A Brief Introduction to Foxology

BMTh Burgering

Laboratory of Physiological Chemistry, University Medical Center Utrecht, Stratenum, Utrecht, The Netherlands

Members of the Forkhead boxO (FOXO) class of domain is sometimes also referred to as the forkhead/ transcription factors are key players in the regulation of winged helix domain. The high degree of sequence cell-fate decisions, such as cell death, cell proliferation and homology within the DNA-binding domain is in cell metabolism. Furthermore, in model organisms, it contrast with an almost complete lack of similarity in has by now been demonstrated that FOXO function the N-terminal and C-terminal transactivation domains. affects the life span of these organisms. Multiple signal Over the last decade, more than 100 members have transduction pathways regulate FOXO function, but most been identified, with roles in development, differentia- importantly, they are negatively regulated by protein tion, proliferation, apoptosis, stress resistance and kinase B (PKB/AKT)-mediated phosphorylation and metabolism (reviewed by Carlsson and Mahlapuu, constitute, therefore, an important downstream com- 2002). During evolution, the number of forkhead genes ponent of insulin signalling. This review issue provides a appears to have increased, with greater numbers timely overview of our understanding of FOXO function identified in vertebrates than in invertebrates; the human and how signalling affects FOXO function. Taken forkhead-box gene family consists of at least 43 together, the reviewed studies on FOXO function and members. Several years ago, a standard nomenclature regulation provide compelling evidence that FOXOs act at for this family of proteins was introduced (Kaestner the crossroad between aging and age-related diseases et al., 2000), and Fox (Forkhead box) was adopted as a including diabetes and cancer. With this perspective, unified symbol for all chordate forkhead/winged helix further studies on FOXO function and regulation may transcription factors. Subclasses are designated by a shed light on how age impacts on the onset and letter, and within each subclass proteins are given a progression of disease. number. Names for the Fox proteins contain all Oncogene (2008) 27, 2258–2262; doi:10.1038/onc.2008.29 uppercase letters for human (for example, FOXA1), only the first letter capitalized for mouse (for example, Keywords: transcription; insulin; aging; cell cycle; cell Foxa1) and the first and subclass letters in uppercase for death; evolution all other chordates (for example, FoxA1). The FoxO class of transcription factors consists of four members: FoxO1, 3a, 4 and 6. The alternative Transcription factors are modular proteins with distinct names for these genes used in earlier studies were FKHR functions contained within defined domains, such as (FoxO1), FKHRL1(FoxO3a) and AFX or Mllt7 DNA-binding and transactivation of transcription. (FoxO4). FoxO2, originally named AF6q21 and cloned Initial sequence comparison between the Drosophila as a novel fusion partner for the AF6 protein, is melanogaster forkhead gene, essential for the proper homologous to FoxO3a and likely not a separate FoxO. formation of terminal structures of the embryo, and the FoxO5 is expressed in Danio rerio only. FoxO1, 3a and 4 hepatocyte-enriched transcription factor HNF-3a are ubiquitously expressed, but between different cell revealed a region of extensive similarity, which mapped types or organs, the expression level of these FoxOs can to the DNA-binding domain of HNF-3a (Weigel and differ considerably. For example, FoxO1 is highly Jackle, 1990). This finding suggested a new class of expressed in adipose tissue, whereas FoxO4 is highly evolutionarily conserved transcription factors with a expressed in muscle and FoxO3a in liver (Furuyama characteristic DNA-binding motif, which was coined the et al., 2000). FoxO6 expression appears restricted to ‘forkhead domain’ after the isolation of the first brain (Jacobs et al., 2003). Splice variants have been member. The Forkhead domain is an approximate described for FoxO1, 3a and 4 (Yang et al., 2002). 100-amino acid, monomeric DNA-binding domain and Because of the shared DNA-binding domain, FoxOs are represents a variant of the helix–turn–helix motif. It is expected to bind to similar sequences within the DNA made upof three a helices and two characteristic large and a core consensus DNA sequence for FOXO binding loops or butterfly-like ‘wings’. Therefore, the forkhead has been determined (50TTGTTTAC30) (Furuyama et al., 2000). However, the details of DNA binding of the FoxOs are still elusive. In this review issue, Tomas Correspondence: Professor BMTh Burgering, Laboratory of Physio- logical Chemistry, University Medical Center Utrecht, Stratenum, Obsil and Veronika Obsilova, further discus the Universiteitsweg 100, Utrecht 3584CG, The Netherlands. structural aspects of FoxO DNA binding and other E-mail: [email protected] structure–function relations. A brief introduction to FOXOlogy BMTh Burgering 2259 Thus, in general, all FoxOs could regulate the same protein action, or physiological events involved in dauer set of genes through binding to this sequence, and formation. Combined with the sequence determination indeed, large overlapin gene expression will be observed of the individual dauer-regulating proteins, this resulted when comparing the transcriptional activity of over- in a number of important observations. First, it was expressing the individual FoxOs. Specificity in function shown that daf-2-dependent increase in life span requires is likely to be obtained through interactions with daf-16 (Kenyon et al., 1993). Second, age-1 was coregulators. In this issue, Kristan van der Vos and identified to act downstream of daf-2 in the regulation Paul Coffer further describe the variety of FoxO protein of daf-16 (Dorman et al., 1995). Third, DAF-2 was interactions and their role in controlling FoxO function. identified as an insulin-type receptor (Kimura et al., Initial interest in the FoxO transcription factors 1997), AGE-1 as an ortholog of the catalytic subunit of stemmed from the fact that they were identified as the lipid kinase phosphoinositide-3 kinase (PI-3K) translocation partners in a number of cancers. The best (Morris et al., 1996), and as already indicated DAF-16 studied example being the t(2;13) and t(1;13) transloca- was shown to be orthologous to FOXO (Ogg et al., tions present in a percentage of alveolar rhabdomyo- 1997). sarcomas resulting in a PAX3–FOXO1 (Galili et al., The identification of DAF-2 as an insulin type of 1993) and PAX7–FOXO1 (Davis et al., 1994) fusion receptor consequently positioned several of the dauer- protein, respectively. Although these observations did regulating genes within the context of a biological not directly imply a role for FOXOs in tumorigenesis, it signalling pathway already extensively studied in mam- has become clear by now that FOXOs are genuine malian (cell) systems. Following the discovery of insulin tumor-suppressors; Fu and Tindall more extensively in 1922 by Banting and Best and its establishment as the review, in this issue, the role of FoxO in cancer. causative link in diabetes, it became essential to under- A different perspective on FOXO function was stand the cellular consequences of insulin action. Initial provided by the observation that FOXOs are homo- characterization of insulin-dependent signalling relied logous to the Caenorhabditis elegans transcription on biochemical analyses in mammalian cells. Insulin was factor, DAF-16 (abnormal DAuer Formation-16) shown to bind to a specific insulin receptor that belongs, (Ogg et al., 1997). The postembryonic life cycle of as the EGF and PDGF receptor, to the class of tyrosine C. elegans consists of four larval stages (L1–L4). When kinase receptors, characterized by an intracellular kinase environmental conditions are unfavorable C. elegans domain that phosphorylates itself and other proteins on can, at the L2 stage, decide to enter an alternate larval tyrosine residues. The insulin receptor consists of a stage, the dauer stage, rather than to progress to L3 dimer and insulin binds to the a subunit of the insulin (Cassada and Russell, 1975). The dauer larva is receptor, which activates the tyrosine kinase activity developmentally arrested and adapted to long-term present in the b subunit. A protein(s) of relative survival. However, when conditions improve, dauer molecular mass between 165 000 and 185 000, collec- larvae resume their life cycle at the L4 stage. tively called pp185, was identified as a tyrosine- The decision to go from L2 to L3 or to enter dauer phosphorylated substrate for the insulin receptor (White has extensively been studied through genetics, and a et al., 1985), and after cloning, it was coined insulin large set of genes controlling dauer formation have been receptor substrate-1 (IRS-1) (Sun et al., 1991). IRS-1, identified (Riddle et al., 1981), one of which is daf-16.A which is phosphorylated on multiple tyrosine residues, developmental arrest such as dauer extends in time the lacks any enzymatic activity but rather acts as a lifecycle of C. elegans, but interestingly a number of multisite ‘docking’ protein to bind to signal-transducing dauer mutations were also shown to extend life span of molecules containing Src-homology 2 (SH2) and adult C. elegans. This was illustrated by the finding that Src-homology-3 (SH3) domains and importantly binds a temperature-sensitive partial loss-of-function allele of to PI-3K (Sun et al., 1991). the daf-2 gene extended life span approximately twofold This observation suggested PI-3K to be an important (Kenyon et al., 1993). At that time point, this observa- downstream component in insulin signalling. Activation tion apparently challenged prevailing (evolutionary) of PI-3K results in the generation of phosphatidylino- theories of ageing, as these would predict no such sitol lipids phosphorylated at the 30 postion of the dramatic effect to be caused by a single-gene mutation. inositol ring (PI3Ps); however, similar to cAMP, PI3Ps Indeed, the theoretical consequences of this and the act as second messengers. Although cAMP is known to subsequent observations made in studying ageing in activate cAMP-dependent protein kinase A (PKA), the model organisms are still debated. Yet, irrespective of identity of the protein activated by PI3Ps remained the outcome of this debate, it is now clear that gene unresolved. The availability of small molecule inhibitors function, even single-gene function, can have great of PI-3K, such as wortmannin and LY294002, enabled impact on organismal aging and as such DAF-16/ analysis of signalling components downstream of PI-3K. FOXO is likely to play an important role in the outcome This led to the identification of the kinases p70S6 kinase of the aging process in most, if not all, organisms. In this (Chung et al., 1994; Ming et al., 1994) and PKB issue, Linda Partridge and Jens Bruning further (Burgering and Coffer, 1995; Franke et al., 1995) as elaborate the role of FoxO in ageing. acting downstream of PI-3K. In addition, it was Analysis of the epistatic relations between these observed that expression of constitutively active PKB dauer-formation genes has ordered these genes into resulted in p70S6kinase activation, suggesting both to be pathways that likely correspond to timely sequences of in a linear pathway downstream of PI-3K (Burgering

Oncogene A brief introduction to FOXOlogy BMTh Burgering 2260 and Coffer, 1995). Yet, how these kinases are activated exclusion of FoxO and inhibition of transcriptional by the PI3Ps generated after PI-3K activation remained activity (Brunet et al., 1999; Brownawell et al., 2001). unclear. PKB harbors a so-called pleckstrin homology This confirmed the genetics in C. elegans, which showed (PH) domain (Mayer et al., 1993; Musacchio et al., activation of DAF-16 through loss of DAF-2 activity. 1993), next to its catalytic domain. It was initially shown Thus, these studies established a core insulin-signalling that a PH domain could bind to PI(4,5)P2 lipids module functional in regulating transcription, which is generated after phospholipase C activation (Harlan conserved through evolution. In line with this, it was et al., 1994), suggesting the possibility of PI3P lipid then shown by genetic analysis that also in C. elegans binding to the PH domain of PKB. Thus, this would PDK-1 regulates PKB (Paradis et al., 1999) and PKB result in subsequent targeting of PKB to the plasma subsequently regulates DAF-16 (Paradis and Ruvkun, membrane at the site of lipid production and possibly its 1998). In addition, in C. elegans, DAF-18 is the ortholog activation. Indeed, it was shown that the PH domain of of the tumor suppressor phosphatase and tensin PKB binds to PI3P lipids with a preference to PI(3,4)P2 homolog (PTEN), which was previously shown to (Franke et al., 1997), and that following growth factor function as a phosphoinositide 3-phosphatase, that stimulation, PKB translocates to the plasma membrane antagonizes PI-3K action. Consistent with this role of (Andjelkovic et al., 1997). However, activation of PTEN, DAF-18 acts as a negative regulator of PKB kinases often also requires a series of phosphorylation activity (Ogg and Ruvkun, 1998). However, although a events, and it was shown that PKB was subject to serine/ strong evolutionary conservation of this pathway is threonine phosphorylation, suggesting phosphorylation clear, details may differ slightly. For example, C. elegans besides membrane localization to be part of the does harbor orthologs of IRS-1 and the PI-3K adaptor activation mechanism (Burgering and Coffer, 1995; p85, named IST-1 and AAP-1, respectively; yet, the role Alessi et al., 1996). These notions culminated in the of IST-1 does not appear identical to that of IRS-1 identification of phosphoinositide-dependent kinase-1 (Wolkow et al., 2002). In contrast, in D. melanogaster, (PDK-1) as an upstream kinase phosphorylating Thr308 an IRS-1 homolog named CHICO is present, and its of PKB (Alessi et al., 1997; Stokoe et al., 1997; Stephens function more resembles that of mammalian IRS-1, and et al., 1998). Thr308 is located in the so-called regulatory consistent with this, chico mutants display correspond- T-loopof the kinase, and this is conserved in all ing changes in life span (Clancy et al., 2001). members of the AGC-kinase family to which PKB Although the above-mentioned facts clearly show that belongs. This characteristic suggested PDK-1 to be a the molecular details of the hardware of insulin general upstream T-loop kinase for all members of the signalling are conserved through evolution, this per se AGC-kinase family. Indeed, PDK-1 has been shown to does not imply that the biological function of this act as a T-loopkinase for many of the AGC kinases pathway is therefore conserved. Studies in model (Mora et al., 2004). Because PDK-1, like PKB, harbors organisms indeed start to reveal that next to the impact a PH domain responsible for binding PI-3P lipids, this on ageing, FoxOs have, in addition, a number of diverse first explains the importance of PI3P-mediated mem- functions and these are further discussed by Arden in brane localization, and second linked PI-3K activation this review issue. In mammals, insulin acts as the main to PDK-1-mediated T-loop phosporylation of the AGC regulator of glucose homeostasis. Studies, both in cell kinase family and activation of these kinases is, thus at lines and in genetically modified mice have shown that least in part, regulated by PI-3K. FoxO acts as an important downstream mediator and Signal transduction pathways appear conserved counterbalance of insulin in regulating glucose home- throughout evolution. A classical example in this respect ostasis, a function that seems to be conserved. Danielle is the signal transduction pathway by which receptors Gros, Pieter van den Heuvel and Morris Birnbaum regulate MAPkinase activation mediated by the small further elaborate upon the role of FoxO in metabolism. GTPase Ras. Studies in D. melanogaster revealed the In addition to glucose homeostasis, insulin also identity of the exchange factor for Ras (Son of Sevenless impacts on cell proliferation and cell survival. In terms (SOS) (Hafen et al., 1993) and studies in C. elegans of cellular responses, these are clearly different, yet identified the role of adaptor proteins (that is, Grb2) in interconnected functions, and more importantly inter- the process of SOS activation (Downward, 1994). dependent on cellular (glucose) metabolism (Plas and Furthermore, genetic analyses in these model organisms Thompson, 2002). Consistent with this, FoxO functions confirmed the RAF/MEK/ERK pathway to act down- in regulating a differential response of halting cell-cycle stream of Ras. Bearing this evolutionary conservation in progression either followed by resumption of cell cycling mind, combined with the notion that three potential or proceeding to a cell-death program. This response is PKB phosphorylation sites are present both in DAF-16 especially relevant under conditions of cellular stress, and FOXOs, this suggested to a number of laboratories including growth factor deprival or lowered glucose. that also in mammalian cells FOXOs could function as Similar to p53, FOXO is instructive in the decision PI-3K/PKB-regulated transcription factors downstream whether stress endured is too strong to progress cellular of insulin. Indeed, all FoxO members were shown to be life without adverse effects, or whether repair may regulated by PI-3K through PKB-mediated phosphor- resolve this. Ho, Myatt and Lam discuss these issues ylation at these three conserved residues (Brunet et al., further. 1999; Kops et al., 1999; Nakae et al., 1999; Rena et al., While the above-described functions of FoxO are in 1999). PKB-mediated phosphorylation results in nuclear line with the regulation of FoxO activity by insulin,

Oncogene A brief introduction to FOXOlogy BMTh Burgering 2261 FoxOs can also function downstream of other receptors within, and parallel to, the insulin-signalling pathway is that regulate PI-3K/PKB and, for example, FoxOs have at present far from complete and thus an area of further also been described as important regulators of immune intense investigation. function, which is probably independent of FoxO acting This review issue aims at providing an overview of the downstream of insulin. Interestingly, the innate immune aforementioned different aspects of FOXOlogy. response plays an important role in the survival of an Although these different aspects are discussed in organism and consequently from the perspective of the individual chapters, there is inevitably some overlap, function of FoxO in aging a role in regulating immune not at least because several studies on FoxO provide response becomes evident. Our current knowledge of compelling examples as to how different aspects in FoxO function in the immune system is in this issue biology can be interconnected. reviewed by Stanford Peng. Taken together, it can be concluded that research on Because of the ability to perform large-scale genetic FOXO is one of the examples that illustrates the power screens in C. elegans and Drosophila, an ever-increasing of combining different scientific approaches, that is, number of novel genetic interactions within PI-3K biochemistry and genetics. It is also clear that analysis of signalling toward DAF-16/FoxO were discovered. FOXO functions now provides strong evidence that Examples are Melted (Teleman et al., 2005), SMK-1 FOXO is acting at the crossroad of life span and age- (Wolff et al., 2006), bar-1/b-catenin (Essers et al., 2005) related diseases such as diabetes and cancer in all model and Sir2/SIRT (Tissenbaum and Guarente, 2001; organisms tested so far. Whether and how this can be Brunet et al., 2004; Motta et al., 2004; van der Horst translated to the human condition is a challenge for the et al., 2004). Furthermore, regulators of DAF-16/ future, but the results summarized in this issue certainly FOXO acting in parallel to insulin signalling were provide a strong enough basis to take up this challenge. discovered, such as the kinases SGK-1 (Brunet et al., 2001) and JNK (Essers et al., 2004; Oh et al., 2005; Wang et al., 2005). Finally, other modes of regulation, Acknowledgements besides phosphorylation, were described most notably Work in my Laboratory is financially supported by the Dutch acetylation and (mono)ubiquitination. The role of all Cancer Foundation (KWF), Netherlands Organization of these different post-translational modifications and Science (NWO), Center of Biomedical Genetics (CBG) and especially the interplay between all these modifications Cancer Genomics Center (CGC). I thank all lab members past are summarized and discussed in this issue by Calnan and present for their support and discussion, Tobias Dansen and Brunet. The understanding, especially at the for critical reading of this manuscript and Cristina Arpesella molecular level, of the function of all these proteins for secretarial assistance in putting together this review issue.

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