Communicative & Integrative Biology

[Cell Cycle 4:7, 908-913; July 2005]; ©2005 Landes Bioscience

Triple Layer Control Review

Phosphorylation, Acetylation and Ubiquitination of FOXO Proteins ABSTRACT FOXO proteins are transcriptional regulators that control cell cycle progression, DNA repair, defense against oxidative damage and apoptosis. These divergent functions of FOXO proteins are regulated by signal-induced, post-translational modifications. Phosphorylation of cytoplasmic FOXO at specific sites by JNK initiates translocation into the nucleus. Acetylation and deacetylation of nuclear FOXO affects the selection of transcriptional programs that are controlled by FOXO proteins. Activation of Akt by growth factors results in phosphorylation of nuclear FOXO at specific sites followed by additional phosphorylations mediated by other kinases. Akt-dependent phosphorylation reduces the DNA-binding activity of FOXO, interferes with binding to the co-activators p300/CBP, and inactivates the FOXO nuclear translocation signal. The Akt-phosphorylated FOXO is exported from the nucleus in a CRM1- and 14-3-3-dependent process. Cytoplasmic, Akt-phosphorylated FOXO interacts with the ubiquitin ligase Skp2 and is targeted for proteasomal degradation. The nuclear-cytoplasmic “FOXO shuttle” is driven by stress signals that result in nuclear import and FOXO transcriptional activity and growth signals that initiate nuclear export and proteasomal degradation of FOXO.

Received 05/02/05; Accepted 05/03/05

Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/abstract.php?id=1796

KEY WORDS FOXO, phosphorylation, acetylation, ubiquitination, Akt, JNK, SIRT1, Skp2

ACKNOWLEDGEMENTS

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ABBREVIATIONS

FOX, forkhead box; FOXO, forkhead box-containing, O subfamily; CDK, cyclin-dependent kinase; JNK, c-Jun NH2-terminal kinase; GADD45, growth arrest and DNA-damageinducible protein 45; SGK, serum and glucocorticoid-regulated kinase; CK1, casein kinase1; DYRK1A, dual-specificity tyrosine-phosphorylated and regulated kinase 1A; PI3K, phosphoinositide 3-kinase; Akt/PKB, protein kinase B; NLS, nuclear localization sequence; CRM1, chromosomal region maintenance protein 1; PAX, paired-box protein; CBP, CREB-binding protein; PCAF, p300/CBP-associated factor; SIRT1, sirtuin-1; SCF, Skp1/ Cul1/F-box protein; TOR, target of rapamycin; ERK, extracellular-signal-regulated kinase.

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Work of the authors is supported by grants from the National Cancer Institute. We thank Karen Arden, Sohye Kang, Andreas G. Bader, and Leyna Zhao for insightful, critical comments on the manuscript. This is manuscript number 17401-MEM of The Scripps Research Institute.

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*Correspondence to: Peter K. Vogt; Department of Molecular and Experimental Medicine; The Scripps Research Institute; 10550 North Torrey Pines Road; La Jolla, California 92037 USA; Email: [email protected]

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of Pharmacology; Kyoto University Graduate School of Medicine; Sakyo-ku, Kyoto Japan

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of Molecular and Experimental Medicine; The Scripps Research Institute; La Jolla, California USA

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Peter K. Vogt1,* Hao Jiang1 Masahiro Aoki2

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When you come to a fork … , take it. Yogi Berra

BACKGROUND AND PERSPECTIVE Gene activity can be regulated at three levels: transcription, translation and post-translational modification of proteins. This review will summarize the post-translational regulation of a group of transcription factors, the FOXO proteins. Their activities and fates are determined by phosphorylation, acetylation, deacetylation and proteolytic degradation. These post-translational modifications direct FOXO proteins to specific sites in the cell: in the nucleus, FOXO proteins control transcriptional programs while in the cytoplasm, FOXO proteins become transcriptionally inactive and undergo proteasomal degradation. FOXO proteins play important roles in the cellular response to growth signals and nutrients, to oxidative stress and to genomic damage. They direct the transcription of genetic programs that set the pace of the cell cycle, contribute to DNA repair, protect from oxidative damage and initiate apoptosis. Each of these specific functions is activated in response to signals that induce the appropriate post-translational modification of FOXO proteins. These modifications translate extracellular signals into activities that govern specific gene expressions.

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Post-Translational Regulation of FOXO Proteins

INTRODUCTION The superfamily of FOX proteins (formerly referred to as forkhead or winged helix proteins) consists of four hundred plus transcriptional regulators, each characterized by a conserved 110 amino acid DNAbinding domain that is referred to as the forkhead box or winged helix domain.1-7 FOX proteins bind to DNA as monomers and perform specific and important regulatory functions during embryonal development and in the adult.8,9 The FOXO proteins constitute a class of the FOX protein family and currently encompass the human proteins FOXO1 (FKHR), FOXO3a (FKHRL1), FOXO4 (AFX), FOXO6 and several proteins from other vertebrates (for a complete list see: www.biology.pomona.edu/fox.html).10 The FOXO proteins are the vertebrate orthologs of the Caenorhabditis elegans DAF16.11,12 FOXO in vertebrates and DAF16 in C. elegans are components of a highly conserved signaling pathway that connects growth and stress signals to transcriptional controls. The functional differences between FOXO isoforms are still largely unexplored. Knock-out experiments suggest non-redundant functions of different FOXO proteins. FOXO1-null mice die on embryonic day 10.5 with defects in vascular development. FOXO3a- and FOXO4- null mice are viable and fertile, showing only minor abnormalities.13,14 Posttranslational regulation mechanisms of different FOXO proteins are also distinct in important details.15-17 The FOXO proteins have attracted increasing interest because of their role in cell cycle regulation, integration of different signaling pathways, cancer and the control of life span.8,9,18-21 FOXO proteins intervene in the cell cycle by activating the cyclin-dependent kinase (CDK) inhibitors p27kip1 and p21cip1.22-24 They also repress transcription of cyclin D1 and D2, which results in an attenuation of CDK4 activity and reduced phosphorylation of the Rb protein, and hence increased binding activity of E2F family members to Rb.25,26 The overall effect of FOXO on cell cycle control is G1 arrest. Transcriptional control by FOXO also extends to the regulation of pro-apoptotic genes Bim and Fas Ligand27,28 and to stress response genes like GADD45, Mn superoxide dismutase and catalase.29-31 These regulatory activities are largely controlled by post-translational modifications of FOXO proteins: phosphorylation, acetylation, deacetylation and ubiquitination. In human cancer, FOXO transcription factors have emerged as cancer-specific fusion proteins that result from chromosomal rearrangements. The childhood tumor alveolar rhabdomyosarcoma shows two recurrent chromosomal translocations in which the C-terminal region of FOXO1 fuses to the N-terminal portion of the Pax3 gene or Pax7 gene.32 These fusions combine the DNA-binding domain of Pax with the transcriptional activation domain of FOXO and cause aberrant regulation of Pax target genes. The PAX-FOXO fusion proteins act as dominant oncoproteins. In cell culture, PAXFOXO fusions are able to induce oncogenic transformation, and that activity requires both DNA-binding and transcriptional regulation.33-35 In acute leukemias, the mixed lineage leukemia gene MLL is fused to FOXO4 or FOXO3a, and again, the potent FOXO-derived transactivation domain is essential for the oncogenic activity of the fusion proteins.36-40 FOXO proteins have also attracted attention through their ability to control the life span of the nematode C. elegans.41-45 The high degree of structural and functional conservation of FOXO proteins throughout evolution suggests that life span-controlling functions of FOXO may also extend to vertebrates. This aspect of FOXO proteins is under active investigation but lies beyond the scope of this article.46 www.landesbioscience.com

Figure 1. Growth-signal initiated downregulation of FOXO proteins. Sequential phosphorylation of nuclear FOXO starts with Akt (IKKβ in the case of FOXO3a) followed by contributions from additional kinases. Akt-dependent phosphorylation of FOXO1 interferes with the function of the FOXO NLS and with DNA and p300/CBP-binding activity. It generates attachment sites for 14-3-3 proteins and facilitates binding to the exportin CRM1. Cytoplasmic Akt-phosphorylated FOXO is recognized by the ubiquitin ligase Skp2 which targets FOXO for proteasomal degradation.

REGULATION OF FOXO BY PHOSPHORYLATION: NUCLEAR-CYTOPLASMIC SHUTTLING The transcriptional regulatory functions of FOXO proteins require nuclear localization. This localization is favored by the absence of growth signals and is correlated with an attenuation of cell replication. Stimulation of cell growth by insulin or growth factors results in cytoplasmic translocation of FOXO proteins. Export from the nucleus into the cytoplasm is regulated by the phosphorylation of FOXO, interferes with FOXO transcriptional activities and promotes proteolytic degradation. The translocation of FOXO into the cytoplasm is the subject of recent comprehensive reviews.15,21 The salient facts are as follows (Fig.1): In response to growth signals, FOXO proteins are phosphorylated by several kinases including Akt (also referred to as PKB), serum and glucocorticoid-regulated kinase (SGK), casein kinase1 (CK1), and DYRK1A (a member of the dualspecificity tyrosine-phosphorylated and regulated kinase group). Akt and SGK target the same substrate motifs, and it is not known whether one could substitute for the other in regulating FOXO. Akt and SGK can both be activated by phosphoinositide 3-kinase (PI3K) but respond to distinct upstream signals. There are three Akt phosphorylation motifs in FOXO: one each at the N and C termini and one in the forkhead-winged helix domain. Phosphorylation is sequential; the site in the forkhead domain initiates the process and acts as a trigger and gatekeeper, followed by phosphorylation on the N- and C-terminal sites. Phosphorylation on the N- and C-terminal sites is essential for cytoplasmic translocation. The Akt phosphorylation motif in the forkhead domain overlaps with the nuclear localization sequence (NLS), and phosphorylation of this site interferes with NLS activities. It also affects DNA-binding activity of FOXO. Phosphorylation of the N-terminal Akt motif disrupts the interaction of this domain with the transcriptional co-activators p300/CBP and facilitates binding of FOXO to a 14-3-3 protein instead. Phosphorylation by Akt is a prerequisite for the subsequent phosphorylation by CK1, which targets two additional sites in the

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ACETYLATION OF FOXO PROTEINS

Figure 2. Stress induced nuclear import, acetylation and deacetylation of FOXO proteins. In response to stress signals, FOXO is phosphorylated by JNK and translocated into the nucleus. The transcriptional activites of FOXO are regulated by p300/CBP-mediated acetylation and SIRT1-mediated deacetylation. SIRT1 has a differential effect on the FOXO-controlled transcriptional program, favoring expression of cell cycle and repair genes (p27kip, GADD45) over pro-apoptotic genes (BIM, FASL).

FOXO sequence. The DYRKIA kinase phosphorylates a single site in FOXO; this specific phosphorylation is independent of Akt or CK1 activity and is not affected by growth signals, although it appears to contribute to efficient nuclear export of FOXO. For FOXO3a, the kinase IKKβ that targets IκB (inhibitor of NF-κB) has been identified as crucial determinant of cellular localization and protein stability.16 FOXO6 deviates from the general pattern of FOXO nuclear export. It lacks the C-terminal phosphorylation sites for Akt, CK1, and DYRKIA and shows nuclear localization after stimulation with growth factors.17 The Akt-dependent phosphorylation in the forkhead domain and on the N-terminal site of FOXO creates two binding motifs for 14-3-3 proteins. The association of 14-3-3 with FOXO also interferes with the DNA-binding activity. A nuclear export signal in the FOXO sequence mediates association with the exportin CRM1 (chromosomal region maintenance protein 1) and is essential for the export from the nucleus. However, binding to CRM1 is not affected by the phosphorylation status of FOXO. In summary, signal-dependent phosphorylation of FOXO inhibits DNA-binding activity, inactivates a putative NLS, interferes with the recruitment of p300/CBP and sets in motion a series of protein-protein interactions that involve 14-3-3 and CRM1 and result in nuclear export. Nuclear-cytoplasmic shuttling of FOXO proteins is, however, a dynamic, two-way process: what goes out must have come in. Whereas nuclear export of FOXO proteins is a response to growth signaling, nuclear import follows stress signals. Oxidative stress, for instance a low concentration of hydrogen peroxide (H2O2), leads to an activation of the small GTPase Ral, which in turn is instrumental in the phosphorylation and activation of the Jun N-terminal kinase JNK (Fig. 2). JNK then phosphorylates cytoplasmic FOXO proteins at the C-terminal sites that are distinct form the Akt phosphorylation sites. JNK-induced phosphorylation initiates nuclear import of FOXO proteins and activation of their transcriptional regulatory potential. Ral, JNK and JNK-induced phosphorylation of FOXO proteins are necessary and sufficient for nuclear import and for the positive control of FOXO proteins.47-50 910

The stress-induced nuclear translocation initiates modification and regulation of FOXO proteins by acetylation.47,51-55 In growing cells, FOXO proteins are predominantly cytoplasmic. Nuclear import triggered by oxidative stress results in association of FOXO proteins with proteins that have histone acetylase activity, such as p300/CBP and p300/CBP-associated factor (PCAF). This interaction is an initial step in assembling the transcriptional activation complex of FOXO, but it also leads to the acetylation of FOXO itself. Paradoxically, acetylation of FOXO appears to reduce transcriptional activity. Acetylated and JNK-phosphorylated nuclear FOXO then recruits SIRT1, a constitutively nuclear, nicotinamide adenine dinucleotide-dependent protein deacetylase. This recruitment, like the nuclear import, is dependent on oxidative stress signals. A FOXO mutant with the three Akt phosphorylation sites mutated to alanine has been shown to be permanently nuclear but does not interact with SIRT1 in the absence of stress signals, suggesting that nuclear translocation of FOXO is necessary but not sufficient for the interaction with SIRT1. The interaction with SIRT1 affects the transcriptional regulatory functions of FOXO. The published studies on this aspect of FOXO acetylation and deacetylation are not in complete agreement, and future work will have to resolve the differences.47,51-55 For this review, we will summarize a scenario that is in accord with most of the available data. As a result of FOXO-SIRT1 interaction, FOXO target genes involved in DNA repair and cell cycle arrest (e.g. p27kip and GADD45) show enhanced expression, while the levels of proapoptotic targets (BIM, FAS Ligand) are either not affected or reduced (Fig. 2). Activation of FOXO3a with LY294002, an inhibitor of phosphoinositide 3-kinase (PI3K), leads to increased expression levels of GADD45. This response is attenuated in SIRT1-/cells and therefore appears to require the participation of SIRT1. The ability of FOXO3a to induce cell cycle arrest is enhanced by extra copies of SIRT1 and diminished in SIRT1-/- cells. As a correlative, overexpression of SIRT1 inhibits stress-triggered apoptosis induced by FOXO3a. The underlying mechanisms for this selective effect of SIRT1 on FOXO3a targets are not known. Acetylation could modify the DNA-binding properties of FOXO, but this modification would have to affect apoptosis-inducing genes differently from genes that intervene in the cell cycle and carry out repair functions.

UBIQUITINATION AND PROTEASOMAL DEGRADATION

Translocation into the cytoplasm exposes FOXO proteins to a third layer of post-translational regulation: ubiquitination-dependent proteasomal degradation.16,56-58 In cells treated with growth factors or insulin, the total levels of FOXO1 are sharply decreased. This degradation of FOXO1 results from ubiquitination and proteolysis. Cytoplasmic, but not nuclear, FOXO1 becomes ubiquitinated, and this modification depends on phosphorylation by Akt and ultimately on signaling from PI3K. In chicken embryo fibroblasts transformed by oncogenic versions of PI3K or Akt, FOXO1 levels are dramatically reduced, typically below the limits of detectability in Western blots. Phosphorylated FOXO1 can be detected in these cells, but at substantially diminished concentrations compared to normal serum starved or growth factor-treated controls. In the presence of the PI3K inhibitors LY294002 and Wortmannin, growth factors fail to affect FOXO1 levels, suggesting a requirement for PI3K in this process. The TOR (target of rapamycin) inhibitor rapamycin and

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the ERK (extracellular signal-regulated protein kinase) inhibitor PD98059 do not interfere with the growth factor-dependent elimination of FOXO. Significantly, inhibitors of proteasomal activity protect FOXO1 from degradation. These observations suggest that the ultimate result of Akt-induced phosphorylation, nuclear export and cytoplasmic ubiquitination is proteasomal degradation of FOXO1. The ubiquitin ligase acting on FOXO1 has been identified as Skp2, a member of the SCF (Skp1, Cul1, F-box protein) ubiquitin ligase complexes.59 Phosphorylation of serine 256 in FOXO1 by Akt creates a binding site for Skp2. Skp2 interacts with cytoplasmic, Akt-phosphorylated FOXO1 and ubiquitinates it, marking it for proteasomal degradation. Expression of exogenous Skp2 reduces the overall FOXO1 levels in the cell, while reduction of cellular Skp2 content through RNA interference elevates the total cellular levels of FOXO1. As a result of Skp2-mediated proteasomal degradation, FOXO1 transcriptional activity is downregulated. The details of proteolytic regulation of FOXO1 are not reproduced in all other FOXO proteins. For FOXO3a, the critical kinase is IKKβ rather than Akt, and the relevant target residue is serine 644, attracting an as yet unidentified ubiquitin ligase.16 However, the essential steps, phosphorylation-dependent ubiquitination followed by proteasomal degradation are the same for FOXO1 and FOXO3a.

POST-TRANSLATIONAL REGULATION OF FOXO PROTEINS AND CANCER

The FOXO-containing fusion proteins identified in alveolar rhabdomyosarcoma and in leukemias act as transcription factors. The FOXO component supplies the transactivation domain, while the other fusion partner contributes the DNA-binding domain. The oncogenic action of these proteins is based on aberrant transcriptional regulation of target genes that are normally regulated by the fusion partners of FOXO. Enhanced transactivation of such target genes and unresponsiveness of the fusion protein to normal controls appear to be critical factors contributing to oncogenic potential. Cancers with recurrent chromosomal translocations constitute a minority of all malignancies. However, FOXO proteins have a broader and perhaps general significance in cancer since they are important negative regulators of cell replication.8,16,18,20,24,60 This inhibitory activity of FOXO is reduced or eliminated during oncogenesis. A significant cancer-related player in the downregulation of FOXO is the ubiquitin ligase Skp2. Skp2 is upregulated in numerous cancers and probably has other, yet to be identified cancer-related targets besides FOXO1. Skp2 has many of the attributes of a dominant transforming oncoprotein.61-65 Various experimental systems underline the importance of FOXO in cellular growth control. A dominant-negative construct of FOXO1 in which the transactivation domain is replaced with a repression domain interferes with FOXO1 transcription and induces oncogenic transformation in cultured chicken embryo fibroblasts.56 Small molecule inhibitors of FOXO nuclear export, identified in a high-content screen, provide further evidence for an important role of FOXO proteins in regulating cell growth.66-68 Some of these inhibitors interfere with the function of CRM1, while others block Akt signaling. Several of these small molecules are highly effective in reducing the growth of tumor cells that show constitutive activation of Akt. Another Fox family protein, FOXG1, functions as a transcriptional repressor and is important in brain development.69-74 It can bind to the same DNA target sequence as FOXO1 and induces oncogenic cellular transformation when ectopically expressed.75-77 www.landesbioscience.com

Figure 3. The FOXO shuttle. Phosphorylation of cytoplasmic FOXO by Ral-activated JNK leads to nuclear import. Acetylation of nuclear FOXO by p300/CBP is followed by deacetylation by SIRT1. Growth factor-stimulated phosphorylation of FOXO results in binding to 14-3-3 and CRM1 and nuclear export. Akt-phosphorylated, cytoplasmic FOXO is ubiquitinated by Skp2 and is degraded by proteasomes.

The dramatic reduction of FOXO1 levels in cells transformed by oncogenic PI3K or Akt is another indicator of the important growth-regulatory role of FOXO proteins. The PI3K gain-of-function mutations recently discovered in a substantial fraction of human solid tumors78-84 are likely to cause a downregulation of FOXO proteins. This process may reveal important new cancer targets and deserves intensive study.

SYNTHESIS AND CONCLUSIONS

The three layers of post-translational regulation of FOXO are not insulated from each other; rather, they are interdependent and interacting. They form a regulatory microcosm that balances the functions and the fates of FOXO proteins with exquisite sensitivity to external signals of growth or stress (Fig. 3). In this system, the line from a single regulatory event branches into multiple effects on distinct functions of FOXO. This kind of regulation gives a new meaning to the term “forkhead”, as indeed many of the control circuits that govern the activities of FOXO proteins show multiple forks. Thus, Akt-mediated phosphorylation corrupts the NLS, reduces DNAbinding, interferes with p300/CBP interaction, facilitates binding to 14-3-3 and initiates an interaction with the nuclear exportation protein CRM1. The alliance of FOXO with the deacetylase SIRT1 is preceded by JNK-induced phosphorylation, nuclear translocation and acetylation. SIRT1 then alters the balance of the FOXO transcriptional program in favor of the genes that induce G1 arrest, perform DNA repair, and defend against oxidative damage as opposed to the genes that initiate apoptosis. The SIRT1-FOXO combination places preservation and healing of the injured over elimination of the defective. The steps involved in nuclear export and proteasomal degradation are known in principle, and to some extent, in satisfying mechanistic detail.9,15,21 In contrast, the signals that initiate and control nuclear translocation and transcriptional activity of FOXO proteins have only recently come to light and there are still significant gaps in our understanding.47-50 Activation and inactivation of FOXO appear to be determined by two competing signaling chains. Prevalence of stress signals favors nuclear translocation and activation, while dominance

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of growth signals leads to inactivation of FOXO. However, this model of two opposing regulatory forces is surely an oversimplification since it does not account for the differential regulation of specific FOXO functions such as the induction of apoptosis, DNA repair, and the control of life span. A complete understanding of the regulation and functions of FOXO proteins remains a fascinating challenge for the future. References 1. Weigel D, Jurgens G, Kuttner F, Seifert E, Jackle H. The homeotic gene fork head encodes a nuclear protein and is expressed in the terminal regions of the Drosophila embryo. Cell 1989; 57:645-58. 2. Weigel D, Jackle H. The fork head domain: a novel DNA binding motif of eukaryotic transcription factors? Cell 1990; 63:455-6. 3. Kaestner KH, Knochel W, Martinez DE. Unified nomenclature for the winged helix/forkhead transcription factors. Genes Dev 2000; 14:142-6. 4. Clark KL, Halay ED, Lai E, Burley SK. 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