A Phosphorylated Intermediate in the Activation of WNK Kinases

Abstract

WNK kinases autoactivate by autophosphorylation. Crystallography of the kinase domain of WNK1 phosphorylated on the primary activating site (pWNK1) in the presence of AMP-PNP reveals a well-ordered but inactive configuration. This new pWNK1 structure features specific and unique interactions of the phosphoserine, less hydration, and smaller cavities compared with those of unphosphorylated WNK1 (uWNK1). Because WNKs are activated by osmotic stress in cells, we addressed whether the structure was influenced directly by osmotic pressure. pWNK1 crystals formed in PEG3350 were soaked in the osmolyte sucrose. Suc-WNK1 crystals maintained X-ray diffraction, but the lattice constants and pWNK1 structure changed. Differences were found in the activation loop and helix C, common switch loci in kinase activation. On the basis of these structural changes, we tested for effects on in vitro activity of two WNKs, pWNK1 and pWNK3. The osmolyte PEG400 enhanced ATPase activity. Our data suggest multistage activation of WNKs.

Compensatory ion transport buffers daily protein rhythms to regulate osmotic balance and cellular physiology

“…In concert with other transport systems, the ubiquitous and essential Na + /K + -ATPase establishes ~10-fold ion gradients over the plasma membrane, with high K + and low Na + /Cl − in the cytosol (Supplementary Table 1). In response to the acute external osmotic challenge, the net movement of water rapidly re-establishes osmotic equilibrium, with resultant volume changes then being quickly countered by solute transport that returns the cell to its original volume 17,58 . This homeostatic mechanism, known as regulatory volume increase/decrease (RVI/D), is common to all mammalian cells, and its molecular mechanisms are very well characterized 17,22,[59][60][61][62] .…”

“…As cytosolic Cl − levels fall, they will eventually begin to limit KCC activity, because Cl − is the essential counterion to K + . However, around this time increased macromolecular crowding acts synergistically with decreased Cl − levels to stimulate WNK activity 58 , which will then stimulate increased import of Na + and Cl − via NKCC. Cytosolic Na + is exported by several mechanisms 32,63,64,66 , whereas Cl − influx provides counterions to sustain KCC activity.…”

Osmosensing by WNK Kinases

“…The WNK1SA Cluster is shown in stereo in Figure 5C and is contrasted with pWNK1 ( Figure 5D ) (PDB file 5W7T; Akella et al. , 2020 ).…”

“…, 2014 ). We also used wt WNK1 (194-483), which is phosphorylated (pWNK1) as expressed in E. coli ( Akella et al. , 2020 ).…”

“…We demonstrated recently that a phosphorylated form of WNK1 does not adopt the canonical structure of an active kinase, and that an osmolyte induced conformational changes toward a more fully active configuration ( Akella et al. , 2020 ).…”