Ceramide Disables 3-Phosphoinositide Binding to the Pleckstrin Homology Domain of Protein Kinase B (PKB)/Akt by a PKCζ-Dependent Mechanism

Abstract

Ceramide is generated in response to numerous stress-inducing stimuli and has been implicated in the regulation of diverse cellular responses, including cell death, differentiation, and insulin sensitivity. Recent evidence indicates that ceramide may regulate these responses by inhibiting the stimulus-mediated activation of protein kinase B (PKB), a key determinant of cell fate and insulin action. Here we show that inhibition of this kinase involves atypical PKCζ, which physically interacts with PKB in unstimulated cells. Insulin reduces the PKB-PKCζ interaction and stimulates PKB. However, dissociation of the kinase complex and the attendant hormonal activation of PKB were prevented by ceramide. Under these circumstances, ceramide activated PKCζ, leading to phosphorylation of the PKB-PH domain on Thr³⁴. This phosphorylation inhibited phosphatidylinositol 3,4,5-trisphosphate (PIP₃) binding to PKB, thereby preventing activation of the kinase by insulin. In contrast, a PKB-PH domain with a T34A mutation retained the ability to bind PIP₃ even in the presence of a ceramide-activated PKCζ and, as such, expression of PKB T34A mutant in L6 cells was resistant to inhibition by ceramide treatment. Inhibitors of PKCζ and a kinase-dead PKCζ both antagonized the inhibitory effect of ceramide on PKB. Since PKB confers a prosurvival signal and regulates numerous pathways in response to insulin, suppressing its activation by a PKCζ-dependent process may be one mechanism by which ceramide promotes cell death and induces insulin resistance.

ACKNOWLEDGMENTS

We are grateful to Dario Alessi, Philip Cohen, and Peter Downes for making available some of the reagents used in our studies. We thank Daan van Aalten for providing the structural image of the isolated PH domain of PKB in complex with the head group of PIP₃, and we thank Alex Gray (DSTT) and Maria Deak (MRC phosphorylation unit) for technical input in some of the experimental work.

This work was supported by the MRC, the Diabetes Research and Wellness Foundation, and Diabetes UK.