TRIM48 Promotes ASK1 Activation and Cell Death through Ubiquitination-Dependent Degradation of the ASK1-Negative Regulator PRMT1.

Apoptosis signal-regulating kinase 1 (ASK1) is an oxidative stress-responsive kinase that is regulated by various interacting molecules and post-translational modifications. However, how these molecules and modifications cooperatively regulate ASK1 activity remains largely unknown. Here, we showed that tripartite motif 48 (TRIM48) orchestrates the regulation of oxidative stress-induced ASK1 activation. A pull-down screen identified a TRIM48-interacting partner, protein arginine methyltransferase 1 (PRMT1), which negatively regulates ASK1 activation by enhancing its interaction with thioredoxin (Trx), another ASK1-negative regulator. TRIM48 facilitates ASK1 activation by promoting K48-linked polyubiquitination and degradation of PRMT1. TRIM48 knockdown suppressed oxidative stress-induced ASK1 activation and cell death, whereas forced expression promoted cancer cell death in mouse xenograft model. These results indicate that TRIM48 facilitates oxidative stress-induced ASK1 activation and cell death through ubiquitination-dependent degradation of PRMT1. This study provides a cell death mechanism fine-tuned by the crosstalk between enzymes that engage various types of post-translational modifications.

Regulation of apoptosis signal-regulating kinase 1 in redox signaling.

Apoptosis signal-regulating kinase 1 (ASK1) is a member of the mitogen-activated protein kinase (MAPK) kinase kinase family and elicits a wide variety of cellular responses to various types of stress through activation of the JNK and p38 MAPK pathways. ASK1 is preferentially activated in response to oxidative stress, but this regulatory mechanism is still not completely understood. In our previous report, thioredoxin (Trx), which is an antioxidant protein and plays pivotal roles in maintaining intracellular redox balance, inhibited ASK1 kinase activity by direct binding to ASK1 under normal conditions. Under oxidative conditions, ASK1 is dissociated from Trx and therefore fully activated. The active site of Trx contains two cysteine residues that undergo reversible oxidation to form a disulfide bond with each other, so that the conformation of Trx is changed by intracellular redox conditions. Thus, the oxidative stress-induced conformational change of Trx is particularly important for interaction with and regulation of ASK1, and elucidation of the regulatory mechanisms of ASK1 by Trx is critical to understanding the intracellular redox signaling. In this chapter, we review the regulatory mechanisms of ASK1 activity by Trx, and describe a method for monitoring in vitro binding between Trx and ASK1 under various redox conditions. In addition, we present methods to detect the oxidative stress-induced activation of ASK1 in the cells by Western blot analysis and in vitro kinase assay. The techniques presented in this chapter will be useful for a range of investigations into intracellular redox signaling.

Ubiquitin-like sequence in ASK1 plays critical roles in the recognition and stabilization by USP9X and oxidative stress-induced cell death.

Ubiquitination is an important posttranslational modification that regulates various cellular processes, including signal transduction. However, physiological roles of ubiquitination in the regulation of MAPK pathways are poorly understood. Here, we identified the deubiquitinating enzyme USP9X as a binding partner of ASK1 that mediates oxidative stress-induced cell death through activation of the JNK and p38 MAPK pathways. In the recognition of ubiquitin by deubiquitinating enzymes, the importance of a tandem glycine-glycine sequence in the ubiquitin C terminus has been suggested. Interestingly, ASK1 contains six amino acids identical to the ubiquitin C terminus (LRLRGG), and the GG sequence of ASK1 was required for the USP9X-ASK1 interaction. We also found that USP9X interacted with oxidative stress-activated ASK1 and prevented it from undergoing ubiquitin-dependent degradation. In USP9X-deficient cells, oxidative stress-induced JNK activation and subsequent cell death were reduced. These results demonstrate that USP9X-dependent stabilization of activated ASK1 plays a crucial role in oxidative stress-induced cell death.

Targeting ASK1 in ER stress-related neurodegenerative diseases.

The accumulation of malfolded proteins in the endoplasmic reticulum (ER) induces ER stress, leading to the disturbance of ER function. To restore ER function and ER homeostasis, cells possess a highly specific ER quality control system termed the unfolded protein response (UPR), which increases the capacity of protein folding and reduces the amount of malfolded proteins. In case of prolonged ER stress or malfunction of the UPR, apoptosis signaling is activated. ER stress-induced apoptosis has recently been implicated in the pathogenesis of various conformational diseases. Apoptosis signal-regulating kinase 1 (ASK1), a member of the MAPK kinase kinase (MAP3K) family, is activated by ER stress and mediates apoptosis. Recent studies have shown that the ASK1 pathway is involved in ER stress-induced neuronal cell death and contributes to the pathogenesis of neurodegenerative diseases. In this review, we summarize the molecular mechanisms of the UPR and ER stress-induced apoptosis and the possible roles of ASK1 activation in neurodegenerative diseases.