The C-terminus of WNK1 kinase is evolutionarily conserved and mediates unique phase behavior

Abstract

With-no-lysine 1 (WNK1) is a serine-threonine kinase with two isoforms, long (L)-WNK1 and kidney-specific (KS)-WNK1. L-WNK1 is an ancient kinase that is activated by hypertonic stress. Alternatively, KS-WNK1 emerged more recently, it is only expressed in the kidney, it is regulated by K+, and it lacks a functional kinase domain. Both WNK1 isoforms contain identical large C-term domains (CTD) with intrinsically disordered regions (IDRs) that drive the protein into discrete puncta. Our research has identified these puncta as membraneless biomolecular condensates that form via phase separation. In cells these condensates are visible at physiologic levels of hypertonic stress and at native levels of protein expression. In the kidney these condensates appear during K+ stress and are termed WNK bodies. We hypothesized that the large CTD of WNK1 mediates its phase behavior and that this function is evolutionarily conserved. To determine whether the CTD is required for condensate formation we performed live cell imaging studies of expressed mRuby2-tagged WNK1 constructs in cells. Full-length L-WNK1 was compared to two truncated constructs (1-494 or 1-1242), revealing that the CTD is required for condensate formation. To screen for the specific regions of the CTD that undergo phase separation, we used an optogenetic approach that mapped two areas within the WNK1 CTD that facilitate phase separation: (480-1242) and (1770-2126). Both regions were intrinsically disordered, exhibited low sequence complexity, and contained coiled-coil and prion-like domains- features typical of proteins that exhibit strong phase behavior. A bioinformatic analysis of human, C. elegan, and Drosophila WNK kinases revealed that these kinases shared nearly identical disorder tendency plots. A deeper analysis of CTD sequence complexity across 27 species ranging from humans to protists revealed that mammalian WNK kinases are enriched in prolines and serines, whereas invertebrates favor glutamine. Thus, the WNK CTD underwent a permissive Q-to-P/S amino acid shift during evolution, likely to preserve phase behavior. This conserved CTD has physiologic implications in the kidney as KS-WNK1 has an identical CTD and undergoes phase separation into WNK bodies during potassium deficiency. Mice that lack KS-WNK1 (KS-WNK1 KO) do not form WNK bodies during potassium deficiency and have a Gitelman-like phenotype with a 33% decrease in phospo-NCC [P = 0.01]. This decrease in pNCC correlated with decreased thiazide sensitivity—KO mice treated with HCTZ (25mg/kg IP) had no significant reduction in blood pressure, whereas WT littermates had a 4.5mmHg reduction in mean arterial pressure [P ≤ 0.05]. Based on these findings we conclude that during evolution KS-WNK1 conserved the intrinsically disordered WNK CTD to assemble WNK bodies to serve as protein scaffolds that enhance NCC activation during K+ stress. R01 DK098145, R01 DK119252, K08 DK118211 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.