The LXXLL motif of murine forkhead transcription factor FoxO1 mediates Sirt1-dependent transcriptional activity
Jun Nakae, … , Yoshihiko Yano, Yoshitake Hayashi
Jun Nakae, … , Yoshihiko Yano, Yoshitake Hayashi
Published September 1, 2006
Citation Information: J Clin Invest. 2006;116(9):2473-2483. https://doi.org/10.1172/JCI25518.
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Research Article Metabolism

The LXXLL motif of murine forkhead transcription factor FoxO1 mediates Sirt1-dependent transcriptional activity

Abstract

The forkhead transcription factor FoxO1 has been identified as a negative regulator of insulin/IGF-1 signaling. Its function is inhibited by phosphorylation and nuclear exclusion through a PI3K-dependent pathway. However, the structure/function relationship of FoxO1 has not been elucidated completely. In this study, we carried out mutation analysis of the FoxO1 coactivator–interacting LXXLL motif (amino acids 459–463). Expression of a 3A/LXXAA mutant, in which 3 Akt phosphorylation sites (T24, S253, and S316) and 2 leucine residues in the LXXLL motif (L462 and L463) were replaced by alanine, decreased both Igfbp-1 and G6Pase promoter activity and endogenous Igfbp-1 and G6Pase gene expression in simian virus 40–transformed (SV40-transformed) hepatocytes. Importantly, mutagenesis of the LXXLL motif eliminated FoxO1 interaction with the nicotinamide adenine dinucleotide–dependent (NAD-dependent) deacetylase sirtuin 1 (Sirt1), sustained the acetylated state of FoxO1, and made FoxO1 nicotinamide and resveratrol insensitive, supporting a role for this motif in Sirt1 binding. Furthermore, intravenous administration of adenovirus encoding 3A/LXXAA FoxO1 into Leprdb/db mice decreased fasting blood glucose levels and improved glucose tolerance and was accompanied by reduced G6Pase and Igfbp-1 gene expression and increased hepatic glycogen content. In conclusion, the LXXLL motif of FoxO1 may have an important role for its transcriptional activity and Sirt1 binding and should be a target site for regulation of gene expression of FoxO1 target genes and glucose metabolism in vivo.

Authors

Jun Nakae, Yongheng Cao, Hiroaki Daitoku, Akiyoshi Fukamizu, Wataru Ogawa, Yoshihiko Yano, Yoshitake Hayashi

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Figure 5

Sirt1 interacts with 3A FoxO1 but not with 3A/LXXAA FoxO1, and disruption of the LXXLL motif enhances acetylation of FoxO1.

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Sirt1 interacts with 3A FoxO1 but not with 3A/LXXAA FoxO1, and disruptio...
(A) SV40-transformed hepatocytes were transduced with adenovirus encoding Flag 3A FoxO1 (lane 1) or Flag 3A/LXXAA FoxO1 (lane 2). After 24 hours serum starvation, cells were cultured in AMEM supplemented with 0.1% bovine serum albumin, 0.5 mM 8-Br-cAMP, 0.5 mM IBMX, and 1 μM dexamethasone for 8 hours. Lysates were immunoprecipitated using anti-Flag monoclonal antibody (M2) and Western blotted with anti-Sir2 polyclonal (first panel) or anti-Flag monoclonal antibody (M2) (fourth panel) or immunoprecipitated using anti-Sir2 polyclonal antibody and Western blotted with anti-Sir2 antibody (second panel) or anti-Flag monoclonal antibody (third panel). (B) SV40-transformed hepatocytes transduced with adenovirus encoding Flag 3A FoxO1 (lane 1) or Flag 3A/LXXAA FoxO1 (lane 2) were incubated with 0.5 mM 8-Br-cAMP, 0.5 mM IBMX, and 1 μM dexamethasone for 8 hours after 24 hours of serum starvation. Lysates were immunoprecipitated using anti-Flag monoclonal antibody (M2) and Western blotted with anti-acetylated lysine polyclonal (top panel) or anti-Flag monoclonal antibody (M2) (bottom panel). (C) Quantification of the data in B. Mean ± SEM of the folds of acetylation of 3A/LXXAA was calculated from 3 independent experiments using NIH Image 1.62 (http://rsb.info.nih.gov/nih-image/). Subsequent blotting with anti-Flag antibody using the same filter normalized acetylation of each FoxO1. (D) HEK293 cells transfected with pCMV5/cMyc/3A FoxO1 (lanes 1–4) or 3A/LXXAA FoxO1 (lanes 5–8) were incubated for 8 hours in the absence or presence of nicotinamide (50 mM) or trichostatin A (TSA) (2 μM). cMyc-tagged FoxO1 was immunoprecipitated, and the acetylation of FoxO1 was assessed by Western blot with the antibody to acetylated lysine (upper panel). Total levels of FoxO1 were assessed with the antibody to cMyc (lower panel).