Ceramide inhibits protein kinase B/Akt by promoting dephosphorylation of serine 473

The second messenger ceramide (N-alkylsphingosine) has been implicated in a host of cellular processes including growth arrest and apoptosis, Ceramide has been reported to have effects on both protein kinases and phosphatases and may constitute an important component of stress response in various ti ssues. We have examined in detail the relationship between ceramide signali ng and the activation of an important signaling pathway, phosphatidylinosit ol (PI) 3-kinase and its downstream target, protein kinase B (PKB). PKB act ivation was observed following stimulation of cells with the cytokine granu locyte-macrophage colony-stimulating factor. Addition of cell-permeable cer amide analogs, C-2- or C-6-ceramide, caused a partial loss (50-60%) of PKB activation. This reduction was not a result of decreased PI(3,4,5)P-3 or PI (3,4)P-2 generation by PI 3-kinase. Two residues of PKB (threonine 308 and serine 473) require phosphorylation for maximal PKB activation. Serine 473 phosphorylation was consistently reduced by treatment with ceramide, wherea s threonine 308 phosphorylation remained unaffected. In further experiments , ceramide appeared to accelerate serine 473 dephosphorylation, suggesting the activation of a phosphatase. Consistent with this, the reduction in ser ine 473 phosphorylation was inhibited by the phosphatase inhibitors okadaic acid and calyculin A. Surprisingly, threonine 308 phosphorylation was abol ished in cells treated with these inhibitors, revealing a novel mechanism o f regulation of threonine 308 phosphorylation, These results demonstrate th at PI 3-kinase-dependent kinase 2-catalyzed phosphorylation of serine 473 i s the principal target of a ceramide-activated phosphatase.