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 glioblasto ma cell line with C2-ceramide resulted in a dose- and time-dependent decrea se in the constitutive phosphorylation of Akt at threonine 308 and serine 4 73. The Ca-ceramide induced dephosphorylation of Akt correlated with a 90-9 5% reduction in the Akt kinase activity. Exposure to Ca-ceramide did not af fect 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 c ells with the PI3-K inhibitor wortmannin and Ca-ceramide resulted in an enh anced rate of Akt dephosphorylation versus either agent alone. Finally, tre atment 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 Ca-ceramide to inhibit the constitutive phosphorylation and activity of Akt in U87MG cells and implica te the activation of ceramide activated protein phosphatase, rather than de creased PI3-K activity, as the mechanism of inhibition. (C) 2001 academic P ress.