WNK kinases sense molecular crowding and rescue cell volume via phase separation

Abstract

When challenged by hypertonicity, dehydrated cells must defend their volume to survive. This process requires the phosphorylation-dependent regulation of SLC12 cation chloride transporters by WNK kinases, but how these kinases are activated by cell shrinkage remains unknown. Within seconds of cell exposure to hypertonicity, WNK1 concentrates into membraneless droplets, initiating a phosphorylation-dependent signal that drives net ion influx via the SLC12 cotransporters to rescue volume. The formation of WNK1 condensates is driven by its intrinsically disordered C-terminus, whose evolutionarily conserved signatures are necessary for efficient phase separation and volume recovery. This disorder-encoded phase behavior occurs within physiological constraints and is activated in vivo by molecular crowding rather than changes in cell size. This allows WNK1 to bypass a strengthened ionic milieu that favors kinase inactivity and reclaim cell volume through condensate-mediated signal amplification. Thus, WNK kinases are physiological crowding sensors that phase separate to coordinate a cell volume rescue response.

Sodium ion regulates liquidity of biomolecular condensates in hyperosmotic stress response

“…Considering that the General Cluster Model defines only the aggregation-prone characteristics for protein units, we addressed the possibility that not only the liquidity of ASK3 condensates but also that of other biomolecular condensates is generally regulated by [Na + ] i . As model proteins, we selected decapping mRNA 1A (DCP1A), WW domain-containing transcription regulator 1 (WWTR1, also known as TAZ) and WNK1, all of which have been reported to rapidly form condensates following hyperosmotic stress (Boyd-Shiwarski et al, 2022; Cai et al, 2019; Jalihal et al, 2020). DCP1A is commonly known as a marker protein of the processing body (p-body) and forms condensates in the cytosol within seconds under hyperosmotic stress (Figure 6A) (Jalihal et al, 2020).…”

“…Wide varieties of interactions, including electrostatic (dipole–dipole, ionic, cation–π), aromatic (π–π) and hydrophobic interactions, drive condensation and determine material properties of biomolecular condensates (Bracha et al, 2019). Considering that hydrophobic residues were suggested to be crucial for WNK1 condensation (Boyd-Shiwarski et al, 2022), material properties including the liquidity of WNK1 condensates may be regulated primarily by hydrophobic interactions. In contrast, intracellular Na + may regulate the liquidity of biomolecular condensates whose liquid-to-solid transition is regulated mainly by charged amino acids.…”

“…Hence, physiological cellular stresses are suitable study subjects to investigate how extrinsic factors regulate condensate liquidity and how the liquidity maintenance of biomolecular condensates contributes to the cellular stress response. In particular, recent studies have revealed that osmotic stress rapidly induces the condensation of multiple biomolecules (Boyd-Shiwarski et al, 2022; Cai et al, 2019; Carrettiero et al, 2022; Dorone et al, 2021; Jalihal et al, 2020; Watanabe et al, 2021; Yasuda et al, 2020). Osmotic perturbation across the plasma membrane evokes cell swelling or shrinkage following osmotic water influx or efflux, respectively (Hoffmann et al, 2009; Lang et al, 1998).…”

“…Cell volume changes accompany various alterations in the intracellular environment, including ionic strength, macromolecular crowding and concentrations of biomolecules, all of which could affect the phase behavior of biomolecules (Ishihara et al, 2021). Although several studies have utilized osmotic stress as a means to investigate biomolecules of interest from the perspective of condensates (Cai et al, 2019;Carrettiero et al, 2022;Jalihal et al, 2020;Yasuda et al, 2020), recent studies have demonstrated that osmoresponsive kinases such as apoptosis signal-regulating kinase 3 (ASK3) and with-nolysine kinase 1 (WNK1) recognize osmotic stress through their intracellular phase transition to mediate signal transduction for volume recovery (Boyd-Shiwarski et al, 2022;Watanabe et al, 2021). In this study, using ASK3 as a model protein, we demonstrate how the material properties of biomolecular condensates are regulated in cells under hyperosmotic stress.…”

Regulation of Distal Nephron Transport by Intracellular Chloride and Potassium

“…In addition to regulation by potassium and chloride, WNKs are activated by increased osmotic pressure [22,48,[92][93][94]. Thus, WNKs are sensors of multiple components of the intracellular ionic and osmotic environment.…”