Ceramide disables 3-phosphoinositide binding to the pleckstrin homology domain of protein kinase B (PKB)/Akt by a PKCzeta-dependent mechanism.
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 PKCzeta, which physically interacts with PKB in unstimulated cells. Insulin reduces the PKB-PKCzeta 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 PKCzeta, leading to phosphorylation of the PKB-PH domain on Thr(34). This phosphorylation inhibited phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) 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 PIP(3) even in the presence of a ceramide-activated PKCzeta and, as such, expression of PKB T34A mutant in L6 cells was resistant to inhibition by ceramide treatment. Inhibitors of PKCzeta and a kinase-dead PKCzeta 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 PKCzeta-dependent process may be one mechanism by which ceramide promotes cell death and induces insulin resistance.
Powell
Darren J
DJ
Division of Molecular Physiology, MRC Protein Phosphorylation Unit, MSI/WTB Complex, Faculty of Life Sciences, University of Dundee, Dundee DD1 5EH, United Kingdom.
Hajduch
Eric
E
Kular
Gursant
G
Hundal
Harinder S
HS
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002518:Ceramides / Q000378:metabolism*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000378:metabolism; D007328:Insulin / Q000378:metabolism; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D020033:Protein Isoforms / Q000378:metabolism; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D015398:Signal Transduction; D000099221:Protein Kinase C zeta
D002518:Ceramides; D004791:Enzyme Inhibitors; D007328:Insulin; D046911:Macromolecular Substances; D010455:Peptides; D018129:Phosphatidylinositol Phosphates; D020033:Protein Isoforms; D011493:Protein Kinase C; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D011993:Recombinant Fusion Proteins; D000099221:Protein Kinase C zeta; C494920:Akt1 protein, rat