A Cysteine‐Rich Hydrophobic Motif in KS‐WNK1 Regulates WNK Body Formation

With‐No‐Lysine kinase 1 (WNK1) coordinates tubular NaCl and K+ transport through the dual action of two major isoforms: a ubiquitously expressed full‐length “ L ong” isoform with intact kinase activity (L‐WNK1), and an N‐terminally truncated kinase‐defective “ Ki dney S pecific” isoform (KS‐WNK1) that is highly expressed in the distal convoluted tubule (DCT). These two isoforms are functionally different, presumably due to differences in their N‐termini, as their C‐termini are identical. In response to changes in dietary potassium, WNK signaling complexes concentrate in the DCT, forming discrete punctate foci of unknown function, which we call “WNK bodies”. The marked expression of KS‐WNK1 in DCT suggests that it may play role in the formation of these structures. We hypothesized that the unique N‐terminus of KS‐WNK1 is critical for WNK body formation in response to potassium stress. C‐terminal antibodies recognizing both L‐ and KS‐WNK1 recognized punctate structures in the DCT of mice maintained on either low or high potassium diets; surprisingly, WNK bodies were not present in mice on control diets. Mice exposed to low K diets developed subnuclear WNK bodies that were twice as abundant and larger than the subapical puncta that appeared in mice on high K diet. Since our WNK1 antibody recognizes both L‐ and KS‐WNK1, we moved to an in vitro system to determine whether L‐ or KS‐WNK1 is important for puncta formation. In cells, KS‐WNK1 collected in similar puncta, whereas L‐WNK1 was distributed diffusely throughout the cytoplasm. Live cell imaging and FRAP studies indicated that the puncta are mobile structures that slowly fill with eGFP‐tagged KS‐WNK1 protein and restrict KS‐WNK1 diffusion into the free cytosol. Mutagenesis studies revealed that KS‐WNK1 puncta formation is dependent on a cysteine‐rich hydrophobic motif harbored in its unique N‐terminus, as ablation of this motif shifted KS‐WNK1 diffusely. Immuno‐TEM and immunofluorescence confocal microscopy studies indicated that both the in vivo WNK bodies and in vitro KS‐WNK1 puncta are non‐membrane bound structures that co‐localize >90% with the 60s core ribosomal exit tunnel protein L22, suggesting that these puncta are similar in nature. Though L22 colocalization was detected under both high and low K stress conditions, low K WNK bodies additionally contained SPAK and WNK4. Coupled with our observations regarding the differences in WNK body positioning and size with different dietary potassium maneuvers, these findings suggest that WNK bodies contain different proteins and have different roles when the DCT is challenged with low or high K stress. Thus, the formation of WNK bodies in the DCT may reflect distinct WNK‐dependent stress responses that are coordinated by KS‐WNK1. Support or Funding Information R01DK098145, P30DK079307, T32DK061296