The transcription factor SKN-1 protects Caenorhabditis elegans from stress and promotes longevity. SKN-1 is regulated by diverse signals that control metabolism, development, and stress responses, but the mechanisms of regulation and signal integration are unknown. We screened the C. elegans genome for regulators of cytoprotective gene expression and identified a new SKN-1 regulatory pathway. SKN-1 protein levels, nuclear accumulation, and activity are repressed by the WD40 repeat protein WDR-23, which interacts with the CUL-4/DDB-1 ubiquitin ligase to presumably target the transcription factor for proteasomal degradation. WDR-23 regulates SKN-1 target genes downstream from p38 mitogen-activated protein kinase, glycogen synthase kinase 3, and insulin-like receptor pathways, suggesting that phosphorylation of SKN-1 may function to modify its interaction with WDR-23 and/or CUL-4/DDB-1. These findings define the mechanism of SKN-1 accumulation in the cell nucleus and provide a new mechanistic framework for understanding how phosphorylation signals are integrated to regulate stress resistance and longevity.In response to xenobiotic and oxidative stress, eukaryotic cells activate conserved pathways that increase the expression of phase II detoxification enzymes that scavenge free radicals, synthesize glutathione, and catalyze conjugation reactions that increase xenobiotic solubility and excretion (20). Phase II detoxification plays a central role in preventing age-related diseases, such as cancer and neurodegeneration (34, 39), and in mediating the multidrug resistance of pathogenic fungi, helminthes, and tumor cells (30,44,57).Phase II detoxification in Caenorhabditis elegans is controlled by the transcription factor SKN-1 (1), which promotes stress resistance and longevity (1,2,31,55). In nonstressed animals, SKN-1 is constitutively localized in the nuclei of hypothalamus-like (ASI) neurons, where it is required for life span extension by dietary restriction (5). SKN-1 is absent from the nuclei of other cell types except during exposure to oxidative stress and xenobiotics, which induces its accumulation in intestinal-cell nuclei, where it activates the expression of phase II detoxification genes (1,2,15,27,55). Despite the central role of SKN-1 in stress resistance and longevity, the mechanisms that control nuclear accumulation of the transcription factor are unknown.Phosphorylation of SKN-1 by glycogen synthase kinase 3 (GSK-3) inhibits nuclear accumulation (2). Nuclear accumulation is also inhibited by phosphorylation via SGK-1, AKT-1, and AKT-2 kinases downstream from the insulin-like receptor DAF-2 (55). Conversely, accumulation of SKN-1 in the nucleus is promoted by phosphorylation by a p38 mitogen-activated protein kinase (MAPK) cascade (23) and the activities of at least four other protein kinases (31). Phosphorylation of SKN-1 by these diverse kinases allows C. elegans to integrate phase II gene expression with metabolism, development, stress, and aging (55). However, the mechanisms by which phosphorylation...

Cells adapt to stressors by activating mechanisms that repair damage and protect them from further injury. Stress-induced damage accumulates with age and contributes to age associated diseases. Increased age attenuates the ability to mount a stress response, but little is known about the mechanisms by which this occurs. To begin addressing this problem, we studied hormesis in the nematode Caenorhabditis elegans. When exposed to a low concentration of the xenobiotic juglone, young worms mount a robust hormetic stress response and survive a subsequent exposure to a higher concentration of juglone that is normally lethal to naïve animals. Old worms are unable to mount this adaptive response. Microarray and RNAi analyses demonstrate that an altered transcriptional response to juglone is responsible in part for the reduced adaptation of old worms. Many genes differentially regulated in young versus old animals are known or postulated to be regulated by the FOXO homologue DAF-16 and the Nrf2 homologue SKN-1. Activation of these pathways is greatly reduced in juglone stressed old worms. DAF-16- and SKN-1-like transcription factors play highly conserved roles in regulating stress resistance and longevity genes. Our studies provide a foundation for developing a molecular understanding of how age affects cytoprotective transcriptional pathways.

SKN-1/Nrf are the primary antioxidant/detoxification response transcription factors in animals and they promote health and longevity in many contexts. SKN-1/Nrf are activated by a remarkably broad-range of natural and synthetic compounds and physiological conditions. Defining the signaling mechanisms that regulate SKN-1/Nrf activation provides insights into how cells coordinate responses to stress. Nrf2 in mammals is regulated in part by the redox sensor repressor protein named Keap1. In C. elegans, the p38 MAPK cascade in the intestine activates SKN-1 during oxidative stress by promoting its nuclear accumulation. Interestingly, we find variation in the kinetics of p38 MAPK activation and tissues with SKN-1 nuclear accumulation among different pro-oxidants that all trigger strong induction of SKN-1 target genes. Using genome-wide RNAi screening, we identify new genes that are required for activation of the core SKN-1 target gene gst-4 during exposure to the natural pro-oxidant juglone. Among 10 putative activators identified in this screen was skr-1/2, highly conserved homologs of yeast and mammalian Skp1, which function to assemble protein complexes. Silencing of skr-1/2 inhibits induction of SKN-1 dependent detoxification genes and reduces resistance to pro-oxidants without decreasing p38 MAPK activation. Global transcriptomics revealed strong correlation between genes that are regulated by SKR-1/2 and SKN-1 indicating a high degree of specificity. We also show that SKR-1/2 functions upstream of the WD40 repeat protein WDR-23, which binds to and inhibits SKN-1. Together, these results identify a novel p38 MAPK independent signaling mechanism that activates SKN-1 via SKR-1/2 and involves WDR-23.