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 cAMP-response 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.

The FOXO family of forkhead transcription factors plays a key role in a variety of biological processes, including metabolism, cell proliferation, and oxidative stress response. We previously reported that Foxo1, a member of the FOXO family, is regulated through reversible acetylation catalyzed by histone acetyltransferase cAMP-response element-binding protein (CREB)-binding protein (CBP) and NAD-dependent histone deacetylase silent information regulator 2, and that the acetylation at Lys-242, Lys-245, and Lys-262 of Foxo1 attenuates its transcriptional activity. However, the molecular mechanism by which acetylation modulates Foxo1 activity remains unknown. Here, we show that the positive charge of these lysines in Foxo1 contributes to its DNA-binding, and acetylation at these residues by CBP attenuates its ability to bind cognate DNA sequence. Remarkably, we also show that acetylation of Foxo1 increases the levels of its phosphorylation at Ser-253 through the phosphatidylinositol 3-kinase–protein kinase B signaling pathway, and this effect was overridden on the acetylation-deficient Foxo1 mutant. Furthermore, in in vitro kinase reactions, the association of wild-type Foxo1 and its target DNA sequence inhibits the protein kinase B-dependent phosphorylation of Foxo1, whereas mutated Foxo1 proteins, which mimic constitutively acetylated states, are efficiently phosphorylated even in the presence of the DNA. These results suggest that acetylation regulates the function of Foxo1 through altering the affinity with the target DNA and the sensitivity for phosphorylation.

Protein arginine methylation is a common posttranslational modification catalyzed by a family of the protein arginine methyltransferases (PRMTs). We have previously reported that PRMT1 methylates Forkhead box O transcription factors at two arginine residues within an Akt consensus phosphorylation motif (RxRxxS/T), and that this methylation blocks Akt-mediated phosphorylation of the transcription factors. These findings led us to hypothesize that the functional crosstalk between arginine methylation and phosphorylation could be extended to other Akt target proteins as well as Forkhead box O proteins. Here we identify BCL-2 antagonist of cell death (BAD) as an additional substrate for PRMT1 among several Akt target proteins. We show that PRMT1 specifically binds and methylates BAD at Arg-94 and Arg-96, both of which comprise the Akt consensus phosphorylation motif. Consistent with the hypothesis, PRMT1-mediated methylation of these two arginine residues inhibits Akt-mediated phosphorylation of BAD at Ser-99 in vitro and in vivo. We also demonstrate that the complex formation of BAD with 14-3-3 proteins, which occurs subsequent to Akt-mediated phosphorylation, is negatively regulated by PRMT1. Furthermore, PRMT1 knockdown prevents mitochondrial localization of BAD and its binding to the antiapoptotic BCL-X _L protein. BAD overexpression causes an increase in apoptosis with concomitant activation of caspase-3, whereas PRMT1 knockdown significantly suppresses these apoptotic processes. Taken together, our results add a new dimension to the complexity of posttranslational BAD regulation and provide evidence that arginine methylation within an Akt consensus phosphorylation motif functions as an inhibitory modification against Akt-dependent survival signaling.

CBP and its homologue p300 play significant roles in cell differentiation, cell cycle, and anti-oncogenesis. We demonstrated that β-catenin, recently known as a potent oncogene, and CBP/p300 are associated through its CH3 region, which is a primary target of adenoviral oncoprotein E1A and various nuclear proteins, such as p53, cyclin E, and AP-1, and both are colocalized in the nuclear bodies. CBP/p300 potentiated Lef-mediated transactivation of β-catenin, and E1A, a potent inhibitor of CBP/p300, repressed its transactivation. Furthermore, overexpression of stable β-catenin mutant competitively suppressed the p53-dependent pathway. These may be a key mechanism of β-catenin involved in oncogenic events underlying disruption of tumor suppressor function through CBP/p300.

FOXO (Forkhead box O) transcription factors constitute an evolutionally conserved subgroup within the large Forkhead family of transcription regulators. FOXO factors are important regulators of the cell cycle, apoptosis, DNA repair, metabolism, oxidative stress resistance and longevity. Genetic studies of Caenorhabditis elegans demonstrated that FOXO factors are major targets of the insulin-like signalling implicated during the regulation of glucose metabolism and lifespan extension. Recently, emerging evidence has also shed light on signalling pathways under oxidative stress that regulate FOXO activity by various post-translational modifications, including phosphorylation, acetylation and ubiquitination. Thus, FOXO factors react to external stimuli by altering their modifications, modulating target gene expression that may be involved in longevity.