Longevity regulatory genes include the Forkhead transcription factor FOXO and the NAD-dependent histone deacetylase silent information regulator 2 (Sir2). Genetic studies demonstrate that Sir2 acts to extend lifespan in Caenorhabditis elegans upstream of DAF-16, a member of the FOXO family, in the insulin-like signaling pathway. However, the molecular mechanisms underlying the requirement of DAF-16 activity in Sir2-mediated longevity remain unknown. Here we show that reversible acetylation of Foxo1 (also known as FKHR), the mouse DAF-16 ortholog, modulates its transactivation function. cAMP-response element-binding protein (CREB)-binding protein binds and acetylates Foxo1 at the K242, K245, and K262 residues, the modification of which is involved in the attenuation of Foxo1 as a transcription factor. Conversely, Sir2 binds and deacetylates Foxo1 at residues acetylated by cAMPresponse element-binding protein-binding protein. Sir2 is recruited to insulin response sequence-containing promoter and increases the expression of manganese superoxide dismutase and p27 kip1 in a deacetylase-activity-dependent manner. Our findings establish Foxo1 as a direct and functional target for Sir2 in mammalian systems.

Forkhead transcription factor FKHR (Foxo1) is a key regulator of glucose homeostasis, cell-cycle progression, and apoptosis. It has been shown that FKHR is phosphorylated via insulin or growth factor signaling cascades, resulting in its cytoplasmic retention and the repression of target gene expression. Here, we investigate the fate of FKHR after cells are stimulated by insulin. We show that insulin treatment decreases endogenous FKHR proteins in HepG2 cells, which is inhibited by proteasome inhibitors. FKHR is ubiquitinated in vivo and in vitro, and insulin enhances the ubiquitination in the cells. In addition, the signal to FKHR degradation from insulin is mediated by the phosphatidylinositol 3-kinase pathway, and the mutation of FKHR at the serine or threonine residues phosphorylated by protein kinase B, a downstream target of phosphatidylinositol 3-kinase, inhibits the ubiquitination in vivo and in vitro. Finally, efficient ubiquitination of FKHR requires both phosphorylation and cytoplasmic retention in the cells. These results demonstrate that the insulin-induced phosphorylation of FKHR leads to the multistep negative regulation, not only by the nuclear exclusion but also the ubiquitination-mediated degradation.