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Unlabelled: Reduction-oxidation (redox) signaling, the translation of an oxidative intracellular environment into a cellular response, is mediated by the reversible oxidation of specific cysteine thiols. The latter can result in disulfide formation between protein hetero- or homodimers that alter protein function until the local cellular redox environment has returned to the basal state. We have previously shown that this mechanism promotes the nuclear localization and activity of the Forkhead Box O4 (FOXO4) transcription factor., Aims: In this study, we sought to investigate whether redox signaling differentially controls the human FOXO3 and FOXO4 paralogs., Results: We present evidence that FOXO3 and FOXO4 have acquired paralog-specific cysteines throughout vertebrate evolution. Using a proteome-wide screen, we identified previously unknown redox-dependent FOXO3 interaction partners. The nuclear import receptors Importin-7 (IPO7) and Importin-8 (IPO8) form a disulfide-dependent heterodimer with FOXO3, which is required for its reactive oxygen species-induced nuclear translocation. FOXO4 does not interact with IPO7 or IPO8., Innovation and Conclusion: IPO7 and IPO8 control the nuclear import of FOXO3, but not FOXO4, in a redox-sensitive and disulfide-dependent manner. Our findings suggest that evolutionary acquisition of cysteines has contributed to regulatory divergence of FOXO paralogs, and that phylogenetic analysis can aid in the identification of cysteines involved in redox signaling.

Significance: Transcription factors of the Forkhead box O class (FOXOs) are associated with lifespan and play a role in age-related diseases. FOXOs, therefore, serve as a paradigm for developing an understanding as to how age-related diseases, such as cancer and diabetes interconnect with lifespan. Understanding the regulatory inputs on FOXO may reveal how changes in these regulatory signaling pathways affect disease and lifespan., Recent Advances: Numerous regulators of FOXO have now been described and a clear and evolutionary conserved role has emerged for phosphoinositide-3 kinase/protein kinase B (also known as c-Akt or AKT) signaling and c-jun N-terminal kinase signaling. Analysis of FOXO function in the context of these signaling pathways has shown the importance of FOXO-mediated transcriptional regulation on cell cycle progression and other cell fates, such as cell metabolism, stress resistance, and apoptosis in mediating disease and lifespan., Critical Issues: Persistent DNA damage is also tightly linked to disease and aging; yet, data on a possible link between DNA damage and FOXO have been limited. Here, we discuss possible connections between FOXO and the DNA damage response in the context of the broader role of connecting lifespan and disease., Future Directions: Understanding the role of lifespan in diseases onset may provide unique and generic possibilities to intervene in disease processes to ensure a healthy lifespan.

Recent compelling data show that reactive oxygen species (ROS) not only are a harmful by-product of aerobic metabolism, but also are used as signaling molecules to regulate various cellular processes. In mammalian cells, ROS are produced transiently in response to many extracellular stimuli, including insulin, and specific inhibition of the ROS suppresses insulin-dependent signaling. Initially, this finding rationalized the concept of ROS acting as insulin mimetics. However, it is becoming evident that ROS are also causal to diabetes, a metabolic disorder characterized by insufficiency of secretion of, or receptor insensitivity to, endogenous insulin. This notion underlines a dual role for ROS in insulin signaling as both deleterious and beneficiary. Moreover, it strongly suggests that a delicate redox balance is required for insulin signaling to remain "healthy" for an organism.

The forkhead box O (FOXO) family of transcription factors regulates a variety of cellular programs, including cell cycle arrest, reactive oxygen species (ROS) scavenging, and apoptosis, and are of key importance in the decision over cell fate. In animal model systems it has been shown that FOXO is involved in the regulation of long lifespan. FOXO activity is tightly controlled by the insulin signaling pathway and by a multitude of ROS-induced posttranslational modifications. In the cell, ROS levels can be sensed by virtue of stimulatory and inhibitory oxidative modification of cysteine residues within proteins that control various signaling cascades. Recently, it was shown that cysteines in FOXO can also act as sensors of the local redox state. In this review we have outlined the cysteine-dependent redox switches that regulate both the insulin and ROS signaling pathways upstream of FOXO. Further, we describe how FOXO controls ROS levels by transcriptional regulation of a multilayered antioxidant system. Finally, we will discuss how cysteine-based redox signaling to FOXO could play a role in fine-tuning the optimal cellular response to ROS to control organismal lifespan.

Forkhead box O (FOXO) transcription factors are involved in various cellular processes, including cell proliferation, stress resistance, metabolism, and longevity. Regulation of FOXO transcriptional activity occurs mainly through a variety of post-translational modifications, including phosphorylation, acetylation, and ubiquitination. Here we describe nemo-like kinase (NLK) as a novel regulator of FOXOs. NLK binds to and phosphorylates FOXO1, FOXO3a, and FOXO4 on multiple residues. NLK acts as a negative regulator of FOXO transcriptional activity. For FOXO4 we show that NLK-mediated loss of FOXO4 activity co-occurs with inhibition of FOXO4 monoubiquitination. Previously, we have shown that oxidative stress-induced monoubiquitination of FOXO4 stimulates its transactivation, which leads to activation of an antioxidant defensive program. Conversely, NLK-dependent inhibition of FOXO4 activity can provide a means to downregulate this defensive program, when oxidative stress reaches a level beyond which repair is no longer feasible and cells need to undergo apoptosis.

Transcription factors are the common convergence points of signal transduction pathways to affect gene transcription. Signal transduction activity results in posttranslational modification (PTM) of transcription factors and the sum of these modifications at any given time point will determine the action of the transcription factor. It has been suggested that these PTMs provide a transcription factor code analogous to the histone code. However, the number and variety of these modifications and the lack of knowledge in general of their dynamics precludes at present a concise view of how combinations of PTMs affect transcription factor function. Also, a single type of PTM such as phosphorylation can have opposing effects on transcription factor activity. Transcription factors of the Forkhead box O (FOXO) class are predominantly regulated through signaling, by phosphoinositide 3-kinase/protein kinase B (also known as AKT) pathway and a reactive oxygen species/c-Jun N-terminal kinase pathway. Both pathways result in increased FOXO phosphorylation yet with opposing result. Whereas PKB-mediated phosphorylation inactivates FOXO, c-Jun N-terminal kinase-mediated phosphorylation results in activation of FOXO. Here we discuss regulation of FOXO transcription factors by phosphorylation as an example for understanding integration of signal transduction at the level of transcription activity.