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Ceramide Activates the Stress-activated Protein Kinases

Westwick, John K., Bielawska, Alicja E., Dbaibo, Ghassan, Hannun, Yusuf A., Brenner, David A.·1995·Journal of Biological Chemistry
DOI 10.1074/jbc.270.39.22689
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FIG. 1 Stimulation of JNK/SAPK activity by sphingomyelinase ( SM ) and D-e-C2-ceramide. A , HL-60 cells were cultured as described (18) and treated with D-e-C 2 -ceramide ( C2 , 5 m M, lane 2 ) or ethanol only ( con , lane 1 ) or the enzymes indicated (300 milliunits/ml, lanes 4-7 ) or vehicle ( con , lane 3 ) or TNF a (10 ng/ml, lane 8 ) for 20 min. The single prominent band visible following 10% SDS-polyacrylamide gel electrophoresis and autoradiography represents phosphorylated GST-c-Jun. Equal substrate loading was confirmed in all cases by Coomassie Blue staining of the gels prior to autoradiography. PL , phospholipase. B , time course of JNK/SAPK induction following D-e-C 2 -ceramide addition (5 m M) to HL-60 cells. The substrate ( sub ) for the kinase assays was GST-cJun or GST alone as control. C , response of SAPK activity to D-e-C 2 -ceramide treatment. All cells received the indicated concentration in an equal volume of ethanol or ethanol alone 5(di), 5 m M dihydroC2-ceramide. Extracts were prepared after 10 min of treatment. The substrate for the kinase assay was wild-type GST-cJun ( lanes 2-9 ) or GST-cJun(AA) ( lane 1 ) in which Ser-63 and Ser-73 are mutated to Ala (10).
FIG. 1 Stimulation of JNK/SAPK activity by sphingomyelinase ( SM ) and D-e-C2-ceramide. A , HL-60 cells were cultured as described (18) and treated with D-e-C 2 -ceramide ( C2 , 5 m M, lane 2 ) or ethanol only ( con , lane 1 ) or the enzymes indicated (300 milliunits/ml, lanes 4-7 ) or vehicle ( con , lane 3 ) or TNF a (10 ng/ml, lane 8 ) for 20 min. The single prominent band visible following 10% SDS-polyacrylamide gel electrophoresis and autoradiography represents phosphorylated GST-c-Jun. Equal substrate loading was confirmed in all cases by Coomassie Blue staining of the gels prior to autoradiography. PL , phospholipase. B , time course of JNK/SAPK induction following D-e-C 2 -ceramide addition (5 m M) to HL-60 cells. The substrate ( sub ) for the kinase assays was GST-cJun or GST alone as control. C , response of SAPK activity to D-e-C 2 -ceramide treatment. All cells received the indicated concentration in an equal volume of ethanol or ethanol alone 5(di), 5 m M dihydroC2-ceramide. Extracts were prepared after 10 min of treatment. The substrate for the kinase assay was wild-type GST-cJun ( lanes 2-9 ) or GST-cJun(AA) ( lane 1 ) in which Ser-63 and Ser-73 are mutated to Ala (10).CC-BY · From the PDF
FIG. 2 TNF a , but not sphingomyelinase or D-e-C2-ceramide, activates NFk B binding activity. A , HL-60 cells were treated with vehicle, 0.1, 1, or 10 m M C2-ceramide ( lanes 1-4 ), or with 10 or 100 milliunits/ml sphingomyelinase ( SM ) ( B. cereus, Boehringer Mannheim) ( lanes 5 and 6 ) or recombinant TNF a ( T , 10 nM, lane 7 ) for 30 min prior to nuclear extract preparation and gel mobility shift analysis using a consensus NFk B binding site probe. ns , nonspecific shifted complex; p50-65 , heterodimeric NFk B. B , extracts from TNF a -treated HL-60 cells were used in binding assays, with antibodies to p50 ( lane 2 ), p65 ( lane 3 ), or c-Rel ( lane 4 ), or with a 50-fold excess of unlabeled wild-type ( wt , lane 5 ) or mutant NFk B binding site ( mut , lane 6 ) included in the reaction. After incubation on ice for 1 h, products were resolved on non-denaturing polyacrylamide gels. SS , supershifted NFk B; ns , nonspecific shifted complex (not competed by cold excess oligonucleotide).
FIG. 2 TNF a , but not sphingomyelinase or D-e-C2-ceramide, activates NFk B binding activity. A , HL-60 cells were treated with vehicle, 0.1, 1, or 10 m M C2-ceramide ( lanes 1-4 ), or with 10 or 100 milliunits/ml sphingomyelinase ( SM ) ( B. cereus, Boehringer Mannheim) ( lanes 5 and 6 ) or recombinant TNF a ( T , 10 nM, lane 7 ) for 30 min prior to nuclear extract preparation and gel mobility shift analysis using a consensus NFk B binding site probe. ns , nonspecific shifted complex; p50-65 , heterodimeric NFk B. B , extracts from TNF a -treated HL-60 cells were used in binding assays, with antibodies to p50 ( lane 2 ), p65 ( lane 3 ), or c-Rel ( lane 4 ), or with a 50-fold excess of unlabeled wild-type ( wt , lane 5 ) or mutant NFk B binding site ( mut , lane 6 ) included in the reaction. After incubation on ice for 1 h, products were resolved on non-denaturing polyacrylamide gels. SS , supershifted NFk B; ns , nonspecific shifted complex (not competed by cold excess oligonucleotide).CC-BY · From the PDF
FIG. 3 MAPkinase activity is stimulated by sphingomyelinase but not ceramide. An immune complex MAP kinase assay was performed with HL-60 cell extracts prepared following cell treatment ( Tx ) with the indicated agonists for 10 min ( lanes 1-5 ) or 2 min ( lanes 6-9 ). C5 and C10 , 5 and 10 m M D-e-C 2 -ceramide; C5(di) , 5 m M DLerythro -dihydro-C 2 -ceramide; SM , 100 milliunits/ml Staphylococcus aureus sphingomyelinase; TNF , 50 ng/ml recombinant human TNF a ; con , control. GST-ElkC was used as the substrate. Equal substrate loading was confirmed in all cases by Coomassie Blue staining of the gels prior to autoradiography.
FIG. 3 MAPkinase activity is stimulated by sphingomyelinase but not ceramide. An immune complex MAP kinase assay was performed with HL-60 cell extracts prepared following cell treatment ( Tx ) with the indicated agonists for 10 min ( lanes 1-5 ) or 2 min ( lanes 6-9 ). C5 and C10 , 5 and 10 m M D-e-C 2 -ceramide; C5(di) , 5 m M DLerythro -dihydro-C 2 -ceramide; SM , 100 milliunits/ml Staphylococcus aureus sphingomyelinase; TNF , 50 ng/ml recombinant human TNF a ; con , control. GST-ElkC was used as the substrate. Equal substrate loading was confirmed in all cases by Coomassie Blue staining of the gels prior to autoradiography.CC-BY · From the PDF
Image 4
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Image 5
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FIG. 4 Stimulation of cjun transcriptional activity. A , cjun mRNA levels are increased following TNF a , sphingomyelinase, and C2-ceramide treatment. HL-60 cells were incubated with TNF a ( TNF , 20 ng/ml), sphingomyelinase ( SM , 100 milliunits/ml), C2-ceramide ( CER , 10 m M), or untreated ( NONE ) for 1 h, and total RNA was prepared followed by electrophoresis and Northern blotting. The blot was hybridized first with a radioactively labeled human cjun probe ( upper panel ) and then with a labeled human glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) probe. Blots were quantitated by phosphor image analysis, and -fold induction of cjun mRNA normalized to GAPDH mRNA ( FOLD ) is indicated. B , activity of luciferase reporter genes in the L929 fibrosarcoma cell line. Jun-luciferase ( Luc ), or 5XGalluciferase plus Gal-ElkC, or (KB)3-luciferase (three copies of the consensus NFk B response element linked to luciferase) was transfected into L929 cells. Cells were then treated with TNF a (20 ng/ml) or sphingomyelinase (100 milliunits/ml) for 5 h, and extracts were prepared for determination of luciferase activity. -Fold activation represents reporter activity relative to cells treated with vehicle only. Results from one representative experiment performed in duplicate are shown ( 6 S.E.). Results were consistent over at least three experiments.
FIG. 4 Stimulation of cjun transcriptional activity. A , cjun mRNA levels are increased following TNF a , sphingomyelinase, and C2-ceramide treatment. HL-60 cells were incubated with TNF a ( TNF , 20 ng/ml), sphingomyelinase ( SM , 100 milliunits/ml), C2-ceramide ( CER , 10 m M), or untreated ( NONE ) for 1 h, and total RNA was prepared followed by electrophoresis and Northern blotting. The blot was hybridized first with a radioactively labeled human cjun probe ( upper panel ) and then with a labeled human glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) probe. Blots were quantitated by phosphor image analysis, and -fold induction of cjun mRNA normalized to GAPDH mRNA ( FOLD ) is indicated. B , activity of luciferase reporter genes in the L929 fibrosarcoma cell line. Jun-luciferase ( Luc ), or 5XGalluciferase plus Gal-ElkC, or (KB)3-luciferase (three copies of the consensus NFk B response element linked to luciferase) was transfected into L929 cells. Cells were then treated with TNF a (20 ng/ml) or sphingomyelinase (100 milliunits/ml) for 5 h, and extracts were prepared for determination of luciferase activity. -Fold activation represents reporter activity relative to cells treated with vehicle only. Results from one representative experiment performed in duplicate are shown ( 6 S.E.). Results were consistent over at least three experiments.CC-BY · From the PDF
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CC-BY · From the PDF

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Metadata

Authors
Westwick, J. K., Bielawska, A. E., Dbaibo, G., Hannun, Y. A., & Brenner, D. A.
Venue
Journal of Biological Chemistry
Publisher
Elsevier BV
Published
1995-09
Volume
270
Issue
39
Pages
22689-22692
ISSN
0021-9258
DOI
10.1074/jbc.270.39.22689
Cited by
303
References
2
Language
en
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CoChat. (2026). Citation check: Ceramide Activates the Stress-activated Protein Kinases. https://app.cochat.ai/papers/10.1074/jbc.270.39.22689
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Westwick, J. K., Bielawska, A. E., Dbaibo, G., Hannun, Y. A., & Brenner, D. A. (1995). Ceramide Activates the Stress-activated Protein Kinases. Journal of Biological Chemistry. https://doi.org/10.1074/jbc.270.39.22689