Oncogene (2008) 27, 2258–2262 & 2008 Nature Publishing Group All rights reserved 0950-9232/08 $30.00 www.nature.com/onc INTRODUCTION 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.