The large repertoire of circadian rhythms in diverse organisms depends on oscillating central clock genes, input pathways for entrainment, and output pathways for controlling rhythmic behaviors. Stress-activated p38 MAP Kinases (p38K), although sparsely investigated in this context, show circadian rhythmicity in mammalian brains and are considered part of the circadian output machinery in Neurospora. We find that Drosophila p38Kb is expressed in clock neurons, and mutants in p38Kb either are arrhythmic or have a longer free-running periodicity, especially as they age. Paradoxically, similar phenotypes are observed through either transgenic inhibition or activation of p38Kb in clock neurons, suggesting a requirement for optimal p38Kb function for normal free-running circadian rhythms. We also find that p38Kb genetically interacts with multiple downstream targets to regulate circadian locomotor rhythms. More specifically, p38Kb interacts with the period gene to regulate period length and the strength of rhythmicity. In addition, we show that p38Kb suppresses the arrhythmic behavior associated with inhibition of a second p38Kb target, the transcription factor Mef2. Finally, we find that manipulating p38K signaling in free-running conditions alters the expression of another downstream target, MNK/Lk6, which has been shown to cycle with the clock and to play a role in regulating circadian rhythms. These data suggest that p38Kb may affect circadian locomotor rhythms through the regulation of multiple downstream pathways.

As organisms are constantly exposed to the damaging effects of oxidative stress through both environmental exposure and internal metabolic processes, they have evolved a variety of mechanisms to cope with this stress. One such mechanism is the highly conserved p38 MAPK (p38K) pathway, which is known to be post-translationally activated in response to oxidative stress, resulting in the activation of downstream antioxidant targets. However, little is known about the role of p38K transcriptional regulation in response to oxidative stress. Therefore, we analyzed the p38K gene family across the genus Drosophila to identify conserved regulatory elements. We found that oxidative stress exposure results in increased p38K protein levels in multiple Drosophila species and is associated with increased oxidative stress resistance. We also found that the p38Kb genomic locus includes conserved AP-1 and lola-PT transcription factor consensus binding sites. Accordingly, over-expression of these transcription factors in D. melanogaster is sufficient to induce transcription of p38Kb and enhances resistance to oxidative stress. We further found that the presence of a putative lola-PT binding site in the p38Kb locus of a given species is predictive of the species' survival in response to oxidative stress. Through our comparative genomics approach, we have identified biologically relevant putative transcription factor binding sites that regulate the expression of p38Kb and are associated with resistance to oxidative stress. These findings reveal a novel mode of regulation for p38K genes and suggest that transcription may play as important a role in p38K-mediated stress responses as post-translational modifications.

In order to understand how oxidative stress signal transduction pathways evolve, we analyzed the molecular evolution of the p38 MAPK (p38K) gene family across the genus Drosophila.p38K family genes play a vital role in oxidative stress resistance and are also important for organismal development and immunity. We find that the p38Ka and p38Kb genes are highly conserved across the genus and that p38Kc is more recently evolved. We further find that the p38Kb genomic locus includes conserved binding sites for the AP-1 and lola-PT transcription factors. Accordingly, over-expression of either AP-1 or lola-PT in D. melanogaster is sufficient to induce transcription of p38Kb under normal conditions, while under oxidative stress only lola-PT over-expression was able to induce p38Kb transcription. In addition, exposure to oxidative stress results in increased p38K protein levels in a number of species. These increased levels are associated with an increased resistance to oxidative stress across species. We also find that the presence of a lola-PT binding site in the p38Kb locus is predictive of the species' survival in response to oxidative stress. Through our comparative genomics approach, we have identified biologically relevant transcription factor binding sites that regulate the expression of p38Kb and are associated with resistance to oxidative stress. These findings reveal a novel mode of regulation for p38 MAP Kinases and suggests that transcription may play as important a role in p38K mediated stress responses as post-translational modifications. Significance StatementOrganisms encounter a variety of environmental stresses such as oxidative stress throughout their lifetime. Therefore, organisms have evolved a number of mechanisms to combat these stresses. In order to understand how these mechanisms evolved, we have compared the genomes of a diverse set of species across the genus Drosophila to examine the p38 MAPK stress response gene family. Our analysis was able to successfully predict transcription factors that not only regulate our target gene, p38Kb, but do so under different conditions to ensure an appropriate stress response. Therefore, we find that in addition to post-translational regulation, transcriptional regulation of signaling pathways may also play an important role in how organisms are able to adapt to stressful environments or respond to stress conditions as they arise. Furthermore, our comparative genomics approach may be utilized to identify transcriptional regulators of other highly conserved signaling pathways.

As organisms age, they often accumulate protein aggregates that are thought to be 55 toxic, potentially leading to age-related diseases. This accumulation of protein 56 aggregates is partially attributed to a failure to maintain protein homeostasis. A variety of 57 genetic factors have been linked to longevity, but how these factors also contribute to 58 protein homeostasis is not completely understood. In order to understand the 59 relationship between aging and protein aggregation, we tested how a gene that 60 regulates lifespan and age-dependent locomotor behaviors, p38 MAPK (p38Kb), 61 influences protein homeostasis as an organism ages. We find that p38Kb regulates age-62 dependent protein aggregation through an interaction with the Chaperone-Assisted 63 Selective Autophagy complex. Furthermore, we have identified Lamin as an age-64 dependent target of p38Kb and the Chaperone-Assisted Selective Autophagy complex. 65 66 Introduction 67 68 Protein turnover is critical for maintaining tissue health as many proteins become 69 damaged or misfolded during normal tissue functions. Therefore, the cell utilizes a 70 variety of protein quality control mechanisms to refold or degrade these damaged 71 proteins, including the ubiquitin proteasome system and macroautophagy. During aging, 72 protein quality control mechanisms become less efficient leading to the accumulation of 73 damaged or misfolded proteins that begin to form protein aggregates 1 . It has been 74 hypothesized that these aggregates are toxic and may lead to the deleterious 75 phenotypes associated with normal aging, such as impaired tissue function 1 . 76Furthermore, decreased protein aggregation has been associated with longevity. For 77 example, over-expression of Foxo leads to an increased lifespan but also a concordant 78 4 decrease in protein aggregation in C. elegans, Drosophila, and mice 2-6 , suggesting that 79 lifespan and protein aggregation are tightly linked processes. However, the molecular 80 mechanisms that underlie the relationship between aging and protein homeostasis have 81 not been fully characterized. 82 83One pathway that has been linked to both aging and protein homeostasis is the 84 stress response p38 MAPK (p38K) pathway. In mammals, there are four p38K genes (α, 85 β, γ, and δ), and p38Kα has been linked to both the inhibition 7,8 and induction 9,10 of 86 macroautophagy, in particular in response to oxidative stress 11,12 . In addition, p38Kα 87 has been linked to regulating macroautophagy in cellular senescence [13][14][15] . However, 88 how p38K signaling may contribute to protein homeostasis in response to natural aging 89is not well understood. The fruit fly Drosophila melanogaster has two canonical p38K 90 genes (p38Ka and p38Kb), and we have previously reported that p38Kb acts in the adult 91 musculature to regulate aging. We found that over-expression of p38Kb leads to 92 increased lifespan while loss of p38Kb results in a short lifespan and age-dependent 93 locomotor behavior defects 16 . In addition, oxidatively damaged ...

Protein turnover is critical for maintaining tissue health as many proteins become damaged or misfolded during normal tissue functions. Therefore, the cell utilizes a variety of protein quality control mechanisms to refold or degrade these damaged proteins, including the ubiquitin proteasome system and autophagy. During aging, protein quality control mechanisms become less efficient leading to the accumulation of damaged or misfolded proteins that begin to form protein aggregates (Taylor & Dillin, 2011). It has been hypothesized that these aggregates are toxic and may lead to the deleterious phenotypes associated with normal aging, such as impaired tissue function (Taylor & Dillin, 2011). Furthermore, decreased protein aggregation has been associated with longevity. For example, over-expression of Foxo leads to an increased lifespan but also a concordant decrease in protein aggregation in C. elegans, Drosophila, and mice (Ben-Zvi et al.,