WNK kinases regulate electro-neutral cotransporters that are controlled by osmotic stress and chloride. We showed previously that autophosphorylation of WNK1 is inhibited by chloride, raising the possibility that WNKs are activated by osmotic stress. Here we demonstrate that unphosphorylated WNK isoforms 3 and 1 autophosphorylate in response to osmotic pressure in vitro, applied with the crowding agent polyethylene glycol 400 or osmolyte ethylene glycol, and that this activation is opposed by chloride. Small Angle X-ray Scattering of WNK3 in the presence and absence of PEG400, static light scattering in ethylene glycol, and crystallography of WNK1 were used to understand mechanism. Osmosensing in WNK3 and WNK1 appear to occur through a conformational equilibrium between an inactive, unphosphorylated, chloride-binding dimer and an autophosphorylation-competent monomer. An improved structure of the inactive kinase domain of WNK1, and a comparison with the structure of a monophosphorylated form of WNK1, suggests that large cavities, greater hydration, and specific bound water may participate in the osmosensing mechanism. Our prior work showed that osmolytes have effects on the structure of phosphorylated WNK1, suggestive of multiple stages of osmotic regulation in WNKs.

Virtually all aspects of cell biology are regulated by a ubiquitin code where distinct ubiquitin chain architectures guide the binding events and itineraries of modified substrates. Various combinations of E2 and E3 enzymes accomplish chain formation by forging isopeptide bonds between the C-terminus of their transiently-linked donor ubiquitin and a specific nucleophilic amino acid on the acceptor ubiquitin, yet it is unknown whether the fundamental feature of most acceptors - the lysine side-chain - affects catalysis. Here, use of synthetic ubiquitins with non-natural acceptor site replacements reveals that the aliphatic side-chain specifying reactive amine geometry is a determinant of the ubiquitin code, through unanticipated and complex reliance of many distinct ubiquitin carrying enzymes on a canonical acceptor lysine.

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.

Previous work has demonstrated that the WNK kinases 1 and 3 are direct osmosensors consistent with their established role in cell volume control. WNK kinases may also be regulated by hydrostatic pressure. Hydrostatic pressure applied to cells in culture with N2 gas or to Drosophila Malpighian tubules by centrifugation induces phosphorylation of downstream effectors of endogenous WNKs. In vitro, the autophosphorylation and activity of the unphosphorylated kinase domain of WNK3 (uWNK3) is enhanced to a lesser extent than in cells by 190 kPa applied with N2 gas. Hydrostatic pressure measurably alters the structure of uWNK3. Data from size exclusion chromatography in line with multi-angle light scattering (SEC-MALS), SEC alone at different back pressures, analytical ultracentrifugation (AUC), NMR, and chemical crosslinking indicate a change in oligomeric structure in the presence of hydrostatic pressure from a WNK3 dimer to a monomer. The effects on the structure are related to those seen with osmolytes. Potential mechanisms of hydrostatic pressure activation of uWNK3 and the relationships of pressure activation to WNK osmosensing are discussed.

Cells respond to hydrostatic pressure to maintain cellular, organ, and organism level functions, yet the direct pressure sensors are largely unknown. Here we show that hydrostatic pressure directly activates With No Lysine(K) kinase-3 (WNK3) 1 , a soluble intracellular protein kinase. Using gel filtration we demonstrate that pressure induces a dimer to monomer transition in a construct of the unphosphorylated kinase domain of WNK3 (uWNK3-KDm or uWNK3). The uWNK3 has not been crystallized, but crosslinking data suggest that the uWNK3 dimer corresponds to crystallographically observed dimer of WNK1 (uWNK1-KDm, or uWNK1) 2,3 . Sequence alignments with WNKs from species living in different pressure environments and mutational analysis lend further support for this idea. Unique features of the uWNK1 structure suggest a mechanism involving bound water. We further show that hydrostatic pressure activates full-length WNK3 in D. melanogaster tubules. PressureK375 F389 Y420 D353 Y420 K375 K351 Cluster E388 K381 T386