Ceramide induces the dephosphorylation and inhibition of constitutively activated Akt in PTEN negative U87mg cells.
In the present study, treatment of the PTEN negative U87MG human glioblastoma cell line with C2-ceramide resulted in a dose- and time-dependent decrease in the constitutive phosphorylation of Akt at threonine 308 and serine 473. The C2-ceramide induced dephosphorylation of Akt correlated with a 90-95% reduction in the Akt kinase activity. Exposure to C2-ceramide did not affect the basal or PDGF activated levels PtdIns-3,4-P(2) and PtdIns-3,4,5-P(3), indicating PI3-K activity was not inhibited. Additionally, treatment of cells with the PI3-K inhibitor wortmannin and C2-ceramide resulted in an enhanced rate of Akt dephosphorylation versus either agent alone. Finally, treatment of cells with the phosphatase inhibitors okadaic acid or calyculin A prevented the C2-ceramide induced dephosphorylation and inhibition of Akt activity. These data demonstrate the ability of C2-ceramide to inhibit the constitutive phosphorylation and activity of Akt in U87MG cells and implicate the activation of ceramide activated protein phosphatase, rather than decreased PI3-K activity, as the mechanism of inhibition.
Zinda
M J
MJ
Department of Cancer Research, Lilly Corporate Center, Indianapolis, Indiana, 46285, USA.
Vlahos
C J
CJ
Lai
M T
MT
D000730:Androstadienes / Q000494:pharmacology; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008387:Marine Toxins; D019319:Okadaic Acid / Q000494:pharmacology; D010080:Oxazoles / Q000494:pharmacology; D051059:PTEN Phosphohydrolase; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011233:Precipitin Tests; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors*; D051057:Proto-Oncogene Proteins c-akt; D012694:Serine / Q000737:chemistry; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D013912:Threonine / Q000737:chemistry; D013997:Time Factors; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins; D000077191:Wortmannin
D000730:Androstadienes; D004791:Enzyme Inhibitors; D008387:Marine Toxins; C064769:N-acetylsphingosine; D010080:Oxazoles; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols; D011518:Proto-Oncogene Proteins; D025521:Tumor Suppressor Proteins; C060974:phosphatidylinositol 3,4,5-triphosphate; C060980:phosphatidylinositol 3,4-diphosphate; D019319:Okadaic Acid; D013912:Threonine; D012694:Serine; C059041:calyculin A; C494918:AKT1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D010744:Phosphoric Monoester Hydrolases; D051059:PTEN Phosphohydrolase; C494929:PTEN protein, human; D013110:Sphingosine; D000077191:Wortmannin