FoxO transcription factors are key regulators of growth, metabolism, life span, and stress resistance. FoxOs integrate signals from different pathways and guide the cellular response to varying energy and stress conditions. FoxOs are modulated by several signaling pathways, e.g., the insulin-TOR signaling pathway and the stress induced JNK signaling pathway. This study reports a genome wide RNAi screen of kinases and phosphatases aiming to find regulators of dFoxO activity in Drosophila S2 cells. By using a combination of transcriptional activity and localization assays several enzymes were identified that modulate dFoxO transcriptional activity, intracellular localization and/or protein stability. Importantly, several currently known dFoxO regulators were found in the screening, confirming the validity of the approach. In addition, several interesting new regulators were identified, including protein kinase C and glycogen synthase kinase 3beta, two proteins with important roles in insulin signaling. Furthermore, several mammalian orthologs of the proteins identified in Drosophila also regulate FOXO activity in mammalian cells. These results contribute to a comprehensive understanding of FoxO regulatory processes (Mattila, 2008).

By using a combination of transcriptional reporter and localization assays, twenty one dFoxO regulators were discovered. Some positive hits from the screen had an effect in dFoxO activity, localization, and protein stability, whereas other hits affected only transcriptional activity, suggesting that more mechanisms beyond subcellular localization and degradation are used by cells to regulate dFoxO activity. In addition to the 18 proteins that affected dFoxO transcriptional activity, the screening produced three more hits. Two of them seem to affect only dFoxO localization (dgkd and ptp69d), and one, neurospecific receptor kinase (nrk), affected exclusively dFoxO protein stability. It is possible that these proteins regulate dFoxO transcription on specific promoters in conjunction with other activators and that such factors are missing in Drosophila S2 cells. This would explain their lack of effect on the dInR promoter. Alternatively, they could affect dFoxO stability resulting in a net effect of dFoxO protein accumulation in the nucleus (Mattila, 2008).

Initially, the screening strategy was designed to identify both positive and negative regulators of dFoxO activity; however, no dFoxO repressors were found. Putative dFoxO repressors were present in the primary hit list of 31 targets, but those were later excluded in the secondary screen. This surprising observation suggests that the screen may be biased against dFoxO repressors. dFoxO is a well known inhibitor of protein biosynthesis in vivo, so under conditions of increased dFoxO activity, a reduction of general translation is expected that could affect GFP and luciferase translation too. Therefore, it is hypothesized that in the case of enhanced dFoxO activity it is possible that the concomitant inhibition of protein biosynthesis overruled a slight increase in reporter accumulation. This would explain the lack of dFoxO repressors among the targets of the screen. Moreover, the design of the screening based on S2 cells excludes the identification of regulatory mechanisms specific for other cell types, and instances where dFoxO is acting as a cofactor thereby regulating transcription indirectly (Mattila, 2008).

The results demonstrate that Drosophila PKC53E isoform is a dFoxO activator. Similar results were obtained in mammalian cells pointing out that the interaction is conserved. PKC isoforms have very important roles in insulin signaling, and each of the isoforms has been shown to be activated by insulin stimulation or conditions important for effective insulin stimulation. Importantly, PKC isoforms can both activate or inhibit insulin signaling: Atypical PKC isoforms are required for insulin-stimulated glucose transport in muscle and adipocytes. In contrast, certain conventional and novel PKC isoforms are known to antagonize insulin signaling in vertebrates. This interaction is implicated in the pathogenesis of free fatty acid mediated insulin resistance. Drosophila possesses six PKC isoforms whose role in this context has not yet been addressed. PKC53E homolog is closest to human conventional PKCα. Interestingly, it has been shown that PKCα inhibits insulin signaling through binding and phosphorylation of IRS1. Thus, PKCα would serve as a constitutively active inhibitory regulator of the insulin cascade through its association with IRS1. On stimulation with insulin, PKCα would dissociate from IRS1, thus releasing this protein from its down-regulated state. This would open the 'gate' for transmission of the insulin signal. It has been found that dFoxO/FOXO1 increases insulin sensitivity by up-regulating insulin receptor transcription. The observation that Drosophila PKCα activates dFoxO adds an additional twist in the complex regulatory network that dFoxO has on insulin signaling. Interestingly, in the experimental system used in this study AKT dependent dFoxO bandshift and AKT Ser-505 phosphorylation was not affected by PKC53E, indicating that PKC53E regulation of dFoxO is independent of AKT signaling (Mattila, 2008).

Another well known enzyme implicated in the control of metabolism identified as a regulator of dFoxO transcriptional activity is the Drosophila ortholog of Glycogen synthase kinase 3β (GSK-3β, Shaggy). GSK-3β is a regulator of glucose metabolism through the phosphorylation and inhibition of glycogen synthase, the rate limiting enzyme of glycogen deposition. GSK-3β is inhibited by AKT, so it was not surprising to see that GSK-3β activates dFoxO. GSK-3β protein level and activity is elevated in type II diabetic skeletal muscle cells reflecting the impairment of whole body glucose uptake characteristic to this disease. In addition, selective inhibition of GSK-3β by lithium chloride represses the expression of g6pase and pepck in rat hepatoma cells, both known targets of FoxO. Taken together, these observations suggest that some of the metabolic effects of GSK-3β are achieved by directly modulating dFoxO activity (Mattila, 2008).

An interesting dFoxO regulator is Polo-like kinase. Polo-like kinases (Plks) are known regulators of cell cycle progression. In addition, Plks have a role in the protection against cellular stress through the transcription factor HSF1. Recently it was proposed that an intricate tradeoff between lifespan and cancer results from opposing effects of enzymes regulating FoxO and p53 activity. Plks are known to inhibit p53 transcriptional activity, so the results raise the possibility that Plks mediate the common but opposing regulators of p53 and FoxO. Interestingly, FoxOs are necessary in the completion of the cell cycle, which is partly mediated by cell cycle dependent activation of Plk expression by FOXO3a. The results show that Drosophila dFoxO is regulated by Polo, suggesting the existence of a positive feedback mechanism that has a role in achieving periodic M-phase gene expression and proper cell cycle exit (Mattila, 2008).

dFoxO localization was affected by eight modulators; however, band shifts demonstrated that none of these proteins phosphorylated dFoxO in the three conserved Ser/Thr amino acids known to regulate nuclear/cytoplasmic status through AKT. This observation raises the possibility that some of the newly identified dFoxO regulators could affect dFoxO nuclear/cytoplasmic localization by phosphorylating dFoxO in additional residues that do not alter its electrophoretic mobility, or that dFoxO regulation by these proteins is indirect. Further studies will be needed to clarify this point (Mattila, 2008).

In summary, this study has identified 21 dFoxO modulators. The results underscore the complexity underlying dFoxO regulation and establish dFoxO as a transcription factor controlled exquisitely by an intricate network of kinases and phosphatases achieving a perfect balance of activity. This balance ensures the correct execution of key cellular processes in metabolism, response to stress, and life span (Mattila, 2008).