Uncoupling of Acetylation from Phosphorylation Regulates FoxO1 Function Independent of Its Subcellular Localization*

The activity of transcription factor FoxO1 is regulated by phosphorylation-dependent nuclear exclusion and deacetylation-dependent nuclear retention. It is unclear whether and how these two post-translational modifications affect each other. To answer this question, we expressed FoxO1 cDNAs with combined mutations of phosphorylation and acetylation sites in HEK-293 cells and analyzed their subcellular localization patterns. We show that mutations mimicking the acetylated state (KQ series) render FoxO1 more sensitive to Akt-mediated phosphorylation and nuclear exclusion and can reverse the constitutively nuclear localization of phosphorylation-defective FoxO1. Conversely, mutations mimicking the deacetylated state (KR series) promote FoxO1 nuclear retention. Oxidative stress and the Sirt1 activator resveratrol are thought to promote FoxO1 deacetylation and nuclear retention, thus increasing its activity. Accordingly, FoxO1 deacetylation was required for the effect of oxidative stress (induced by H2O2) to retain FoxO1 in the nucleus. H2O2 also inhibited FoxO1 phosphorylation on Ser-253 and Thr-24, the key insulin-regulated sites, irrespective of its acetylation. In contrast, the effect of resveratrol was independent of FoxO1 acetylation and its phosphorylation on Ser-253 and Thr-24, suggesting that resveratrol acts on FoxO1 in a Sirt1- and Akt-independent manner. The dissociation of deacetylation from dephosphorylation in H2O2-treated cells indicates that the two modifications can occur independently of each other. It can be envisaged that FoxO1 exists in multiple nuclear forms with distinct activities depending on the balance of acetylation and phosphorylation.

FoxO1 and its closely related isoforms FoxO3A and FoxO4 are transcription factors characterized by a conserved winged helix ("forkhead") DNA binding domain. Genetic epistasis experiments in Caenorhabditis elegans demonstrated a role for these proteins in insulin receptor signaling, spawning studies of their contribution to mammalian metabolism, cellular differentiation, and transformation (1). It is now recognized that FoxOs are critical regulators of hepatic gluconeogenesis (2,3) and pancreatic ␤-cell function (4 -8), in addition to differentiation of myotubes (9 -11) and adipocytes (12). Moreover, the C. elegans FoxO ortholog DAF-16 is required for life extension caused by DAF-2 (insulin receptor) mutations, suggesting that FoxO has a role in longevity (13,14).
The effects of phosphorylation and acetylation on FoxO function have been studied extensively but separately. However, these two modifications are likely to occur concurrently in vivo and to reciprocally affect each other. In this study, we generated an allelic series of FoxO1 mutants containing changes to both acetylation and phosphorylation sites and analyzed their regulation in response to physiologic (insulin) and pathophysiologic cues (oxidative stress, resveratrol) to explore the reciprocal regulation of acetylation and phosphorylation and their combined effects on FoxO1 cellular localization and biological functions.
Protein Analyses-We harvested cells and prepared protein extracts in buffer containing 20 mM Tris, pH 7.4, 150 mM NaCl, 10% glycerol, 2% Nonidet P-40, 1 mM EDTA, pH 8.0, 0.2% semidehydroascorbate, 0.5% sodium deoxycholate supplemented with protease and phosphatase inhibitors (Boston Bioproducts). We fractionated 40 g of protein by gel electrophoresis, followed by Western blot. Immunoprecipitation was carried out by standard methods. FLAG immunoprecipitation was carried out according to the manufacturer's instructions, and bound proteins were eluted using a FLAG peptide.
Effects of Acetylation Site Mutations on FoxO1 Subcellular Localization-We studied the effect of mutating acetylation sites on insulin-induced FoxO1 subcellular translocation. To this end, we transfected wild type (WT), KQ, or KR FoxO1-GFP fusion proteins into HEK-293 cells. WT localized to the nucleus in serum-free medium and translocated to the cytoplasm upon insulin stimulation. The Akt inhibitor Akti-1/2 inhibited this process (33) (Fig. 1B). Conversely, the KQ mutant was predominantly cytoplasmic, regardless of whether cells were incubated in serum-free medium or in the presence of insulin and Akt inhibitor. The KR mutant translocated to the cytoplasm after insulin stimulation in a WT-like fashion and was retained in the nucleus after Akti-1/2 treatment, whereas the S253A mutant was constitutively nuclear under all conditions tested ( Fig. 1B) (24).
We next examined whether acetylation trumps phosphorylation as a signal for FoxO1 retention in the nucleus. To this end, we compared the phosphorylation-defective mutant ADA (12) with combined phosphorylation/acetylation site mutants, ADA-KQ or ADA-KR. ADA was constitutively nuclear regardless of the culture conditions ( Fig. 2A). Surprisingly, ADA-KQ had predominantly cytoplasmic localization, whereas ADA-KR was retained in the nucleus ( Fig. 2A). These data indicate that acetylation trumps phosphorylation as a signal regulating FoxO1 cellular localization. Inhibition of nuclear export by leptomycin B resulted in nuclear accumulation of both KQ and ADA-KQ, indicating that acetylation doesn't prevent nuclear targeting of FoxO1 but likely accelerates its export to the cytoplasm or retards its nuclear import (Fig. 2B).
To rule out that mutation of the phosphorylation sites affected FoxO1 acetylation, we measured acetyl-FoxO1 levels in the phosphorylation site mutants, S253A and T24A. As the former is unaffected by insulin treatment, we measured only basal acetylation in the absence of insulin; in the latter, we compared acetylation levels in the absence and presence of insulin. However in neither case did we observe changes to FoxO1 acetylation (Fig. 2C).
Effects of Acetylation Site Mutations on Insulin-induced FoxO1 Phosphorylation-To understand why acetylation promotes FoxO1 nuclear exclusion, we examined whether it affects phosphorylation of Ser-253, the site required for insulin-dependent nuclear translocation (24). To avoid the potential confounding effects of Thr-24 phosphorylation on subcellular localization (24), we measured Ser-253 phosphorylation in T24A-KQ and T24A-KR mutants following exposure of cells to different doses of insulin. Insulin-induced Ser-253 phosphorylation of the T24A mutant paralleled Akt phosphorylation in a dose-dependent manner, with an ED 50 ϳ 0.3 nM (Fig. 3). In contrast, the ED 50 for Ser-253 phosphorylation decreased to Ͻ0.15 nM in the T24A-KQ mutant and rose to Ͼ1.5 nM in the T24A-KR mutant, resulting in a 10-fold difference between the two mutants. Interestingly, levels of the T24A-KQ mutant decreased in insulin-treated cells. Based on prior studies, this is  AUGUST 27, 2010 • VOLUME 285 • NUMBER 35 likely to reflect increased protein degradation through the proteasome (8). This process was reversed by Akt inhibition, as was Ser-253 phosphorylation (Fig. 3). These data indicate that acetylation increases FoxO1 sensitivity to Akt phosphorylation and degradation, suggesting that FoxO1 nuclear exclusion and protein turnover are integrated through acetylation-based mechanisms.

FoxO1 Acetylation and Phosphorylation
Uncoupling of Acetylation from Phosphorylation following H 2 O 2 -induced Oxidative Stress-Oxidative stress and the polyphenol resveratrol promote FoxO1 nuclear retention (30). Their effects have been ascribed to FoxO1 deacetylation (27,28,34). However, the data in Fig. 3 raise the possibility that they also inhibit FoxO1 phosphorylation or promote its dephosphorylation. To answer this question, we used acetylation site FoxO1 mutants to examine FoxO1 localization and phosphor-ylation following incubation of cells with insulin and H 2 O 2 , a chemical agent used to mimic oxidative stress (35), or insulin and resveratrol, a Sirt1 and AMP-activated protein kinase agonist (29,30). Addition of H 2 O 2 to insulin-treated cells prevented FoxO1 nuclear export. This effect was reversed by the constitutively acetylated KQ mutant but not by the deacetylated KR mutant, indicating that H 2 O 2 promotes FoxO1 deacetylation or requires that FoxO1 be deacetylated to keep it in the nucleus (Fig. 4A). Resveratrol also prevented FoxO1 nuclear exclusion in response to insulin but, unlike H 2 O 2 , failed to prevent nuclear exclusion of either KQ or KR mutants (Fig.  4A), indicating that its effects are independent of FoxO1 acetylation.
Next we compared the effects of H 2 O 2 and resveratrol on insulin-dependent phosphorylation of Ser-253 and Thr-24. Insulin promoted FoxO1 phosphorylation on both sites. Addition of H 2 O 2 to insulin decreased phosphorylation of both sites. The effect of H 2 O 2 was preserved in the KQ and KR mutants, indicating that it is independent of FoxO1 acetylation (Fig. 4B). Addition of resveratrol to insulin also decreased insulin-dependent Ser-253 and Thr-24 phosphorylation in WT FoxO1, but not in the KQ and KR mutants (Fig. 4B). Neither H 2 O 2 nor resveratrol affected insulin-induced Akt phosphorylation to a significant extent (Fig. 4B), and their effect on Ser-253 phosphorylation was independent of changes in FoxO1 protein levels, as indicated by the fact that they retained their ability to decrease Ser(P)-253 in the presence of the protein synthesis inhibitor, cycloheximide (Fig. 4C).
From these experiments, we concluded that the effects of resveratrol are mediated neither by changes in FoxO1 acetylation nor by dephosphorylation of Ser-253 and Thr-24. In contrast, H 2 O 2 promotes FoxO1 nuclear retention through a dual  mechanism: deacetylation and reduced insulin-dependent phosphorylation of Ser-253 and Thr-24. Interestingly, the two effects can occur independently. Thus, despite their apparent similarities, resveratrol and H 2 O 2 affect FoxO1 activity in mechanistically distinct fashions. H 2 O 2 might promote FoxO1 dephosphorylation either by preventing access of the relevant kinases to these sites or by easing access by the relevant phosphatases. To address this question, we examined whether the ability of H 2 O 2 to prevent FoxO1 phosphorylation on Ser-253 was reversed by inhibition of PP2A, a FoxO1 Ser-253 phosphatase (36). H 2 O 2 decreased Ser-253 phosphorylation, and its effect was partly reversed by the PP2A inhibitor microcystin-LR (Fig. 5A). The ability of microcystin-LR to offset H 2 O 2 inhibition of Ser-253 phosphor-ylation was preserved in the KQ, but not in the KR, mutant (Fig.  5A). Given that the KR mutant is predominantly nuclear, these data are consistent with the interpretation that PP2A-dependent FoxO1 dephosphorylation occurs outside the nucleus. The fact that the effect of microcystin-LR is partial indicates that H 2 O 2 also regulates other phosphatases or prevents access of Akt to FoxO1.
The ability of H 2 O 2 to promote FoxO1 nuclear retention was preempted, but not reversed, by the deacetylase inhibitor nicotinamide (8). Thus, cell pretreatment with nicotinamide blocked H 2 O 2 -induced nuclear translocation, but addition of nicotinamide after H 2 O 2 treatment was unable to reverse this effect (Fig. 5B), indicating that FoxO1 acetylation can prevent its nuclear entry but cannot promote its nuclear exclusion.
The effect of resveratrol, like that of H 2 O 2 , appears to entail reduced FoxO1 phosphorylation on Ser-253 and Thr-24. Decreased Ser-253 phosphorylation in resveratrol-treated cells was partly reversed by microcystin-LR (Fig. 5C), indicating that part of the effect of resveratrol is PP2A-dependent. Because 14-3-3 participates in nucleocytoplasmic shuttling of FoxO by binding phosphorylated Thr-24 and Ser-253 (37,38) and given the tight relationship between the phosphorylation of these sites and acetylation, we asked whether acetylation  affected binding of 14-3-3 to the Ser-253 site. Using immunoprecipitation of FLAG-tagged T24A, T24A-KQ, and T24A-KR mutants, followed by immunoblotting with anti-14-3-3 antibody, we observed that insulin-induced phosphorylation of Ser-253 was associated with increased binding of T24A to 14-3-3 (Fig. 6). Constitutively deacetylated T24A-KR bound 14-3-3 more efficiently than T24A (Fig. 6, lanes WT and KR under ϩInsulin), even as its phosphorylation on Ser-253 was reduced. The constitutively acetylated T24A-KQ mutant also showed increased 14-3-3 binding, but, unlike the KR mutant, it was associated with increased Ser-253 phosphorylation (Fig. 6, compare lanes WT and KQ under ϪInsulin with the same lanes under ϩInsulin). These data indicate that FoxO1 binding to 14-3-3 is also modulated by its acetylation state, lending further support to the idea that acetylation affects FoxO1 nucleocytoplasmic shuttling.
Conclusions-The goal of this study was to examine the reciprocal regulation of two primary posttranslational modifications of FoxO1, acetylation and phosphorylation, and their combined effects on FoxO1 function. Using constitutively deacetylated (KR) and acetylated (KQ) mutants, we show that acetylation causes a leftward shift in the dose-response curve for insulin-induced FoxO1 phosphorylation, whereas deacetylation causes a rightward shift. As a result, the two mutants differ by ϳ10-fold in their insulin sensitivity, suggesting that acetylation is a major determinant of FoxO1 activity in vivo.
A new finding of the present study is that the two modifications, acetylation and phosphorylation, can be uncoupled from each other. Using H 2 O 2 to mimic oxidative stress and induce FoxO1 deacetylation, we show that H 2 O 2 can antagonize insulin signaling by promoting either FoxO1 deacetylation or dephosphorylation of Ser-253 and Thr-24, the former in part through the serine/threonine phosphatase PP2A. In either case, the expectation is that FoxO1 will be retained in the nucleus.
In contrast, and somewhat surprisingly, resveratrol promotes FoxO1 nuclear localization independent of acetylation, as well as of Ser-253 and Thr-24 phosphorylation. These data support the recent observation that resveratrol acts by deacetylation-independent mechanisms (e.g. AMP-activated protein kinase activation) (39). We cannot exclude the possibility that resveratrol is unable to induce dephosphorylation of the KQ and KR mutants, because of structural alterations caused by the replacement of acetyllysine with arginine or glutamine.
The uncoupling of FoxO1 phosphorylation from acetylation in H 2 O 2 -treated cells has important ramifications for FoxO1 nuclear function. In fact, it has been shown that acetylation affects FoxO1 affinity to bind its DNA targets (8,26) and may thus favor DNA binding-independent modalities of FoxO1 function, e.g. cell differentiation versus replication and metabolism (10). Likewise, it has previously been demonstrated that phosphorylation affects transactivation properties of FoxO1 (32), making it theoretically possible that FoxO1 be nuclear and inactive.
In conclusion, the findings that phosphorylated FoxO1 can be retained in the nucleus by decreasing its acetylation and, conversely, that dephosphorylated FoxO1 can be targeted to the cytoplasm through increased acetylation underscore that phosphorylation and acetylation are regulated through partly overlapping, and partly independent, mechanisms and suggest new research directions to develop agents that modulate pleiotropic functions of FoxOs.