Kazuki Hattori

The roles of ASK family proteins in stress responses and diseases

Apoptosis signal-regulating kinase 1 (ASK1) is a member of the mitogen-activated protein kinase kinase kinase family, which activates c-Jun N-terminal kinase and p38 in response to a diverse array of stresses such as oxidative stress, endoplasmic reticulum stress and calcium influx. In the past decade, various regulatory mechanisms of ASK1 have been elucidated, including its oxidative stress-dependent activation. Recently, it has emerged that ASK family proteins play key roles in cancer, cardiovascular diseases and neurodegenerative diseases. In this review, we summarize the recent findings on ASK family proteins and their implications in various diseases.

ASK1 signalling regulates brown and beige adipocyte function

Recent studies suggest that adult humans have active brown or beige adipocytes, the activation of which might be a therapeutic strategy for the treatment of diverse metabolic diseases. Here we show that the protein kinase ASK1 regulates brown and beige adipocytes function. In brown or white adipocytes, the PKA-ASK1-p38 axis is activated in response to cAMP signalling and contributes to the cell-autonomous induction of genes, including Ucp1. Global and fat-specific ASK1 deficiency leads to impaired metabolic responses, including thermogenesis and oxygen consumption, at the cell and whole-body levels, respectively. Our data thus indicate that the ASK1 signalling axis is a regulator of brown and beige adipocyte gene expression and function.

The regulatory and signaling mechanisms of the ASK family

Apoptosis signal-regulating kinase 1 (ASK1) was identified as a MAP3K that activates the JNK and p38 pathways, and subsequent studies have reported ASK2 and ASK3 as members of the ASK family. The ASK family is activated by various intrinsic and extrinsic stresses, including oxidative stress, ER stress and osmotic stress. Numerous lines of evidence have revealed that members of the ASK family are critical for signal transduction systems to control a wide range of stress responses such as cell death, differentiation and cytokine induction. In this review, we focus on the precise signaling mechanisms of the ASK family in response to diverse stressors.

Pleiotropic properties of ASK1

BACKGROUND Apoptosis signal-regulating kinase 1 (ASK1), also known as mitogen-activated protein kinase kinase kinase 5 (MAP3K5), has the potential to induce cellular apoptosis under various physiological conditions. It has long been suggested that ASK1 is highly sensitive to oxidative stress and contributes substantially to apoptosis. However, recent studies have indicated that ASK1 has pleiotropic roles in living organisms through other mechanisms in addition to apoptosis. SCOPE OF THE REVIEW This review describes the physiological functions of ASK1 in living organisms, focusing on the regulatory mechanisms of ASK1 activity and its importance in the pathogenesis of various diseases. We also highlight recent works published within the past few years. MAJOR CONCLUSIONS ASK1 forms a high-molecular-mass complex within the cell, designated as the ASK1 signalosome. Soon after the discovery of ASK1, several regulatory components of the ASK1 signalosome have been revealed, including thioredoxin (Trx), tumor-necrosis factor α receptor-associated factors (TRAFs) and 14-3-3s. In parallel with the precise analyses unveiling the molecular basis of ASK1 regulation, the physiological or pathophysiological significance of ASK1 in diverse organs has been elucidated. In addition to the generation of global knockout mice or tissue-specific knockout mice, ASK1-specific inhibitors have illuminated the biological roles of ASK1. GENERAL SIGNIFICANCE The multi-faceted features of the function of ASK1 have been discovered over the past two decades, revealing that ASK1 is a crucial molecule for maintaining cellular homeostasis, especially under conditions of stress. Based on the results that ASK1 deficiency provides beneficial effects for several diseases, modulating ASK1 activity is a promising method to ameliorate a subset of diseases.

Cold stress‐induced ferroptosis involves the ASK1‐p38 pathway

A wide variety of cell death mechanisms, such as ferroptosis, have been proposed in mammalian cells, and the classification of cell death attracts global attention because each type of cell death has the potential to play causative roles in specific diseases. However, the precise molecular mechanisms leading to cell death are poorly understood, particularly in ferroptosis. Here, we show that continuous severe cold stress induces ferroptosis and the ASK1‐p38 MAPK pathway in multiple cell lines. The activation of the ASK1‐p38 pathway is mediated by critical determinants of ferroptosis: MEK activity, iron ions, and lipid peroxide. The chemical compound erastin, a potent ferroptosis inducer, also activates the ASK1‐p38 axis downstream of lipid peroxide accumulation and leads to ASK1‐dependent cell death in a cell type‐specific manner. These lines of evidence provide mechanistic insight into ferroptosis, a type of regulated necrosis.

The Ablation of Mitochondrial Protein Phosphatase Pgam5 Confers Resistance Against Metabolic Stress.

Phosphoglycerate mutase family member 5 (PGAM5) is a mitochondrial protein phosphatase that has been reported to be involved in various stress responses from mitochondrial quality control to cell death. However, its roles in vivo are largely unknown. Here, we show that Pgam5-deficient mice are resistant to several metabolic insults. Under cold stress combined with fasting, Pgam5-deficient mice better maintained body temperature than wild-type mice and showed an extended survival rate. Serum triglycerides and lipid content in brown adipose tissue (BAT), a center of adaptive thermogenesis, were severely reduced in Pgam5-deficient mice. Moreover, although Pgam5 deficiency failed to maintain proper mitochondrial integrity in BAT, it reciprocally resulted in the dramatic induction of fibroblast growth factor 21 (FGF21) that activates various functions of BAT including thermogenesis. Thus, the enhancement of lipid metabolism and FGF21 may contribute to the cold resistance of Pgam5-deficient mice under fasting condition. Finally, we also found that Pgam5-deficient mice are resistant to high-fat-diet-induced obesity. Our study uncovered that PGAM5 is involved in the whole-body metabolism in response to stresses that impose metabolic challenges on mitochondria.

ASK family kinases mediate cellular stress and redox signaling to circadian clock

Significance The cellular stress response and circadian clock system are fundamental functions in homeostatic regulation in almost all organisms. However, whether these two mechanisms are interlocked with each other, and the key molecule that links cellular stress and the circadian clock, remain unclear. Here we identify ASK family kinases that are essential for the circadian clock to respond to cellular stress, and report that Ask1 transcription is rhythmically controlled by the circadian clock. Moreover, LC-MS/MS–based proteomic analysis provides insight into a molecular mechanism in which dephosphorylation-triggered changes to the ASK complex mediate cellular stress to the circadian clock. From the perspective of cell signaling, our present findings expand previously reported roles of stress signaling toward regulation of the circadian clock. Daily rhythms of behaviors and physiologies are generated by the circadian clock, which is composed of clock genes and the encoded proteins forming transcriptional/translational feedback loops (TTFLs). The circadian clock is a self-sustained oscillator and flexibly responds to various time cues to synchronize with environmental 24-h cycles. However, the key molecule that transmits cellular stress to the circadian clockwork is unknown. Here we identified apoptosis signal-regulating kinase (ASK), a member of the MAPKKK family, as an essential mediator determining the circadian period and phase of cultured cells in response to osmotic changes of the medium. The physiological impact of ASK signaling was demonstrated by a response of the clock to changes in intracellular redox states. Intriguingly, the TTFLs drive rhythmic expression of Ask genes, indicating ASK-mediated association of the TTFLs with intracellular redox. In behavioral analysis, Ask1, Ask2, and Ask3 triple-KO mice exhibited compromised light responses of the circadian period and phase in their activity rhythms. LC-MS/MS–based proteomic analysis identified a series of ASK-dependent and osmotic stress-responsive phosphorylations of proteins, among which CLOCK, a key component of the molecular clockwork, was phosphorylated at Thr843 or Ser845 in the carboxyl-terminal region. These findings reveal the ASK-dependent stress response as an underlying mechanism of circadian clock flexibility.

Apoptosis signal-regulating kinase 1 in regulated necrosis

“Classification” is a major point of contention in the field of cell death. Ferroptosis is a regulated form of necrosis that was identified in 2012 and is specified by the accumulation of lipid peroxide within the cell. Several studies have demonstrated that the pharmacological inhibition of ferroptosis is beneficial for ischemia reperfusion injury models in multiple tissues, thus suggesting the relevance of ferroptosis to some human diseases. A growing number of studies have recently uncovered the mechanism by which detrimental levels of lipid peroxide accumulate; however, the molecular mechanisms that link lipid peroxide accumulation to the execution of cell death remain largely unknown [1]. Reactive oxygen species (ROS), such as lipid peroxide, are vital signal mediators. ROS induce a diverse array of signaling cascades, including the thioredoxin (Trx) apoptosis signalregulating kinase 1 (ASK1) axis [2]. We recently revealed that ASK1-p38 MAPK cascade is activated through ferroptosis induced by cold stress (Fig. 1) [3]. Importantly, the ASK1 axis lies downstream of lipid peroxide and has no effect on lipid peroxide accumulation. Precise analyses revealed that the ASK1-p38 axis is only partially involved in canonical ferroptosis induced by erastin or RSL3 in a cell-type-specific manner; however, the same axis plays critical roles in cold-stress-induced ferroptosis [3]. Aberrant fluctuations of environmental cues are harmful for cellular homeostasis. An environmental temperature change is a vital cue and is known to affect the cellular functions in a diverse manner, i.e., enzyme kinetics and membrane fluidity. Our recent dataset revealed that prolonged severe cold stress induces the accumulation of lipid peroxide, which activates the ASK1-p38 axis and promotes ferroptosis [3]. Although lipid peroxide accumulation mediates membrane destruction, we demonstrate that signaling pathways downstream of lipid peroxide may be involved in the execution of cell death, i.e., by disrupting lipid membrane homeostasis. This is a rare example wherein an environmental stress induces ferroptosis, but it is worth mentioning that heat stress also induces ferroptosis-like cell death in plants [4]. Although numerous studies have demonstrated that thermal stress induces ROS production, the precise mechanism through which lipid peroxide accumulates remains unknown. ROS signaling mediates not only ferroptosis but also apoptosis and parthanatos; intensity, spatiotemporal dynamics, and other features of ROS presumably determine cell fate. One plausible explanation is that multiple signaling pathways are activated in parallel but that only one signal predominates to execute a specific type of cell death in a situation-dependent manner. Our previous study demonstrated that high concentrations of H2O2 induce receptor-interacting protein 1 (RIP1)independent necrosis via the ASK1-p38 MAPK axis [5]. For cold stress-induced cell death, our preliminary data suggest that the parthanatos pathway is also evoked because PARP1 inhibition also attenuates cold-induced cell death (Hattori, K., Ishikawa, H. and Ichijo, H., unpublished observation). This evidence raises the possibility that ferroptosis and parthanatos intersect and crosstalk in the course of death-inducing signaling mechanisms. In fact, each form of cell death possesses some common features. Nicotinamide adenine dinucleotide (NAD), an essential factor in cell metabolism, controls various cell death pathways [6]. Of note, reduced NAD levels are induced both in ferroptosis [7] and parthanatos [6], suggesting that a common NAD-dependent mechanism is involved in the execution of necrotic cell death. Given that numerous NADdependent enzymes exist, NAD depletion must lead to the disruption of overall cellular homeostasis, including lipid homeostasis on the plasma membranes. The classification of cell death opens the possibility that manipulating a specific type of cell death may contribute to the development of unique therapeutic interventions because each type of cell death may be involved in a specific type of disease. Nevertheless, we should not overlook the fact that all types of cell death are potentially intertwined. In addition, cell death induced by the fluctuation of environmental cues might be specifically modulated via diverse mechanisms because cells typically sense environmental variation using multiple signaling pathways. ASK1, as the name suggests, has long been considered an apoptosis-regulating factor [2]. We here suggest that ASK1 is involved in a wider range of ROS-dependent cell death processes, including ferroptosis, as a hub of multiple signaling cascades [3,5].

β-TrCP-dependent degradation of ASK1 suppresses the induction of the apoptotic response by oxidative stress

Apoptosis signal-regulating kinase 1 (ASK1) is a key player in the homeostatic response of many organisms. Of the many functions of ASK1, it is most well-known for its ability to induce canonical caspase 3-dependent apoptosis through the MAPK pathways in response to reactive oxygen species (ROS). As ASK1 is a regulator of apoptosis, its proper regulation is critical for the well-being of an organism. To date, several E3 ubiquitin ligases have been identified that are capable of degrading ASK1, signifying the importance of maintaining ASK1 expression levels during stress responses. ASK1 protein regulation under unstimulated conditions, however, is still largely unknown. Using tandem mass spectrometry, we have identified beta-transducin repeat containing protein (β-TrCP), an E3 ubiquitin ligase, as a novel interacting partner of ASK1 that is capable of ubiquitinating and subsequently degrading ASK1 through the ubiquitin-proteasome system (UPS). This interaction requires the seven WD domains of β-TrCP and the C-terminus of ASK1. By silencing the β-TrCP genes, we observed a significant increase in caspase 3 activity in response to oxidative stress, which could subsequently be suppressed by silencing ASK1. These findings suggest that β-TrCP is capable of suppressing oxidative stress-induced caspase 3-dependent apoptosis through suppression of ASK1, assisting in the organisms ability to maintain homeostasis in an unstable environment.