Kengo Watanabe

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.

Mitogen-activated protein kinases as key players in osmotic stress signaling

BACKGROUNDnOsmotic stress arises from the difference between intracellular and extracellular osmolality. It induces cell swelling or shrinkage as a consequence of water influx or efflux, which threatens cellular activities. Mitogen-activated protein kinases (MAPKs) play central roles in signaling pathways in osmotic stress responses, including the regulation of intracellular levels of inorganic ions and organic osmolytes.nnnSCOPE OF REVIEWnThe present review summarizes the cellular osmotic stress response and the function and regulation of the vertebrate MAPK signaling pathways involved. We also describe recent findings regarding apoptosis signal-regulating kinase 3 (ASK3), a MAP3K member, to demonstrate its regulatory effects on signaling molecules beyond MAPKs.nnnMAJOR CONCLUSIONSnMAPKs are rapidly activated by osmotic stress and have diverse roles, such as cell volume regulation, gene expression, and cell survival/death. There is significant cell type specificity in the function and regulation of MAPKs. Based on its activity change during osmotic stress and its regulation of the WNK1-SPAK/OSR1 pathway, ASK3 is expected to play important roles in osmosensing mechanisms and cellular functions related to osmoregulation.nnnGENERAL SIGNIFICANCEnMAPKs are essential for various cellular responses to osmotic stress; thus, the identification of the upstream regulators of MAPK pathways will provide valuable clues regarding the cellular osmosensing mechanism, which remains elusive in mammals. The elucidation of in vivo MAPK functions is also important because osmotic stress in physiological and pathophysiological conditions often results from changes in the intracellular osmolality. These studies potentially contribute to the establishment of therapeutic strategies against diseases that accompany osmotic perturbation.

A PP6-ASK3 Module Coordinates the Bidirectional Cell Volume Regulation under Osmotic Stress

Cell volume regulation is a vital system for cellular activities. When perturbed by hypoosmotic or hyperosmotic stress, cells immediately induce the cell volume recovery system, regulatory volume decrease (RVD) or regulatory volume increase (RVI), respectively. In contrast to the knowledge about effector molecules, the molecular mechanisms linking osmosensing to RVD/RVI induction remain unknown. Additionally, few reciprocal responders in the bidirectional osmotic stress response have been identified. We previously reported that ASK3 bidirectionally switches its kinase activity under osmotic stress. Herein we demonstrate that ASK3 controls both RVD and RVI under osmotic stress. Using a high-content genome-wide small interfering RNA (siRNA) screen, we identify PP6 as a direct ASK3 inactivator. Furthermore, PP6 rapidly interacts with ASK3 in an osmolality-dependent manner, and it inactivates ASK3 to induce RVI and, thereby, cell survival under hyperosmotic stress. These findings suggest that the PP6-ASK3 interaction is a core module in the bidirectional osmotic stress response.

Cryo-EM structure of the volume-regulated anion channel LRRC8

Maintenance of cell volume against osmotic change is crucial for proper cell functions, such as cell proliferation and migration. The leucine-rich repeat-containing 8 (LRRC8) proteins are anion selective channels, and were recently identified as pore components of the volume-regulated anion channels (VRACs), which extrude anions to decrease the cell volume upon cell-swelling. Here, we present the human LRRC8A structure, determined by a single-particle cryo-electron microscopy analysis. The sea anemone-like structure represents a trimer of dimers assembly, rather than a symmetrical hexameric assembly. The four-spanning transmembrane region has a gap junction channel-like membrane topology, while the LRR region containing 15 leucine-rich repeats forms a long twisted arc. The channel pore is along the central axis and constricted on the extracellular side, where the highly conserved polar and charged residues at the tip of the extracellular helix contribute to the anion and other osmolyte permeability. Comparing the two structural populations facilitated the identification of both compact and relaxed conformations, suggesting that the LRR region is flexible and mobile with rigid-body motions, which might be implicated in structural transitions upon pore opening. Overall, our structure provides a framework for understanding the molecular mechanisms of this unique class of ion channels.