FoxO (forkhead box O; forkhead members of the O class) are transcription factors that function under the control of insulin/insulin-like signalling. FoxO factors have been associated with a multitude of biological processes, including cell-cycle, cell death, DNA repair, metabolism and protection from oxidative stress. Central to the regulation of FoxO factors is a shuttling system, which confines FoxO factors to either the nucleus or the cytosol. Shuttling of FoxO requires protein phosphorylation within several domains, and association with 14-3-3 proteins and the nuclear transport machinery. Description of the FoxO-shuttling mechanism contributes to the understanding of FoxO function in relation to signalling and gene regulation.

Forkhead transcription factors of the FoxO-group are associated with cellular processes like cell cycle progression and DNA-repair. FoxO function is regulated by protein kinase B (PKB) via the phosphatidylinositol 3-kinase/PKB survival pathway. Phosphorylation of serine and threonine residues in specific PKB phosphorylation motifs leads to exclusion of FoxO-proteins from the nucleus, which excludes them from exerting transactivating activity. Members of the FoxO-group have three highly conserved regions containing a PKB phosphorylation motif. This study describes the cloning and characterization of a novel forkhead domain gene from mouse that appeared to be highly related to the FoxO group of transcription factors and was therefore designated FoxO6. The FoxO6 gene was mapped in region D1 on mouse chromosome 4. In humans, FOXO6 is located on chromosomal region 1p34.1. Embryonic expression of FoxO6 is most apparent in the developing brain, and FoxO6 is expressed in a specific temporal and spatial pattern. Therefore it is probably involved in regulation of specific cellular differentiation. In the adult animal FoxO6 expression is maintained in areas of the nucleus accumbens, cingulate cortex, parts of the amygdala, and in the hippocampus. Structure function analysis of FoxO6 compared with its group members shows that the overall homology is high, but surprisingly a highly conserved region containing multiple phosphorylation sites is lacking. In transfection studies, FoxO6 coupled to GFP showed an unexpected high nuclear localization after stimulation with growth factors, in contrast to the predominant cytosolic localization of FoxO1 and FoxO3. We also show that nuclear export of FoxO6 is mediated through the phosphatidylinositol 3-kinase/PKB pathway. Furthermore, we show using a chimeric approach that we can fully restore the ability of FoxO6 to shuttle between nucleus and cytosol. In conclusion, the data presented here gives a new view on regulation of FoxOfunction through multiple phosphorylation events and other mechanisms involved in the nuclear exclusion of FoxO-proteins.Transcription factors of the forkhead family have an important role in development and function of an organism (1). Since the discovery of the winged helix structure (forkhead domain) in Drosophila, more than 90 genes containing the forkhead domain have been identified, in species ranging from yeast to humans (1). Daf-16, a forkhead transcription factor in Caenorhabditis elegans has been extensively studied for its role in controlling longevity and dauer formation (2). Transcriptional activity is negatively regulated via an insulin-like signal transduction cascade. In humans Daf-16 has four described orthologues, FOXO1 (FKHR), FOXO2, (AF6q21), FOXO3a (FKHRL1), and FOXO4 (AFX). Together, these proteins form the FOXO-class of forkhead transcription factors in humans. Also in mice, Daf-16 orthologues are identified and are designated FoxO1, FoxO3, and FoxO4 (3).A subset of FOXO genes has been associated with disorders like tumorogenesis and rhabd...

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Circadian rhythms are responsive to external and internal cues, light and metabolism being among the most important. In mammals, the light signal is sensed by the retina and transmitted to the suprachiasmatic nucleus (SCN) master clock [1], where it is integrated into the molecular oscillator via regulation of clock gene transcription. The SCN synchronizes peripheral oscillators, an effect that can be overruled by incoming metabolic signals [2]. As a consequence, peripheral oscillators can be uncoupled from the master clock when light and metabolic signals are not in phase. The signaling pathways responsible for coupling metabolic cues to the molecular clock are being rapidly uncovered [3-5]. Here we show that insulin-phosphatidylinositol 3-kinase (PI3K)-Forkhead box class O3 (FOXO3) signaling is required for circadian rhythmicity in the liver via regulation of Clock. Knockdown of FoxO3 dampens circadian amplitude, an effect that is rescued by overexpression of Clock. Subsequently, we show binding of FOXO3 to two Daf-binding elements (DBEs) located in the Clock promoter area, implicating Clock as a transcriptional target of FOXO3. Transcriptional oscillation of both core clock and output genes in the liver of FOXO3-deficient mice is affected, indicating a disrupted hepatic circadian rhythmicity. Finally, we show that insulin, a major regulator of FOXO activity [6-9], regulates Clock levels in a PI3K- and FOXO3-dependent manner. Our data point to a key role of the insulin-FOXO3-Clock signaling pathway in the modulation of circadian rhythms.