FoxO6, a Novel Member of the FoxO Class of Transcription Factors with Distinct Shuttling Dynamics
2003; Elsevier BV; Volume: 278; Issue: 38 Linguagem: Inglês
10.1074/jbc.m302804200
ISSN1083-351X
AutoresFrank M. J. Jacobs, Lars P. van der Heide, Patrick J. Wijchers, J. Peter H. Burbach, Marco F.M. Hoekman, Marten P. Smidt,
Tópico(s)Circular RNAs in diseases
ResumoForkhead 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 FoxO-function through multiple phosphorylation events and other mechanisms involved in the nuclear exclusion of FoxO-proteins. 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 FoxO-function 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 (1Kaufmann E. Knochel W. Mech. Dev. 1996; 57: 3-20Crossref PubMed Scopus (574) Google Scholar). 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 (1Kaufmann E. Knochel W. Mech. Dev. 1996; 57: 3-20Crossref PubMed Scopus (574) Google Scholar). Daf-16, a forkhead transcription factor in Caenorhabditis elegans has been extensively studied for its role in controlling longevity and dauer formation (2Ogg S. Paradis S. Gottlieb S. Patterson G.I. Lee L. Tissenbaum H.A. Ruvkun G. Nature. 1997; 389: 994-999Crossref PubMed Scopus (1543) Google Scholar). 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 (3Biggs III, W.H. Cavenee W.K. Arden K.C. Mamm. Genome. 2001; 12: 416-425Crossref PubMed Scopus (173) Google Scholar). A subset of FOXO genes has been associated with disorders like tumorogenesis and rhabdomyosarcomas. Genetic analysis of a type of acute lymphocytic leukemia revealed that the cause of the disorder is a translocation between chromosome 11 and chromosome X [t(X,11)]. This translocation involves fusion of the general transcription factor HTRX1 with the forkhead gene FOXO4 on the X chromosome (4Parry P. Wei Y. Evans G. Genes Chromosomes Cancer. 1994; 11: 79-84Crossref PubMed Scopus (139) Google Scholar). A form of rabdomyosarcoma is caused by a translocation between chromosome 2 or chromosome 1 and chromosome 13 [t (1Kaufmann E. Knochel W. Mech. Dev. 1996; 57: 3-20Crossref PubMed Scopus (574) Google Scholar, 13Alessi D.R. Caudwell F.B. Andjelkovic M. Hemmings B.A. Cohen P. FEBS Lett. 1996; 39: 333-338Crossref Scopus (550) Google Scholar) or t (2Ogg S. Paradis S. Gottlieb S. Patterson G.I. Lee L. Tissenbaum H.A. Ruvkun G. Nature. 1997; 389: 994-999Crossref PubMed Scopus (1543) Google Scholar, 13Alessi D.R. Caudwell F.B. Andjelkovic M. Hemmings B.A. Cohen P. FEBS Lett. 1996; 39: 333-338Crossref Scopus (550) Google Scholar)], which leads to fusion of the PAX7 or PAX3 gene with the forkhead FOXO1 (5Galili N. Davis R.J. Fredericks W.J. Mukhopadhyay S. Rauscher III, F.J. Emanuel B.S. Rovera G. Barr F.G. Nat. Genet. 1993; 5: 230-235Crossref PubMed Scopus (774) Google Scholar, 6Davis R.J. Bennicelli J.L. Macina R.A. Nycum L.M. Biegel J.A. Barr F.G. Hum. Mol. Genet. 1995; 4: 2355-2362Crossref PubMed Scopus (52) Google Scholar). The fusion product turned out to be a stronger activator compared with PAX3 or PAX7, which function as inhibitors of myogenic differentiation of migrating limb myoblasts (7Epstein J.A. Lam P. Jepeal L. Maas R.L. Shapiro D.N. J. Biol. Chem. 1995; 270: 11719-11722Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). Blockage of this terminal differentiation pathway by the PAX-FOXO1 fusion product is the direct cause of this disorder. Since their discovery, FOXO-members have been subject of intensive investigation, especially their place in the phosphatidylinositol 3 (PI3) 1The abbreviations used are: PI3, phosphatidylinositol 3; PKB, protein kinase B; hiFCS, heat-inactivated fetal calf serum; GFP, green fluorescent protein; EGFP, enhanced green fluorescent protein; IGF-1, insulin-like growth factor; DBE, DAF-16 binding elements; HEK, human embryonic kidney-cells; MAPK, mitogen-activated protein kinase; NES, nuclear export signal; EST, expressed sequence tag.-kinase/protein kinase B (PKB) pathway and the identification of the transcriptional targets. Binding of insulin-like substrates to the insulin receptor leads via PI3-kinase to phosphorylation and activation of PKB. As demonstrated in mammalian cell-lines, PKB phosphorylates specific motifs within FOXO proteins, inducing translocation to the cytosol, thereby preventing their transcriptional activity (8Kops G.J. Burgering B.M. J. Mol. Med. 1999; 77: 656-665Crossref PubMed Scopus (253) Google Scholar, 9Kops G.J. de Ruiter N.D. De Vries-Smits A.M. Powell D.R. Bos J.L. Burgering B.M. Nature. 1999; 398: 630-634Crossref PubMed Scopus (952) Google Scholar). To elucidate cellular functions of FOXO proteins, many studies focused on identification of their transcriptional targets. FOXO3a has been demonstrated to play an important part in cell cycle progression of fibroblast cells by regulating expression of the mitotic genes cyclin B and polo-like-kinase. Interference with FOXO3a transcriptional activity induces defective cytokinesis, a delayed transition from M to G1, and finally accumulation of cells in the G2/M stage (10Alvarez B. Martinez A.C. Burgering B.M. Carrera A.C. Nature. 2001; 413: 744-747Crossref PubMed Scopus (237) Google Scholar). FOXO3a triggers DNA repair through the Gadd45 protein, which was shown to be a direct transcriptional target of this forkhead protein (11Tran H. Brunet A. Grenier J.M. Datta S.R. Fornace Jr., A.J. DiStefano P.S. Chiang L.W. Greenberg M.E. Science. 2002; 296: 530-534Crossref PubMed Scopus (710) Google Scholar). Recently FOXO3a has been shown to protect quiescent cells from oxidative stress by inducing transcription of manganese superoxide dismutase (12Kops G.J. Dansen T.B. Polderman P.E. Saarloos I. Wirtz K.W. Coffer P.J. Huang T.T. Bos J.L. Medema R.H. Burgering B.M. Nature. 2002; 419: 316-321Crossref PubMed Scopus (1273) Google Scholar). Taken together, these findings indicate that FOXO proteins are of crucial importance for the ability of a cell to respond to environmental changes. Processes of proliferation, differentiation, and responsiveness to extracellular changes are highly relevant in the nervous system. The properties of FoxO proteins render them candidates to play an important role in neuronal regulatory processes. For this reason we eluded on the identification of FoxO proteins in the central nervous system. In this study we describe the cloning and characterization of a novel member of the FoxO class and detail structural and functional properties related to gene regulation. This novel protein, FoxO6, clearly differs from FoxO1 and FoxO3 in its shuttling properties. Through mutation analysis and the generation of chimeric proteins this difference is identified as a domain absent in FoxO6 located just behind the forkhead domain in FoxO3 and FoxO1. PCR, Cloning, and Sequencing—From adult C57/Bl6 mouse brain, we dissected the tissue in the ventral midbrain. Total RNA was isolated and subjected to reverse transcription-PCR for cDNA synthesis using reverse transcriptase Superscript II and both oligo(dt) and random hexamer primers. Degenerate primers (forward, 5′-MGGCTSAMHYTSKCBCAGAT-3′; reverse, 5′-TTGTGVCGRTAKGARTYCTTCCA-3′) were designed to identify (novel) members of a subset of the forkhead family of transcription factors. This set of primers amplifies part of the forkhead domain of members of the FoxO group. The annealing temperature was 45 °C, and PCR products were separated on a 2% agarose gel by gel electrophoresis. Fragments of the expected length of 110 bp were purified (Qiagen PCR Purification kit), ligated in pGemT Easy (Promega), and transformed to Escherichia coli DH5alpha. Resulting colonies were subjected to colony PCR. Fragments of appropriate length were purified (Qiagen PCR-Purification kit) and sequenced on a Beckman Coulter CEQ 2000 sequencer under standard conditions. In all other PCR reactions we used the Long Range PCR kit (Roche) with the following modifications: denaturation and extension temperature were 98 and 68 °C, respectively. RNA Probe Synthesis—For the generation of a specific FoxO6 RNA probe, EST clone IMAGp998p163044q2 was subjected to PCR, and the amplified fragment was purified and sequenced. The fragment of 900 bp in length (200 bp coding sequence upstream the stopcodon and 700 bp 3′ untranslated region) did not contain the forkhead domain. T3-and T7-RNA polymerase were used in combination with a DIG RNA Labeling kit (Roche) to synthesize a sense and antisense DIG-labeled cRNA probe. In Situ Hybridization—In situ hybridization was performed as follows. Cryostat sections cut at 16 μm were thaw-mounted onto Super-frost+ slides, dried, and fixed for 10 min in fresh 4% paraformaldehyde in phosphate-buffered saline. After washing with phosphate-buffered saline, sections were acetylated for 10 min in a solution containing 245 ml H2O, 3.3 ml triethanolamine, 438 μl HCl (37%), and 625 μl acetic anhydride. Sections were washed with phosphate-buffered saline and prehybridized for 2 h in a prehybridization solution (50% deionized formamide, 5× SSC, 5× Denhardt's solution, 250 μg/ml baker's yeast, and 500 μg/ml sonificated salmon sperm DNA). Hybridization was performed overnight at 72 °C with 400 ng/ml DIG-labeled probe added to 150 μl hybridization solution each slide, covered with nescofilm. The nescofilm was removed in 2× SSC, and sections were placed in 0.2× SSC for 2 h and washed in a solution containing 100 mm Tris/HCl, pH 7.4, 150 mm NaCl (buffer 1). Preincubation with 1.5 ml of buffer 1 with 10% heat-inactivated fetal calf serum (hiFCS) was performed for 1 h at room temperature in a humidified chamber. Sections were incubated overnight at 4 °C with alkaline phosphatase-conjungated mouse anti-DIG Fab fragment (Roche), 1:5000 diluted in buffer 1 with 1% heat inactivated fetal calf serum. Sections were washed the next day in buffer 1 and equilibrated with a solution containing 100 mm Tris/HCl, pH 9.5, 50 mm MgCl2, 100 mm NaCl. Subsequently 200 μl NBT/BCIP solution (Roche) and 2.4 mg/10 ml final volume levamisole was added to a 100 mm Tris/HCl, pH 9.5, 50 mm MgCl2, 100 mm NaCl solution, and the color reaction was performed in the dark for about 8 h. The color reaction was stopped by adding 10 mm Tris/HCL, 5 mm EDTA, pH 8.0, and slides were dehydrated with ethanol and mounted using entellan (Merck). FoxO6-GFP Translational Fusion—Primers were designed to amplify the coding sequence of FoxO1, FoxO3, and FoxO6, introducing restriction sites, leaving the methionine intact and removing the stop-codon. Both PCR products and the EGFP-N1 vector (Clontech) were cut with appropriate restriction enzymes and purified. After 1 h ligation of the FoxO1, FoxO3, and FoxO6 cDNA fragments into the EGFP-N1 vector, the resulting constructs were transformed to E. coli DH5α. Colonies were subjected to colony PCR, and products were sequenced. A colony carrying the correct construct was selected and grown and plasmids were purified (Qiagen). The final construct encoded FoxO1, FoxO3, or FoxO6, immediately followed by EGFP. Mutations of either Thr-26 or Ser-184 to alanine residues were generated using site-directed mutagenesis. Resulting mutant DNA fragments were ligated in EGFP-N1 and sequenced. For the construction of the chimeric FoxO6[4Ser] we undertook a PCR-based strategy using the FoxO6-GFP construct in which we replaced FoxO6 amino acids 243–259 for FoxO3 amino acids 303–327. In a similar way we constructed FoxO6[NES2], in which we replaced FoxO6 amino acids 314–355 for FoxO3 amino acids 381–433. For the PCRs we used FoxO6 sequence-based primers with FoxO3 sequence overhang, and vice versa. Both constructs were sequenced. Cell Culture and Transfection of HEK-293 Cells—HEK-293 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) hiFCS, 100 units/ml penicillin, 100 units/ml streptomycin, and 2 mm l-glutamine in a humidified atmosphere with 5% CO2 at 37 °C. Cells were seeded in 12-well plates and grown for 24 h on glass coverslips. Cells were transfected with calcium phosphate precipitates containing 1.9 μg/well plasmid DNA. (0.12 μg target construct/1.78 μg pBlueScript carrier DNA). Forkhead Translocation Protocol—Twenty hours after transfection cells were serum-starved for 24 h. Translocation was induced by replacing the serum-free medium with medium supplemented with hiFCS (10% (v/v)), insulin (100 nm), or IGF-1 (1 ng/ml). After 2 h of incubation cells were fixed using 4% PFA in phosphate-buffered saline for 10 min at room temperature. Slides were embedded in Dabco-Mowiol and analyzed by fluorescent-microscopy. In experiments using inhibitors, cells were preincubated with either PD98059 (25 μm), LY294002 (25 μm) (TOCRIS), or leptomycin B (2 ng/ml) for 1 h. Subsequent stimuli were in the presence of inhibitors. Luciferase Assays—Cells were grown in 6-well plates and transfected with 5 μg of plasmid DNA/well, including 1 μg 6× DBE-Luc (kindly provided by B. M. Burgering), with or without 0.3 μg FoxO-GFP or empty vector and the appropriate amount of carrier plasmid. After transfection, cells were lysed and total GFP fluorescence was measured in 96-well plates using a FujiFilm FLA-5000 image reader to normalize the samples for transfection efficiency. Linearity of the measurements was checked with an EGFP standard curve. Luciferase activity of each sample was measured and corrected for total FoxO-GFP. Each experiment was at least performed in triplicate. Isolation and Characterization of FoxO6 mRNA—We used a degenerate PCR strategy to screen for (novel) members of the FoxO group of forkhead transcription factors expressed in the mouse ventral midbrain. Primers were designed to amplify part of the forkhead domain, a region with high sequence homology. Using this strategy we cloned PCR fragments encoding FoxO1, FoxO3, and FoxO4. Interestingly, we cloned a PCR fragment that showed high similarity with these genes, but differed in 10 of 110 bp compared with its closest family member. Data base analysis of this sequence in mouse genomic DNA databases led to characterization of the putative 3′ and 5′ part of a novel gene of the FoxO-group of transcription factors. Initially 7 mouse-derived ESTs from mouse tissue were identified, originating from the 3′ region (BI686281, AA656491, BF581745, AI593097, D21486, AI425281, and BF461725), and only recently a mouse brain-derived EST was released originating from the 5′ region (CA316065). Based on genomic DNA-sequence information, primers (forward; gcgggaccatggctgcgaagc, reverse; acttcaaccatccctcccagac) were designed to amplify the total coding region from mouse ventral midbrain cDNA. The resulting PCR fragment was cloned and sequenced. Primary sequence analysis revealed that the amplified cDNA contained a large open reading frame predicted to encode a 559 amino acid protein. The presence of a forkhead domain and overall similarity to FoxO1, FoxO3, and FoxO4, identified the protein as a novel member of the FoxO class of forkhead transcription factors (Fig. 1). Because FoxO5 is already designated in zebrafish (3Biggs III, W.H. Cavenee W.K. Arden K.C. Mamm. Genome. 2001; 12: 416-425Crossref PubMed Scopus (173) Google Scholar), we named this gene FoxO6. Noteworthy are the recently submitted "genome scan" gene predictions (XM284000 and XM143959) based on genomic and EST sequence data. These predictions are incomplete and incorrect for the fact that part of the genomic sequence of FoxO6 is not yet present in the databases. Comparison of the deduced amino acid sequence of FoxO6, FoxO1, FoxO3, and FoxO4, demonstrated that FoxO6 is 34% identical to FoxO1, 38% identical to FoxO3, and 36% identical to FoxO4 over their shared lengths. Within the forkhead domain this identity is increased to 90% for FoxO1, 89% for FoxO3, and 90% for FoxO4 (Fig. 1). Chromosomal Structure and Localization—In the murine genome FoxO6 is located on chromosome 4, region D1 between chromosomal markers 1283756 and X59556 (within 20 kb of marker 1283756), according to the MGSC v3 data base of the Sanger Institute. Mouse genomic data base analysis revealed that the open reading frame of FoxO6 is divided by a large intron of ∼18 kb long, resulting in 2 putative exons of 414 and 1266 bp in length. A polyadenylation signal (AATAAA) is found 818 bp downstream from the stopcodon. This 3′ end corresponds to 3′ EST sequences, which indicates that the FoxO6 mRNA contains a 3′ untranslated region of at least 818 bp long and that this is in fact the last exon of the FoxO6 gene. The startcodon (GGCGGGACCATG G) of the mapped FoxO6 amino acid sequences lies within a proper Kozak sequence. In addition, the 5′ EST contains no upstream startcodons in either frame. These facts and the homology to FoxO1, FoxO3, and FoxO4 indicate that the mapped methionine is the correct startcodon. Based on the 5′ EST, FoxO6 contains a 5′ untranslated region of at least 98 bp. Comparison of mouse FoxO6 to human genomic databases revealed that the human FoxO6 orthologue is located at chromosomal region 1p34.1. Within this regions several diseases have been mapped, but no clear indication for FoxO6 dysfunction related disease could be identified. In the human EST data base four different 3′ ESTs were found, originating from brain tissue and tumor-cell lines (AI361654, AI341823, M85901, and AA927741). All human ESTs showed ∼95% sequence identity to mouse FoxO6. Expression Pattern of FoxO6 in Murine Tissue—To elucidate the possible function of FoxO6 we examined the spatial and temporal expression pattern in murine tissues. In situ hybridizations using DIG-labeled probes specific for FoxO6 transcripts were performed in adult mouse brain (Fig. 2). In rostral sections the FoxO6 transcript was detected in the ependyma, the medial part of the anterior olfactory nucleus, and diffuse in the cingulate cortex (Fig. 2A). More caudal, expression was detected in the shell of the nucleus accumbens, the claustrum, the dorsal endopiriform nucleus, and the cingulate cortex (Fig. 2B). The transcript was dominantly present in the hippocampus, especially CA1 and CA3 areas, and to a lesser extent in the dentate gyrus and CA2 area (Fig. 2, C and D). Furthermore, the transcript was detected in the posteroventral part of the medial amygdaloid nucleus, portions of the amygdalo-hippocampal area, and dorsal and ventral endopiriform nuclei. In E12.5 embryos, a high level of expression of FoxO6 was detected in the trigeminal ganglion and tissue surrounding the lateral portion of the fourth ventricle that forms the cerebellum (Fig. 3A). The olfactory epithelium showed high amounts of the transcript, as well as the dorsal root ganglia along the embryo's spine (Fig. 3B). Lower amounts of the transcript were found in striatal areas and in the neopallial cortex, which forms the cerebral cortex. The level of expression in the olfactory epithelium and the dorsal root ganglions was sustained in embryos of E14.5 and E18.5, whereas expression in the trigeminal ganglion and developing cerebellum was diminished and expression in the striatal area was slightly increased. In addition, embryos of E14.5 showed a markedly increased expression in the neopallial cortex (Fig. 3C). Expression in the neopallial cortex was most apparent in the outermost layer, which represents the layer of cells that migrated most recently (Fig. 3D). In the cerebral cortex of E18.5 mouse, the transcript was still abundantly detected. This was also the case in the developing hippocampal areas (Fig. 3E), especially the inner layer that also contains the most recently migrated cells. In the periphery, the FoxO6 transcript was detected in the thymus (Fig. 3F), the cortical region of the kidney (Fig. 3G), the whiskers and dents (data not shown). These data show that the FoxO6 gene is dominantly present in the developing and adult murine brain, indicative for a function of FoxO6 during development and in the adult functional central nervous system. FoxO6 Lacks a Region Containing a PKB, CK1, and DYRK1A Phosphorylation Motif—In FoxO1, FoxO3, and FoxO4, three PKB phosphorylation motifs (13Alessi D.R. Caudwell F.B. Andjelkovic M. Hemmings B.A. Cohen P. FEBS Lett. 1996; 39: 333-338Crossref Scopus (550) Google Scholar) have been reported (8Kops G.J. Burgering B.M. J. Mol. Med. 1999; 77: 656-665Crossref PubMed Scopus (253) Google Scholar). The first PKB phosphorylation motif is located in the region just downstream the startcondon, a second in the forkhead domain, and a third in a region just downstream the forkhead domain. (Fig. 4A). The first and second regions containing a PKB phosphorylation motif are present in FoxO6 as well. Strikingly, the third region containing a motif for PKB catalyzed phosphorylation is absent in FoxO6. Besides a PKB phosphorylation motif, this region contains a stretch of 3 additional serine residues, present in the other members of the FoxO group (Fig. 4B). In FOXO1, Ser-319 is substrate for PKB, Ser-322 and Ser-325 are phosphorylated by CK1 (14Rena G. Woods Y.L. Prescott A.R. Peggie M. Unterman T.G. Williams M.R. Cohen P. EMBO J. 2002; 21: 2263-2271Crossref PubMed Scopus (201) Google Scholar), and Ser-329 is phosphorylated by DYRK1A (15Woods Y.L. Rena G. Morrice N. Barthel A. Becker W. Guo S. Unterman T.G. Cohen P. Biochem. J. 2001; 355: 597-607Crossref PubMed Scopus (219) Google Scholar). Although homology of FoxO6 to FoxO3 and FoxO1 is high just upstream of this region, the conserved PKB site including the stretch of serine residues is not conserved. Noteworthy is the fact that a third Arg-Xaa-Arg-Xaa-Xaa-Thr motif is found in the far C terminus of FoxO6, in a region that shows no similarity to the other FoxO proteins and Daf16. In addition, no CK1 or DYRK1A motifs are found in this region. Therefore it is not certain whether the threonine residue in this region is a natural substrate for PKB. Translocation of FoxO6 Is Dramatically Decreased Compared with FoxO1 and FoxO3—Previous studies (8Kops G.J. Burgering B.M. J. Mol. Med. 1999; 77: 656-665Crossref PubMed Scopus (253) Google Scholar) in mammalian cell lines have shown that in response to stimulation with insulin-like growth factors, PKB phosphorylates FOXO-proteins. This results in translocation of the forkhead protein from the nucleus to the cytosol (8Kops G.J. Burgering B.M. J. Mol. Med. 1999; 77: 656-665Crossref PubMed Scopus (253) Google Scholar). To test whether FoxO6 responds in a similar manner to growth factor stimulation, we transfected HEK-293 cells with FoxO1-, FoxO3-, and FoxO6-GFP constructs. Twenty-four hours after transfection FoxO1 and FoxO3 displayed a predominant cytosolic localization in virtually 100% of transfected cells for FoxO1 and ∼80% for FoxO3. In strong contrast to FoxO1/FoxO3, FoxO6 was fully localized in the nucleus 24 h after transfection (Fig. 5, 1st column). Subsequent serum starvation for 20 h resulted in a predominant nuclear localization for FoxO1 and FoxO3, although some cytoplasmic fluorescence was still apparent. FoxO6 however had an exclusive nuclear localization (Fig. 5, 2nd column). When stimulated with serum, IGF-1, or insulin, FoxO1 and FoxO3 were excluded from the nucleus and showed a predominant cytosolic localization (Fig. 5, 3–5th columns). Under these conditions FoxO6 displayed a predominant nuclear localization. Although translocation of FoxO6 was significantly less as compared with FoxO1 and FoxO3, a general increase in cytoplasmic fluorescence was detected. This indicates that some protein export from the nucleus had occurred. Translocation of FoxO6 Is Mediated by a PI3-kinase-dependent Mechanism—To assess whether nuclear export of FoxO6 is regulated in a PI3-kinase-dependent manner, we preincubated cells with LY294002, a PI3-kinase inhibitor, before treatment with either IGF-1 or insulin. PI3-kinase inhibition resulted in a significant decrease in cytosolic localization of FoxO6 in cells treated with either IGF-1 or insulin (Fig. 6). Besides the PI3-kinase pathway, IGF-1 and insulin can activate the MAPK pathway as well. Cells preincubated with PD98059, an inhibitor of the MAPK pathway, displayed no difference in IGF-1/insulin-induced translocation. These findings clearly indicate that translocation of FoxO6 upon IGF-1 or insulin stimulation is mediated by the PI3-kinase pathway. These results are in perfect agreement with results from similar studies done with other FoxO proteins (16Biggs III, W.H. Meisenhelder J. Hunter T. Cavenee W.K. Arden K.C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7421-7426Crossref PubMed Scopus (942) Google Scholar, 17Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5434) Google Scholar, 18Rena G. Guo S. Cichy S.C. Unterman T.G. Cohen P. J. Biol. Chem. 1999; 24: 17179-17183Abstract Full Text Full Text PDF Scopus (606) Google Scholar). Mutation of Thr-26 or Ser-184 Blocks Nuclear Exclusion of FoxO6 —Mutation analysis in FOXO1 has shown that substitution of Thr-24 or Ser-256 by alanine residues (mimicking a non-phosphorylated state) results in a blocked nuclear exclusion (16Biggs III, W.H. Meisenhelder J. Hunter T. Cavenee W.K. Arden K.C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7421-7426Crossref PubMed Scopus (942) Google Scholar, 19Rena G. Prescott A.R. Guo S. Cohen P. Unterman T.G. Biochem. J. 2001; 354: 605-612Crossref PubMed Scopus (219) Google Scholar). As stated before, the regions containing Thr-24 and Ser-256 in FOXO1 are highly conserved in all members. Thr-26 and Ser-184 are the equivalent residues in FoxO6 and are therefore potentially phosphorylated by PKB as well, resulting in nuclear export. To verify this possibility, we constructed mutant FoxO6 proteins, where either the Thr-26 or the Ser-184 residue was substituted by an alanine. Both mutan
Referência(s)