Figure 1.

dFOXO is the only Drosophila FOXO/DAF-16 homolog. A TBLASTN search of the Drosophila genome for known and predicted genes encoding forkhead transcription factors retrieved 16 genes. (a) A phylogenetic tree calculated from a multiple sequence alignment of the forkhead domains of these 16 proteins and of the human FOXO proteins FOXO1 (FKHR), FOXO3a (FKHRL1) and FOXO4 (AFX), the C. elegans DAF-16 and mouse Foxa3 (HNF-3γ; protein names on the figure are from GenBank). The similarity of dFOXO to FOXO proteins is highlighted in blue. (b) dFOXO has three PKB phosphorylation sites in the same orientation as those of mammalian FOXO proteins. The sites are indicated above the protein; PEST (destruction), nuclear localization (NLS), nuclear export (NES) and DNA-binding sequences are also shown. (c) A multiple amino-acid sequence alignment of the dFOXO, human FOXO and DAF-16 forkhead domains illustrates the high degree of sequence conservation especially within the DNA-binding domain. The secondary structure is indicated above the alignment. Similar and identical amino-acid residues are shaded in gray and black, respectively. The region encoding helix 3 of the forkhead domain, which is the DNA-recognition helix contacting the major groove of the DNA double helix, is identical in the five proteins. Given the high structural similarity between the DNA-binding domains of FOXO4 (AFX) and HNF-3γ [85], it is likely that FOXO proteins contact insulin response elements through helix 3. Two EMS-induced point mutations described in this study are shown in red. (d) The dFOXO gene spans a genomic region of 31 kilobases (kb) and contains 11 exons (blue bars). The EP35-147 transposable element is inserted in the second intron upstream of the open reading frame, allowing GAL4-induced expression of endogenous dFOXO.