Protein kinase Cδ regulates nuclear export of FOXO1 through phosphorylation of the chaperone 14-3-3ζ

Abstract

Aims/hypothesis Forkhead box protein O1 (FOXO1) is a transcription factor essential for beta cell fate. Protein kinase B-dependent phosphorylation of FOXO1 at S256 (P-FOXO1) enables its binding to 14-3-3 dimers and nuclear export. Dephosphorylated FOXO1 enters nuclei and activates pro-apoptotic genes. Since our previous observations suggest that protein kinase C delta (PKCδ) induces nuclear accumulation of FOXO1, the underlying mechanism was examined. Methods In human islets, genetically modified mice and INS-1E cells apoptosis was assessed by TUNEL staining. Subcellular translocation of proteins was examined by confocal microscopy and signalling pathways were analysed by western blotting and overlay assay. Results In PKCδ-overexpressing (PKCδ-tg) mouse islet cells and INS-1E cells FOXO1 accumulated in nuclei, surprisingly, as P-FOXO1. PKCδ-tg decelerated IGF-1-dependent stimulation of nuclear export, indicating that changes in export caused nuclear retention of P-FOXO1. Nuclear accumulation of P-FOXO1 was accompanied by increased phosphorylation of 14-3-3ζ at S58 and reduced dimerisation of 14-3-3ζ. Palmitic acid further augmented phosphorylation of 14-3-3ζ and triggered nuclear accumulation of FOXO1 in both INS-1E and human islet cells. Furthermore, the overexpression of a phosphomimicking mutant of 14-3-3ζ (S58D) enhanced nuclear FOXO1. In accordance with the nuclear accumulation of P-FOXO1, PKCδ overexpression alone did not increase apoptotic cell death. Additionally, insulin secretion and glucose homeostasis in PKCδ-overexpressing mice remained unaffected. Conclusions/interpretation These results suggest that PKCδ-mediated phosphorylation of 14-3-3ζ contributes to the nuclear retention of FOXO1, even when FOXO1 is phosphorylated as under non-stress conditions. P-FOXO1 does not induce pro-apoptotic genes, but may rather exert beneficial effects on beta cells.

Proteomic Profiling for Identification of Novel Biomarkers Differentially Expressed in Human Ovaries from Polycystic Ovary Syndrome Patients

“…Expression of 14-3-3ε is linked with insufficient insulin secretion and reduced β-cell mass [40]. The 14-3-3γ dimers and exportin-1 (chromosomal region maintenance-1)-dependent nuclear export binds to phosphorylated factor forkhead box protein O1 (FOXO1) by protein kinase B [41, 42].…”

“…Accordingly, reduced 14-3-3ζ levels induce mitochondrial-linked β-cell death [45]. Gerst et al suggested that nuclear 14-3-3ζ binds to P-FOXO1, thereby promoting nuclear extrusion of the transcription factor [40]. This additional role underlines the importance of 14-3-3ζ for β-cell function [45].…”

Moonlighting chaperone‐like activity of the universal regulatory 14‐3‐3 proteins

“…). In line with these influences on their targets, 14‐3‐3 proteins have often been referred to as chaperones . We suggest that this definition is not completely correct.…”

Glucose Responsiveness of β-Cells Depends on Fatty Acids

“…INS-1E cells (a kind gift of P. Maechler and C. B. Wollheim, University of Geneva, Switzerland) are derived from a rat insulinoma and were cultured as described previously [22]. Briefly, the cells were cultured in RPMI1640 containing 11 mM glucose and supplemented with 10 % FCS, 10 mM Hepes, 2 mM L-glutamine, 1 mM Na pyruvate and 10 μM β-mercaptoethanol (Sigma, München, Germany) but without addition of antibiotics.…”