FoxO6, a Novel Member of the FoxO Class of Transcription Factors with Distinct Shuttling Dynamics

Abstract

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...

Long live FOXO: unraveling the role of FOXO proteins in aging and longevity

“…FOXO1, FOXO3, and FOXO4 are highly related, share the same DNA‐binding motifs, present similar patterns of expression, and seem to have overlapping functions (Anderson et al ., 1998; Biggs et al ., 2001; Jacobs et al ., 2003; Obsil & Obsilova, 2011). FOXO6 is mainly expressed in the brain and has been shown to be regulated by distinct mechanisms.…”

Post-translational modifications of FOXO family proteins

“…In mammalian species, the FOXO subfamily includes FOXO1, FOXO3, FOXO4 and FOXO6. FOXO1, FOXO3 and FOXO4 are widely expressed in various tissues and organs (8), however the expression of FOXO6 is most often detected in the developing brain (9), and recent studies have demonstrated that FOXO6 is involved in cell growth and transformation of liver cancer and lung cancer (10,11). FOXO homolog genes, DAF-16 and dFOXO, exists in lower organisms, including Caenorhabditis elegans, Drosophila and sponges (12).…”

Many forks in the path: cycling with FoxO

“…The PI3K-PKB signalling cascade is a key pathway by which cells may respond to a wide range of stimuli, which may be generated intrinsically (for example, growth factors, cytokines and cell-cell contact) or from an external source (for example, irradiation, and physical or genotoxic stress) (Leevers et al, 1999;Hennessy et al, 2005;Wymann and Marone, 2005;Samuels and Ericson, 2006). At the molecular level, many of the PI3K-PKB-mediated mitogenic responses are achieved through the direct repression of FoxO transcription factors by PKB-mediated phosphorylation, promoting nuclear export, proteosomal degradation and a decrease in transactivation activity (Brunet et al, 1999(Brunet et al, , 2001Kops et al, 2002b;Jacobs et al, 2003). FoxO is also a target of many other kinases, including serum and glucocorticoid-regulated kinase, c-Jun N-terminal kinase, a dual specificity tyrosinephosphorylated and regulated kinase (DYRK1A), CDK2 and IkB kinase (Brunet et al, 1999(Brunet et al, , 2001Kops et al, 2002b;Jacobs et al, 2003;Greer and Brunet, 2005) and the deregulation of PKB and these kinases is frequently observed in proliferative disorders and can contribute significantly to tumorigenesis (Hennessy et al, 2005;Samuels and Ericson, 2006).…”

“…At the molecular level, many of the PI3K-PKB-mediated mitogenic responses are achieved through the direct repression of FoxO transcription factors by PKB-mediated phosphorylation, promoting nuclear export, proteosomal degradation and a decrease in transactivation activity (Brunet et al, 1999(Brunet et al, , 2001Kops et al, 2002b;Jacobs et al, 2003). FoxO is also a target of many other kinases, including serum and glucocorticoid-regulated kinase, c-Jun N-terminal kinase, a dual specificity tyrosinephosphorylated and regulated kinase (DYRK1A), CDK2 and IkB kinase (Brunet et al, 1999(Brunet et al, , 2001Kops et al, 2002b;Jacobs et al, 2003;Greer and Brunet, 2005) and the deregulation of PKB and these kinases is frequently observed in proliferative disorders and can contribute significantly to tumorigenesis (Hennessy et al, 2005;Samuels and Ericson, 2006). The intimate link between FoxO signalling and regulation of cell cycle control is highlighted by the fact that in these instances the re-introduction of FoxO expression, for example in PTEN-deficient tumours, can induce cell cycle arrest followed by cell death (Nakamura et al, 2000).…”