Phosphorylation-dependent charge switching coordinates WNK1 phase behavior and volume regulatory function

With-No-Lysine (WNK) kinases are ancient cell volume regulators that control salt transport in the kidney, brain, and other organs. Recently, we uncovered a novel role for WNK kinases as molecular crowding sensors that mediate cell volume recovery via phase separation (PS). During acute cell shrinkage, WNK1 rapidly concentrates in membraneless biomolecular condensates. Within these dynamic liquid-like signaling hubs, WNK1 activates the downstream kinases SPAK and OSR1, which in turn directly phosphorylate NKCC and KCC salt cotransporters to promote net ion influx and regulatory volume increase (RVI). Following fluid volume recovery, the condensates dissolve. While this finding highlights a physiological role for phase separation in RVI, it remains unclear how these WNK droplets disperse, allowing activated SPAK and OSR1 to reach the plasma membrane and phosphorylate the NKCC/KCC system. Homotypic WNK1 PS is mediated by its C-terminal domain, a large serine-enriched intrinsically disordered region (IDR) that undergoes phosphorylation for unclear reasons. We hypothesized that phosphorylation of these residues alters WNK charge at key PS-driving sites, resulting in condensate disassembly via electrostatic repulsion. To test this, we generated mRuby2-tagged WNK1 mutant constructs, substituting all 57 known phosphorylation sites with either aspartic acid (phosphomimic S≥D) or alanine (phosphonull S≥A). These constructs were then expressed and characterized in a CRISPR-engineered HEK293 WNK1/WNK3 double knockout cell line lacking background WNK expression. Live-cell imaging studies conducted at physiologic hypertonic stress (50 mOsm sorbitol) revealed that both phosphomutant constructs exhibited altered phase behavior compared to wild-type WNK1: While the phosphomimic S≥D mutant formed short-lived droplets that dissolved rapidly, the phosphonull S≥A mutant formed long-lived assemblies that failed to leave the condensed phase efficiently. In both cases, the phosphomutants exhibited impaired downstream SPAK/OSR1 activation, as assessed by biochemical analysis of phosphorylation within a 15-min timecourse. In preliminary 4D live cell imaging studies, the S≥D and S≥A mutants were unable to mediate RVI as efficiently as wild-type WNK1. Furthermore, the kinase activity of WNK1 itself appeared to be required for optimal condensate dynamics, as adding the pan-WNK kinase inhibitor WNK463 to wild-type WNK1, or introducing a kinase-dead WNK1 mutation (K233M) caused the kinase to form inert inclusions under baseline isotonic conditions that failed to disperse with hypotonic stress. In summary, these data underscore the importance of phosphorylation-mediated IDR charge switching on WNK1 phase behavior and function. This process may be mediated by autophosphorylation, thereby allowing the WNK-SPAK/OSR1 pathway to self-disassemble its condensed phase following activation. NIH R01DK098145, R01DK119252, U54DK137329, TL1DK143271 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.