Oncogene (2008) 27, 2263–2275 & 2008 Nature Publishing Group All rights reserved 0950-9232/08 $30.00 www.nature.com/onc REVIEW Structure/function relationships underlying regulation of FOXO transcription factors T Obsil1,2 and V Obsilova2 1Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University, Prague, Czech Republic and 2Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic The FOXO subgroup of forkhead transcription factors their forkhead DBD and constitute a discrete family plays a central role in cell-cycle control,differentiation, within the ‘winged helix protein’ superfamily that occurs metabolism control,stress response and apoptosis. There- in both eukaryote and prokaryote genomes (Clark et al., fore,the function of these important molecules is tightly 1993; Gajiwala and Burley, 2000). controlled by a wide range of protein–protein interactions Among the forkhead box family of transcription and posttranslational modifications including phospho- factors (a group of more than 100 proteins identified so rylation,acetylation and ubiquitination. The mechanisms far), the ‘O’ subgroup consists of four members by which these processes regulate FOXO activity are (FOXO1, FOXO3, FOXO4 and FOXO6). The FOXO mostly elusive. This review focuses on recent advances in proteins were initially identified in humans at chromo- structural studies of forkhead transcription factors and somal rearrangements in certain tumors (Galili et al., the insights they provide into the mechanism of DNA 1993; Davis et al., 1994; Parry et al., 1994; Borkhardt recognition. On the basis of these data,we discuss et al., 1997; Hillion et al., 1997; Anderson et al., 1998). structural aspects of protein–protein interactions and They are the vertebrate orthologs ofthe Caenorhabditis posttranslational modifications that target the forkhead elegans DAF-16 transcription factor and constitute key domain and the nuclear localization signal of FOXO components ofa highly conserved signaling pathway proteins. that connects growth and stress signals to transcrip- Oncogene (2008) 27, 2263–2275; doi:10.1038/onc.2008.20 tional control (Lin et al., 1997; Ogg et al., 1997). Molecules ofFOXO proteins consist offourdomains: a Keywords: FOXO; forkhead transcription factors; highly conserved forkhead DBD, a nuclear localization 14-3-3 protein; phosphorylation; acetylation; signal (NLS) located just downstream ofDBD, a ubiquitination nuclear export sequence (NES) and a C-terminal transactivation domain (Figure 1). FOXO1, FOXO3 and FOXO6 proteins have similar length ofapproxi- mately 650 amino-acid residues, whereas FOXO4 Introduction sequence is shorter and contains about 500 amino-acid residues. Analysis ofmultiple sequence alignment shows The forkhead box (FOX) transcription factors contain that several regions ofFOXO proteins are highly approximately 110-amino-acid-long winged helix DNA- conserved (Figure 2). The regions showing the highest binding domain (DBD) known as the forkhead domain sequence conservation include the N-terminal region (Weigel and Jackle, 1990; Kaestner et al., 2000; Mazet surrounding first AKT/protein kinase B (PKB) phos- et al., 2003). The FOX proteins display large functional phorylation site, the forkhead DBD, the region contain- diversity and play a wide range ofroles in development, ing NLS and the part ofthe C-terminal transactivation proliferation, differentiation, stress resistance, apoptosis domain. and control ofmetabolism (recently reviewed by Transcriptional activity ofFOXO proteins is regu- Carlsson and Mahlapuu, 2002; Lehmann et al., 2003; lated through insulin–phosphatidylinositol 3-kinase– Greer and Brunet, 2005; and van der Horst and AKT/PKB signaling pathway. Phosphorylation by Burgering, 2007). Their name is derived from the AKT/PKB creates two binding sites for the 14-3-3 Drosophila melanogaster forkhead gene, the first identi- proteins and induces phosphorylation ofadditional sites fied FOX transcription factor, which is important for by casein kinase-1 (CK1) and dual-specificity tyrosine- the correct formation of the anterior and posterior gut regulated kinase-1A. The AKT/PKB-mediated phos- ofthe embryo (Weigel et al., 1989). All FOX proteins phorylation ofFOXO induces binding of14-3-3 show high degree ofamino-acid sequence identity in proteins, and the resulting complex is then translocated to the cytosol where the bound 14-3-3 protein prevents reentry ofFOXO into the nucleus likely by interfering Correspondence: Professor T Obsil, Department of Physical and Macromolecular Chemistry, Faculty ofScience, Charles University; with the function of their NLS (Brunet et al., 1999; 12843 Prague, Czech Republic. Brownawell et al., 2001; Cahill et al., 2001; Zhao E-mail: [email protected] et al., 2004; Obsilova et al., 2005). In addition to Structure/function relationships in FOXO proteins T Obsil and V Obsilova 2264 Figure 1 Schematic representation ofprimary structure ofFOXO proteins. All FOXO proteins have the same domain organization and contain forkhead DNA-binding domain (DBD), nuclear localization signal (NLS), nuclear export sequence (NES) and transactivation domain (TA). Only the AKT/protein kinase B (PKB) phosphorylation sites are shown. Figure 2 Sequence alignment and posttranslational modifications ofFOXO subgroup members. Residues that are conserved at least in three sequences are shaded in gray. Residue color coding: brown, AKT/protein kinase B (PKB)/serum- and glucocorticoid-inducible kinase (SGK) phosphorylation sites; red, non-AKT/PKB phosphorylation sites; and green, acetylation sites. Symbol ( + ) denotes monoubiquitination sites. Regions ofnuclear localization signal (NLS) and nuclear export sequence (NES) motifsare labeled by yellow boxes. Question marks indicate unknown acetylating enzymes. Symbol (#) denotes oxidative stress-induced phosphorylation events (Brunet et al., 2004). Symbol (*) denotes sites phosphorylated by mitogen-activated kinases ERK and p38 (Asada et al., 2007). CBP, cyclic-AMP responsive element binding (CREB)-binding protein; cGK1, cyclic GMP-dependent kinase-1; DIRK1, dual-specificity tyrosine-regulated kinase-1A; JNK, c-JUN N-terminal kinase. AKT/PKB-mediated phosphorylation, the function of FOXO molecule (Figure 2). The precise mechanisms by FOXO proteins is also controlled by other types of which posttranslational modifications regulate FOXO posttranslational modifications including non-AKT/ functions are mostly elusive, but in many cases they PKB-mediated phosphorylation, acetylation and ubi- seem to affect DNA-binding potential of FOXO quitination. Sites ofthese posttranslational modifica- proteins, function of their NLS and NES, or interac- tions are often located within the conserved regions of tions ofFOXO with other proteins. Oncogene Structure/function relationships in FOXO proteins T Obsil and V Obsilova 2265 This review focuses on recent advances in structural However, different classes of forkhead proteins recog- studies of forkhead transcription factors and the insights nize diverse DNA sequences adjacent to the core they provide into the mechanism ofDNA recognition. sequence through still not fully understood mechanism. On the basis ofthese data, we discuss structural aspects To date, six structures offorkheaddomains bound to ofprotein–protein interactions and posttranslational DNA have been solved: FoxA3ÀDNA (Clark et al., modifications that target the forkhead domain and the 1993), FoxD3ÀDNA (Jin and Liao, 1999), FOXP2À NLS ofFOXO proteins. DNA (Stroud et al., 2006), NFATÀFOXP2ÀDNA (Wu et al., 2006), FOXK1aÀDNA (Tsai et al., 2006) and FOXO3aÀDNA (Tsai et al., 2007). Structure of FoxA3ÀDNA complex provided first details about Structure of forkhead DNA-binding domain interactions between forkhead DBD and the core sequence, showing that the helix H3 represents the main Structural studies offorkheadproteins began with the DNA recognition site (Clark et al., 1993). The forkhead solution ofco-crystal structure ofthe DBD ofa FoxA3 domain ofFoxA3 binds to the DNA duplex as a (HNF-3g) DNA complex (Clark et al., 1993). FoxA3 is À monomer with the helix H3 docked into the major a liver-specific transcription factor that plays important groove roughly perpendicular to the DNA axis provid- roles in cell differentiation and tissue-specific gene ing extensive interactions with the core sequence. expression (Lai et al., 1993). Its structure showed that Residues that are involved in these interactions the forkhead/winged helix motif is a compact structure (Asn165 and His169) are highly conserved among all containing about 110 amino-acid residues that fold forkhead proteins (Figure 3c). Contacts between fork- into three a-helices (H1, H2 and H3), three b-strands head domain and DNA involve direct hydrogen bonds (S1, S2 and S3) and two wing-like loops (W1 and W2) between side chains and bases, water-mediated side (Figure 3). Topologically, the arrangement ofthe chainÀbase interactions and van der Waals contacts domain is H1ÀS1ÀH2ÀH3ÀS2ÀW1ÀS3ÀW2. The with both the bases and the backbone ofDNA (Figures strand S1, inserted between helices H1 and H2, interacts 5a and 6a). Other regions ofFoxA3 DBD that make with strands S2 and S3 to form a three-stranded, important interactions with DNA, in addition to twisted, antiparallel b-sheet. While the N-terminal part recognition helix H3, are both wings W1 and W2 ofthe domain is formed by a cluster ofthree a-helices, flanking the helix H3. Mainly the wing W2 was shown to the C-terminal halfconsists of