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.

Figure 1.

Structure of pWNK1. (A) Overall structure of pWNK1. Helices are pink and β-strands are cyan, AMP-PNP (ANP) and the pS382 are shown in sticks. (B) Overlay of the structure of pWNK1 (PDB file 5W7T) with PKA (PDB file 1ATP). pWNK is magenta and its activation loop red; PKA is green, and it activation loop blue. (C) Unique conformation of the pWNK1 activation loop and unique interactions of pS382 with R376 in hAL, rather than R348 from the catalytic loop. (D) Interactions of the phosphorylation site in PKA from the same perspective as (C) showing canonical interactions with basic residues from the catalytic loop (R165), helix C (H87) and the activation loop (K189). Diagrams drawn in PyMOL

Figure 2.

Bound water in pWNK1 associated with the inactive configuration of hAL. (A) Waters in pWNK1 near helix AL. Cartoon rendering of pWNK1 in magenta (main cartoon) and red (activation loop). Waters are shown in yellow; electron density contoured at 0.8 σ in blue. (B) Cavities are depicted in gray in the same orientation as (A). (C) Waters between hC and β4 and extending from the V-shaped linker (V) into the back of the active site (below and β4 in the orientation shown). (D) Closeup of the cavity near the V-shaped linker and active site. (Surfaces calculated in PyMOL using Cavity_cull default settings).

Figure 3.

Effects of sucrose on pWNK1 and pWNK3. (A) Overlay of the AMP-PNP complexed pWNK1 structure in the presence (magenta) and absence (gray) of sucrose (0.6 M). Activation loop, helix C and β4-β5 of suc-pWNK1 are shown in cyan. (B) Placement of conformational changes along the sequence (WNK1 numbering) between pWNK1 and PKA (green), pWNK1 and uWNK1 (pink), and pWNK1 and suc-pWNK1 with 0.6 M sucrose (cyan). (C) DSF melt temperatures (Tm) of pWNK1 in the absence (blue) and presence (cyan) of 0.6 M sucrose and pWNK3 in the absence (purple) and presence (violet) of 0.6 M sucrose. Note that the DSF conditions differ from the crystallographic study in A. * Trace is the negative first derivative of fluorescence from Sypro orange. Note that sucrose stabilizes pWNK1 and pWNK3. (D) Progress curves for the activity of pWNK1 on gOSR1 in increasing sucrose concentrations (0 mM, 50 mM, 100 mM, 200 mM, 400 mM, and 600 mM sucrose shown in colors shifting from black to cyan) tracking the disappearance of ATP using Kinase-Glo^®. (E) Progress curves as in (D) following the incorporation of ³²P into total protein (4 μM pWNK1 and 40 μM gOSR1) using [γ-³²]P ATP with increasing sucrose concentrations (same concentrations and coloring as in (D)). Representative data from three replicates shown in (C). Progress curves in (D) reflect 5 simultaneous experiments, each having 3 independent replicates. Error bars are standard error. Panel E shows 5 simultaneous experiments, with autoradiography shown in Figure S2A. * “Additional DSF experiments with Mg⁺² and AMP-PNP added as controls could not be conducted at submission on account of the COVID-19 lockdown.”

Figure 4.

Effects of PEG400 on pWNK1 and pWNK3. (A) DSF Melt temperatures (Tm) of pWNK1 (blue/cyan) and pWNK3 (purple/violet) with and without 15% PEG400. Note the reduced thermal stability in PEG400. (B) Progress curves for the activity of pWNK1 on gOSR1 in increasing PEG400 concentrations (0 mM, 50 mM, 100 mM, 200 mM, 400 mM, 600 mM in shades of blue from black to cyan) tracking the disappearance of ATP using Kinase-Glo^®. (C) Progress curves for pWNK3 activity, PEG400 concentrations as in (B) with colors shifting from black to violet with increasing sucrose. (D) Progress curves for the activity of pWNK1 on gOSR1 with (cyan) and without (blue) 15%PEG400, tracking the disappearance of ATP using Kinase-Glo^®. (E) Progress curves for the activity of pWNK3 on gOSR1 with (violet) and without (purple) 15%PEG400, as in (D). (F) Progress curves for the activity of pWNK1 on gOSR1 with (cyan) and without (blue) 15%PEG400, following the incorporation of ³²P into total protein. Autoradiography in Figure S2B. (G) Progress curves for the activity of pWNK3 on gOSR1 with (violet) and without (purple) 15%PEG400, as in (F) Autoradiography in Figure S2C. Representative data from three replicates shown in (A). Panels B and C represent 5 simultaneous experiments. Panels D, E, F, and G reflect data from 3 experimental replicates. Error bars are standard error.