Direct Binding to Ceramide Activates Protein Kinase Cζ before the Formation of a Pro-apoptotic Complex with PAR-4 in Differentiating Stem Cells

Abstract

We have reported that ceramide mediates binding of atypical protein kinase C (PKC) to its inhibitor protein, PAR-4 (prostate apoptosis response-4), thereby inducing apoptosis in differentiating embryonic stem cells. Using a novel method of lipid vesicle-mediated affinity chromatography, we showed here that endogenous ceramide binds directly to the PKC⅐PAR-4 complex. Ceramide and its analogs activated PKC prior to binding to PAR-4, as determined by increased levels of phosphorylated PKC and glycogen synthase kinase-3␤ and emergence of a PAR-4-to-phosphorylated PKC fluorescence resonance energy transfer signal that co-localizes with ceramide. Elevated expression and activation of PKC increased cell survival, whereas expression of PAR-4 promoted apoptosis. This suggests that PKC counteracts apoptosis, unless its ceramide-induced activation is compromised by binding to PAR-4. A luciferase reporter assay showed that ceramide analogs activate nuclear factor (NF)-B unless PAR-4-dependent inhibition of PKC suppresses NF-B activation. Taken together, our results show that direct physical association with ceramide and PAR-4 regulates the activity of PKC. They also indicate that this interaction regulates the activity of glycogen synthase kinase-3␤ and NF-B.

High glucose activates PKC-ζ and NADPH oxidase through autocrine TGF-β1 signaling in mesangial cells

“…PKC-immune complex kinase assays have been commonly used to monitor PKC-activity (12,39,49,58). As demonstrated in Fig.…”

“…Instead, atypical PKCs are regulated by phosphatidylinositol 3,4,5-trisphosphate produced by phosphatidylinositol (PI)3-kinase, ceramide, and by other proteins (20,49,58). PI3 kinase phosphorylates the D-3 position of the inositol ring of phosphoinositides, recruiting proteins with pleckstrin homology domains to membranes, such as PKB/kt and PKC-/ .…”

Ceramide Regulates Atypical PKCζ/λ-mediated Cell Polarity in Primitive Ectoderm Cells

“…In Vitro Lipid-Protein Polarity Complex-The in vitro reconstitution of a lipid-protein polarity complex was performed following the LIMAC (lipid vesicle-mediated affinity chromatography) procedure as published previously (5). In brief, phosphatidylserine (PS, 420 g) and C 16 -ceramide (107 g) were dried from organic solvent and resuspended under sonication in 500 l of buffer (50 mM Tris/HCl, pH 7.5, 150 mM NaCl).…”

“…Our studies have shown that the membrane sphingolipid ceramide regulates apoptosis during embryonic stem (ES) 3 cell differentiation (1)(2)(3)(4)(5). This regulation is initiated by direct physical interaction of ceramide with atypical PKC or (5).…”

“…This regulation is initiated by direct physical interaction of ceramide with atypical PKC or (5). Homozygous knock-out of PKC in mice results in lethality at embryonic day 9 because of severe defects of post-gastrulation morphology (6).…”