Dirk Bohmann

Ubiquitin-dependent c-Jun degradation in vivo is mediated by the δ domain

Summary The role of the ubiquitin-dependent proteolysis system in c-Jun breakdown was investigated. Using in vitro experiments and a novel in vivo assay that utilizes molecularly-tagged ubiquitin and c-Jun proteins, it was shown that c-Jun, but not its transforming counterpart, retroviral v-Jun, can be efficiently multiubiquitinated. Consistently, v-Jun has a longer half-life than c-Jun. Mutagenesis experiments indicate that the reason for the escape of v-Jun from multiubiquitination and its resulting stabilization is the deletion of the δ domain, a stretch of 27 amino acids that is present in c-Jun but not in v-Jun. c-Jun sequences containing the δ domain, when transferred to the bacterial β-galactosidase protein, function as a cis-acting ubiquitination and degradation signal. The correlation between transforming ability and the escape from ubiquitin-dependent degradation described here suggests a novel route to oncogenesis.

Diverse functions of JNK signaling and c-Jun in stress response and apoptosis

c-Jun/AP-1 activation has been implicated in various, often opposing cellular responses. For example, although there is considerable evidence that c-Jun activation can be a positive step in the events leading a cell towards apoptosis, there are also many reports stating the opposite: that under certain circumstances c-Jun can inhibit apoptosis and promote proliferation or differentiation instead – and that these responses are important for normal mammalian development. It is clear that the effects of c-Jun on cellular responses depend strongly on the cell type and the context of other regulatory influences that the cell is receiving. This review focuses on recent developments in understanding how activation of JNK and c-Jun contributes to different cellular responses.

JNK Extends Life Span and Limits Growth by Antagonizing Cellular and Organism-Wide Responses to Insulin Signaling

Aging of a eukaryotic organism is affected by its nutrition state and by its ability to prevent or repair oxidative damage. Consequently, signal transduction systems that control metabolism and oxidative stress responses influence life span. When nutrients are abundant, the insulin/IGF signaling (IIS) pathway promotes growth and energy storage but shortens life span. The transcription factor Foxo, which is inhibited by IIS, extends life span in conditions of low IIS activity. Life span can also be increased by activating the stress-responsive Jun-N-terminal kinase (JNK) pathway. Here we show that JNK requires Foxo to extend life span in Drosophila. JNK antagonizes IIS, causing nuclear localization of Foxo and inducing its targets, including growth control and stress defense genes. JNK and Foxo also restrict IIS activity systemically by repressing IIS ligand expression in neuroendocrine cells. The convergence of JNK signaling and IIS on Foxo provides a model to explain the effects of stress and nutrition on longevity.

Stress-Activated Cap'n'collar Transcription Factors in Aging and Human Disease

The oxidative stress response is an attractive target for treating human diseases and extending the healthy life span. Cap’n’collar (Cnc) proteins form a family of basic leucine zipper transcription factors. Some Cnc factors have important functions in development, whereas others are critical for maintaining homeostasis in the face of environmental stresses. The “electrophile counterattack” is a conserved cellular response to oxidative stressors and electrophilic xenobiotics. In this adaptive process, Cnc factors transcriptionally activate protective genes through antioxidant response elements (AREs) in their regulatory sequences. In vertebrates, the electrophile counterattack is largely mediated by Nrf2. By defending animals against oxidative stress, Nrf2 prevents DNA and protein damage and protects against the development of cancer and many other oxidative stress-related disorders, including respiratory and neurodegenerative diseases. In animal models of disease, pharmacological activation of Nrf2 can prevent cancer and other pathologies linked to oxidative stress. In humans, inherited DNA sequence polymorphisms that decrease Nrf2 abundance have been linked to various diseases of the skin, lung, stomach, and intestine, which are all organs that are subjected to environmental stressors. Thus, individuals genetically predisposed to a suboptimal antioxidant response may be more susceptible to disorders caused or exacerbated by oxidative stress. Like many diseases, the aging process is also linked to oxidative stress, and studies in model organisms show that Cnc transcription factors promote longevity. Paradoxically, in aging and in advanced human respiratory and neurodegenerative disease, the Cnc antioxidant response is suppressed. Thus, Nrf2-activating compounds could be beneficial in the treatment of human diseases and might help extend the healthy life span. However, hyperactivation of the antioxidant response is detrimental in model organisms and has been linked to chemoresistant cancers in humans. These observations caution that sustained induction of Cnc factors can be deleterious and indicate that the antioxidant response system must be tightly controlled; moreover, they imply that compounds inhibiting Nrf2 may also be useful therapeutics as chemotherapy sensitizers. This Review, with 3 figures, 2 tables, and 321 citations, describes the stress-activated Cnc transcription factors, their regulatory mechanisms, and their roles in aging and human disease. Cap’n’collar (Cnc) transcription factors are conserved in metazoans and have important developmental and homeostatic functions. The vertebrate Nrf1, Nrf2, and Nrf3; the Caenorhabditis elegans SKN-1; and the Drosophila CncC comprise a subgroup of Cnc factors that mediate adaptive responses to cellular stress. The most studied stress-activated Cnc factor is Nrf2, which orchestrates the transcriptional response of cells to oxidative stressors and electrophilic xenobiotics. In rodent models, signaling by Nrf2 defends against oxidative stress and aging-associated disorders, such as neurodegeneration, respiratory diseases, and cancer. In humans, polymorphisms that decrease Nrf2 abundance have been associated with various pathologies of the skin, respiratory system, and digestive tract. In addition to preventing disease in rodents and humans, Cnc factors have life-span–extending and anti-aging functions in invertebrates. However, despite the pro-longevity and antioxidant roles of stress-activated Cnc factors, their activity paradoxically declines in aging model organisms and in humans suffering from progressive respiratory disease or neurodegeneration. We review the roles and regulation of stress-activated Cnc factors across species, present all reported instances in which their activity is paradoxically decreased in aging and disease, and discuss the possibility that the pharmacological restoration of Nrf2 signaling may be useful in the prevention and treatment of age-related diseases.

Differential regulation of c-Jun by ERK and JNK during PC12 cell differentiation.

The two MAP kinases JNK and ERK direct distinct cellular activities even though they share a number of common substrates, including several transcription factors. Here we have compared JNK and ERK signalling during PC12 cell differentiation and investigated how activation of c‐Jun by the MAPKs contributes to this cellular response. Exposure to nerve growth factor, or expression of constitutively active MEK1—two treatments which cause differentiation of PC12 cells into a neuronal phenotype—result in activation of ERK‐type MAP kinases and phosphorylation of c‐Jun on several sites including Ser63 and Ser73. Constitutively activated c‐Jun, which mimics the MAPK‐phosphorylated form of the protein, can induce neuronal differentiation of PC12 cells independently of upstream signals. Conversely, expression of dominant‐negative c‐JunbZIP prevents neurite outgrowth induced by activated MEK1. Activation of MEKK1, which stimulates the JNK pathway, is not sufficient for PC12 cell differentiation but can induce apoptosis. However, neurite outgrowth is triggered when c‐Jun is co‐expressed with activated MEKK1 or SEK1. Consistently, MEK‐induced ERK activation in PC12 cells induces c‐Jun expression, while JNK signalling does not. Therefore, dual input of expression and phosphorylation of c‐Jun provided by the ERK pathway is required to direct neuronal differentiation in PC12 cells.

Keap1/Nrf2 Signaling Regulates Oxidative Stress Tolerance and Lifespan in Drosophila

Keap1/Nrf2 signaling defends organisms against the detrimental effects of oxidative stress and has been suggested to abate its consequences, including aging-associated diseases like neurodegeneration, chronic inflammation, and cancer. Nrf2 is a prominent target for drug discovery, and Nrf2-activating agents are in clinical trials for cancer chemoprevention. However, aberrant activation of Nrf2 by keap1 somatic mutations may contribute to carcinogenesis and promote resistance to chemotherapy. To evaluate potential functions of Keap1 and Nrf2 for organismal homeostasis, we characterized the pathway in Drosophila. We demonstrate that Keap1/Nrf2 signaling in the fruit fly is activated by oxidants, induces antioxidant and detoxification responses, and confers increased tolerance to oxidative stress. Importantly, keap1 loss-of-function mutations extend the lifespan of Drosophila males, supporting a role for Nrf2 signaling in the regulation of longevity. Interestingly, cancer chemopreventive drugs potently stimulate Drosophila Nrf2 activity, suggesting the fruit fly as an experimental system to identify and characterize such agents.

JNK signaling confers tolerance to oxidative stress and extends lifespan in Drosophila.

Changes in the genetic makeup of an organism can extend lifespan significantly if they promote tolerance to environmental insults and thus prevent the general deterioration of cellular function that is associated with aging. Here, we introduce the Jun N-terminal kinase (JNK) signaling pathway as a genetic determinant of aging in Drosophila melanogaster. Based on expression profiling experiments, we demonstrate that JNK functions at the center of a signal transduction network that coordinates the induction of protective genes in response to oxidative challenge. JNK signaling activity thus alleviates the toxic effects of reactive oxygen species (ROS). In addition, we show that flies with mutations that augment JNK signaling accumulate less oxidative damage and live dramatically longer than wild-type flies. Our work thus identifies the evolutionarily conserved JNK signaling pathway as a major genetic factor in the control of longevity.

JNK- and Fos-regulated Mmp1 expression cooperates with Ras to induce invasive tumors in Drosophila

Loss of the epithelial polarity gene scribble in clones of Drosophila imaginal disc cells can cooperate with Ras signaling to induce malignant tumors. Such mutant tissue overproliferates, resists apoptosis, leaves its place of origin and invades other organs, ultimately causing lethality. We show that increased Jun N‐terminal kinase (JNK) signaling resulting from the loss of scribble promotes the movement of transformed cells to secondary sites. This effect requires Fos‐dependent transcriptional activation of a matrix metalloprotease gene mmp1 downstream of JNK. Expression of the Mmp inhibitor Timp or Mmp RNAi knockdown suppresses cell invasiveness. The proinvasive function of the JNK pathway is revealed in a tumor context when active Ras signaling prevents the apoptotic response to JNK activity as it occurs in nontransformed cells. Based on these results, we present a model that explains the oncogenic cooperation between JNK and Ras, and describes how aberrant regulation of cell survival, proliferation and mobilization cooperate to incite malignant tumor formation.

Redox regulation by Keap1 and Nrf2 controls intestinal stem cell proliferation in Drosophila

In Drosophila, intestinal stem cells (ISCs) respond to oxidative challenges and inflammation by increasing proliferation rates. This phenotype is part of a regenerative response, but can lead to hyperproliferation and epithelial degeneration in the aging animal. Here we show that Nrf2, a master regulator of the cellular redox state, specifically controls the proliferative activity of ISCs, promoting intestinal homeostasis. We find that Nrf2 is constitutively active in ISCs and that repression of Nrf2 by its negative regulator Keap1 is required for ISC proliferation. We further show that Nrf2 and Keap1 exert this function in ISCs by regulating the intracellular redox balance. Accordingly, loss of Nrf2 in ISCs causes accumulation of reactive oxygen species and accelerates age-related degeneration of the intestinal epithelium. Our findings establish Keap1 and Nrf2 as a critical redox management system that regulates stem cell function in high-turnover tissues.

Biochemical analysis of transcriptional activation by Jun: Differential activity of c- and v-Jun

The human proto-oncogene product, c-Jun, is a member of the AP-1 family of transcription factors, which mediate the regulation of gene expression in response to extracellular signaling. Comparison of c-Jun and v-Jun by in vitro transcription assays revealed that v-Jun has significantly greater transcriptional activity than c-Jun. Analysis of Jun mutants expressed in bacteria indicates that this difference in transcriptional activity is due to the presence of a regulatory domain located at the N-terminal region of c-Jun. Other Jun mutants identify an activation domain rich in acidic and proline residues toward the C-terminal end of the molecule, in a region near the DNA binding domain. These findings suggest that during retroviral transduction, a constitutively active Jun protein has been generated by deleting a negatively acting domain. This putative repressor domain may also play a role in the signal-dependent induction of c-Jun activity.