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Ceramide Induces the Dephosphorylation and Inhibition of Constitutively Activated Akt in PTEN Negative U87MG Cells

https://doi.org/10.1006/bbrc.2000.4248Get rights and content

Abstract

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-P2 and PtdIns-3,4,5-P3, 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.

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    To whom correspondence should be addressed at Department of Cancer Research, Lilly Research Labs, Lilly Corporate Center DC0424, Indianapolis, IN 46285. Fax: 317-276-9086. E-mail: [email protected].

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