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

Abstract

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.

Cells recognize osmotic stress through liquid-liquid phase separation lubricated with poly(ADP-ribose)

“…1B) and gradually diminished several dozen minutes after hyperosmotic stress (Fig. S1A and B), which corresponds to the time range of cell volume recovery ( 10, 17 ). In addition to mannitol-supplemented medium, sodium chloride-supplemented medium induced a similar pattern of ASK3 localization (Fig.…”

“…2D and S3A), suggesting that ASK3 condensation is required for its inactivation. In fact, we discovered the unique relationship between ASK3 condensates and one of the PP6 subunits ANKRD52 ( 17, 26 ); ANKRD52 condensates are not completely colocalized with ASK3 condensates, but they move around and grow while sharing their phase boundaries (Fig. 2E and Movie S4).…”

“…Previously, we elucidated that ASK3 is inactivated a few minutes after hyperosmotic stress by protein phosphatase 6 (PP6) ( 17 ). Since ASK3 condensation occurs prior to its inactivation, we evaluated the inactivation of ASK3 mutants lacking condensation ability.…”

“…To reveal the details of ASK3 condensation and inactivation, we investigated the candidate regulators of ASK3 inactivation identified by our genome-wide siRNA screen ( 17 ). Among them, we focused on the highest-ranked and unexpected candidate nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the mammalian nicotinamide adenine dinucleotide (NAD) salvage pathway ( 27 ) (Fig.…”

“…We previously reported that apoptosis signal-regulating kinase 3 (ASK3; also known as MAP3K15) is phosphorylated and activated under hypoosmotic stress and conversely dephosphorylated and inactivated under hyperosmotic stress ( 16 ). Furthermore, in addition to the rapid, sensitive and reversible nature, this bidirectional response of ASK3 orchestrates proper cell volume recovery under both hypoosmotic and hyperosmotic stresses ( 17 ). Therefore, we conceived that the elucidation of ASK3 regulation under osmotic stress would lead to the clarification of a general mammalian osmosensing system.…”