A phosphorylated intermediate in the activation of WNK kinases.

WNK kinases auto-activate by autophosphorylation. Crystallography of the kinase domain of WNK1 phosphophorylated on the primary activating site (pWNK1) and 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 as compared with unphophorylated WNK1 (uWNK1). Since 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 occurred in the activation loop and helix C, common switch loci in kinase activation. Based on these structural changes, we tested for effects on in vitro activity of two WNKs, pWNK1 and pWNK3. The osmolyte PEG400 enhanced ATPase activity. The data presented suggest multistage activation of WNKs.