Regulation of Drosophila FKH/FOXA by TOR Figure 7. FKH and TOR impact on the expression of the dFOXO target d4E-BP. Realtime qPCR was applied to quantify mRNAs in larval extracts. (A) Compared to wildtype and unspecific control animals, transcript levels of d4E-BP are low in larvae expressing FKH dsRNA (w; arm-Gal4/+; UAS-FKH- RNAi/+) and high in larvae overexpressing FKH (w;; ppl-Gal4/UAS-FKH). d4E-BP is upregulated in larvae treated with rapamycin, and the elevated d4E-BP transcription resulting from rapamycin feeding is completely suppressed by FKH RNAi. (B) d4E-BP is upregulated in dTOR mutants (y w; dTORDP/+ and y w; dTORDP/dTORDP) and the expression is suppressed in larvae transheterozygous for dTOR and FKH mutations (y w; dTORDP/+; fkh1/+ and y w; dTORDP/+; fkh6/+). This suggests that d4E-BP is a transcriptional target not only of the insulin pathway and dFOXO, but also of the TOR pathway and FKH. Significance was tested using an unpaired 2-tailed Student’s t-test. * = p,0,05 ** = p,0,01 *** = p,0,001. Error bars represent SEM. doi:10.1371/journal.pone.0015171.g007 nuclei. In the field of mammalian forkhead transcription factors, the insulin-dependent nucleocytoplasmic shuttling of FOXO proteins is well established [3]. Also mouse Foxa2 has been shown to translocate to the cytoplasm under conditions of high insulin signaling in cultured cells as well as in tissues such as liver and hypothalamus [32,33,34], but there have been conflicting reports arguing that hepatic Foxa2 is constitutively nuclear, irrespective of metabolic state or insulin levels [35,36]. It remains to be investigated whether the regulation of Drosophila FKH localization and activity by TORC1 described here is conserved in mammalian orthologs such as Foxa2. Conversely, it remains to be investigated whether the TOR-dependent nuclear exclusion of FKH is a consequence of protein phosphorylation. Cytoplasmic sequestration of mouse Foxa2 is linked to phosphory- lation of T156 by AKT, a residue within a sequence motif that is conserved in Drosophila FKH [34]. In the light of our study and the interaction of FoxA/PHA-4 with TOR and S6K in C. elegans [11], it is also tempting to speculate that FoxA proteins may be regulated through phosphorylation by TOR or S6K. d4E-BP has been previously described as a target of dFOXO [30,31]. We have demonstrated that d4E-BP expression is silenced Figure 8. A simplified working model of nutrient-dependent gene expression by TOR and FKH. (A) Under conditions of dietary protein abundance, the TOR signaling module is active and exerts a negative regulation on FKH, which is consequently sequestered in the cytoplasm and unable to modulate gene transcription. (B) When TOR complex 1 activity is inhibited by rapamycin or protein deprivation, the repression of FKH activity is diminished. A significant fraction of the cellular FKH pool accumulates in the nucleus and activates expression of the growth-inhibiting genes CG6770, cabut and d4E-BP. doi:10.1371/journal.pone.0015171.g008 PLoS ONE | www.plosone.org 9 December 2010 | Volume 5 | Issue 12 | e15171