tumor cells, whereas knocking down ACER2 had the opposite effects. ACER2 overexpression, along with treatment with GT11, another DES inhibitor, markedly increased cellular DHS, leading to tumor cell death, whereas ACER2 overexpression or GT11 treatment alone failed to do so, suggesting that both ACER2 up-regulation and DES inhibition are necessary and sufficient to mediate 4-HPR-induced DHS accumulation, cytotoxicity, and death in tumor cells. Taken together, these results suggest that up-regulation of the ACER2/DHS pathway mediates the cytotoxicity of 4-HPR in tumor cells and that up-regulating or activating ACER2 may improve the anti-cancer activity of 4-HRR and other DHC-inducing agents.
Sphingosine-1-phosphate (S1P), a sphingolipid metabolite, promotes cell proliferation and survival whereas its precursor, sphingosine, has the opposite effects. However, much remains unknown about their regulation. Here we identify a novel human ceramidase (haCER2) that regulates the levels of both sphingosine and S1P by controlling the hydrolysis of ceramides. haCER2 is localized to the Golgi complex and is highly expressed in the placenta. High ectopic expression of haCER2 caused fragmentation of the Golgi complex and growth arrest in HeLa cells due to sphingosine accumulation. Low ectopic expression of haCER2 increased S1P without sphingosine accumulation, promoting cell proliferation in serum-free medium. This proliferative effect was suppressed by dimethylsphingosine, an inhibitor of the S1P formation, or by the RNA interference (RNAi) -mediated inhibition of S1P(1,) a G-protein-coupled receptor for S1P. The RNAi-mediated down-regulation of haCER2 enhanced the serum deprivation-induced growth arrest and apoptosis of HeLa cells, which was inhibited by addition of exogenous S1P. Serum deprivation up-regulated both haCER2 mRNA and activity in HeLa cells. haCER2 mRNA is also up-regulated in some tumors. Taken together, these results suggest that haCER2 is important for the generation of S1P and S1P-mediated cell proliferation and survival, but that its overexpression may cause cell growth arrest due to an accumulation of sphingosine.
Any process that results in a change in state or activity of a cell or an organism (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of a retinoic acid stimulus.
The polypeptide core of the integrin beta1 subunit (beta1) is glycosylated sequentially in the endoplasmic reticulum and the Golgi complex to form beta1 precursor and mature beta1, respectively. The beta1 precursor to mature beta1 conversion, termed beta1 maturation, regulates the cell surface levels and function of beta1-containing integrins, beta1 integrins. Here we demonstrate that the human alkaline ceramidase 2 (ACER2), a Golgi enzyme, regulates beta1 maturation by controlling the generation of sphingosine. ACER2 overexpression inhibited beta1 maturation, thus leading to a decrease in the levels of mature beta1 in T-REx HeLa cells, whereas RNA interference-mediated knockdown of ACER2 enhanced beta1 maturation in MCF-7 cells. ACER2 overexpression decreased the cell surface levels of beta1 integrins, thus inhibiting cell adhesion to fibronectin or collagen, whereas ACER2 knockdown has the opposite effects. Treatment with all-trans retinoic acid (ATRA) increased both the expression of ACER2 and the generation of sphingosine in HeLa cells and inhibited beta1 maturation. ACER2 knockdown attenuated the inhibitory effects of ATRA on both beta1 maturation and cell adhesion. In contrast, treatment with phorbol myristate acetate (PMA), a protein kinase C activator, decreased the expression of ACER2 and sphingosine in T-REx HeLa cells, thus enhancing beta1 maturation. ACER2 overexpression inhibited the stimulatory effects of PMA on both beta1 maturation and cell adhesion. These results suggest that the ACER2/sphingosine pathway plays an important role in regulating beta1 maturation and cell adhesion mediated by beta1 integrins.
The chemical reactions and pathways resulting in the formation of sphingosine (sphing-4-enine), trans-D-erytho-2-amino-octadec-4-ene-1,3-diol, a long chain amino diol sphingoid base that occurs in most sphingolipids in animal tissues.
Sphingosine-1-phosphate (S1P), a sphingolipid metabolite, promotes cell proliferation and survival whereas its precursor, sphingosine, has the opposite effects. However, much remains unknown about their regulation. Here we identify a novel human ceramidase (haCER2) that regulates the levels of both sphingosine and S1P by controlling the hydrolysis of ceramides. haCER2 is localized to the Golgi complex and is highly expressed in the placenta. High ectopic expression of haCER2 caused fragmentation of the Golgi complex and growth arrest in HeLa cells due to sphingosine accumulation. Low ectopic expression of haCER2 increased S1P without sphingosine accumulation, promoting cell proliferation in serum-free medium. This proliferative effect was suppressed by dimethylsphingosine, an inhibitor of the S1P formation, or by the RNA interference (RNAi) -mediated inhibition of S1P(1,) a G-protein-coupled receptor for S1P. The RNAi-mediated down-regulation of haCER2 enhanced the serum deprivation-induced growth arrest and apoptosis of HeLa cells, which was inhibited by addition of exogenous S1P. Serum deprivation up-regulated both haCER2 mRNA and activity in HeLa cells. haCER2 mRNA is also up-regulated in some tumors. Taken together, these results suggest that haCER2 is important for the generation of S1P and S1P-mediated cell proliferation and survival, but that its overexpression may cause cell growth arrest due to an accumulation of sphingosine.
Increased generation of dihydrosphingosine (DHS), a bioactive sphingolipid, has been implicated in the cytotoxicity of the synthetic retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) in tumor cells. However, how 4-HPR increases DHS remains unclear. Here we demonstrate that 4-HPR increases the expression of ACER2, which catalyzes the hydrolysis of dihydroceramides to generate DHS, and that ACER2 up-regulation plays a key role in mediating the 4-HPR-induced generation of DHS as well as the cytotoxicity of 4-HPR in tumor cells. Treatment with 4-HPR induced the accumulation of dihydroceramides (DHCs) in tumor cells by inhibiting dihydroceramide desaturase (DES) activity, which catalyzes the conversion of DHCs to ceramides. Treatment with 4-HPR also increased ACER2 expression through a retinoic acid receptor-independent and caspase-dependent manner. Overexpression of ACER2 augmented the 4-HPR-induced generation of DHS as well as 4-HPR cytotoxicity, and 4-HPR-induced death in tumor cells, whereas knocking down ACER2 had the opposite effects. ACER2 overexpression, along with treatment with GT11, another DES inhibitor, markedly increased cellular DHS, leading to tumor cell death, whereas ACER2 overexpression or GT11 treatment alone failed to do so, suggesting that both ACER2 up-regulation and DES inhibition are necessary and sufficient to mediate 4-HPR-induced DHS accumulation, cytotoxicity, and death in tumor cells. Taken together, these results suggest that up-regulation of the ACER2/DHS pathway mediates the cytotoxicity of 4-HPR in tumor cells and that up-regulating or activating ACER2 may improve the anti-cancer activity of 4-HRR and other DHC-inducing agents.
Human alkaline ceramidase 2 (ACER2) plays an important role in cellular responses by regulating the hydrolysis of ceramides in cells. Here we report its biochemical characterization, membrane topology, and activity regulation. Recombinant ACER2 was expressed in yeast mutant cells (Deltaypc1Deltaydc1) that lack endogenous ceramidase activity, and microsomes from ACER2-expressiong yeast cells were used to biochemically characterize ACER2. ACER2 catalyzed the hydrolysis of various ceramides and followed Michaelis-Menten kinetics. ACER2 required Ca(2+) for both its in vitro and cellular activities. ACER2 has 7 putative transmembrane domains, and its amino (N) and carboxyl (C) termini were found to be oriented in the lumen of the Golgi complex and cytosol, respectively. ACER2 mutant (ACER2DeltaN36) lacking the N-terminal tail (the first 36 amino acid residues) exhibited undetectable activity and was mislocalized to the endoplasmic reticulum, suggesting that the N-terminal tail is necessary for both ACER2 activity and Golgi localization. ACER2 mutant (ACER2DeltaN13) lacking the first 13 residues was also mislocalized to the endoplasmic reticulum although it retained ceramidase activity. Overexpression of ACER2, ACER2DeltaN13, but not ACER2DeltaN36 increased the release of sphingosine 1-phosphate from cells, suggesting that its mislocalization does not affect the ability of ACER2 to regulate sphingosine 1-phosphate secretion. However, overexpression of ACER2 but not ACER2DeltaN13 or ACER2DeltaN36 inhibited the glycosylation of integrin beta1 subunit and Lamp1, suggesting that its mistargeting abolishes the ability of ACER2 to regulation protein glycosylation. These data suggest that ACER2 has broad substrate specificity and requires Ca(2+) for its activity and that ACER2 has the cytosolic C terminus and luminal N terminus, which are essential for its activity, correct cellular localization, and regulation for protein glycosylation.
Human alkaline ceramidase 2 (ACER2) plays an important role in cellular responses by regulating the hydrolysis of ceramides in cells. Here we report its biochemical characterization, membrane topology, and activity regulation. Recombinant ACER2 was expressed in yeast mutant cells (Deltaypc1Deltaydc1) that lack endogenous ceramidase activity, and microsomes from ACER2-expressiong yeast cells were used to biochemically characterize ACER2. ACER2 catalyzed the hydrolysis of various ceramides and followed Michaelis-Menten kinetics. ACER2 required Ca(2+) for both its in vitro and cellular activities. ACER2 has 7 putative transmembrane domains, and its amino (N) and carboxyl (C) termini were found to be oriented in the lumen of the Golgi complex and cytosol, respectively. ACER2 mutant (ACER2DeltaN36) lacking the N-terminal tail (the first 36 amino acid residues) exhibited undetectable activity and was mislocalized to the endoplasmic reticulum, suggesting that the N-terminal tail is necessary for both ACER2 activity and Golgi localization. ACER2 mutant (ACER2DeltaN13) lacking the first 13 residues was also mislocalized to the endoplasmic reticulum although it retained ceramidase activity. Overexpression of ACER2, ACER2DeltaN13, but not ACER2DeltaN36 increased the release of sphingosine 1-phosphate from cells, suggesting that its mislocalization does not affect the ability of ACER2 to regulate sphingosine 1-phosphate secretion. However, overexpression of ACER2 but not ACER2DeltaN13 or ACER2DeltaN36 inhibited the glycosylation of integrin beta1 subunit and Lamp1, suggesting that its mistargeting abolishes the ability of ACER2 to regulation protein glycosylation. These data suggest that ACER2 has broad substrate specificity and requires Ca(2+) for its activity and that ACER2 has the cytosolic C terminus and luminal N terminus, which are essential for its activity, correct cellular localization, and regulation for protein glycosylation.
Protein involved in the biochemical reactions of lipids. Lipids are a diverse class of compounds which are insoluble in water but soluble in organic solvents. They include fats, oils, triacylglycerols, fatty acids, glycolipids, phospholipids and steroids.
Enzyme which catalyzes hydrolysis reaction, i.e. the addition of the hydrogen and hydroxyl ions of water to a molecule with its consequent splitting into two or more simpler molecules.
A reference proteome is a set of protein sequences derived from a complete proteome which constitutes a defined standard for a particular user community. Reference proteomes are manually defined according to a number of criteria. They cover the proteomes of well- studied model organisms and other proteomes of interest for biomedical and biotechnological research. Reference proteomes have been selected to provide broad coverage of the tree of life, and constitute a representative cross-section of the taxonomic diversity to be found within UniProtKB.
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