Identifying Novel Functions of the WNK Pathway

The with no lysine [K] (WNK) pathway consists of WNK kinases, their downstream target kinases, oxidative stress responsive (OSR)1 and SPS/Ste20-related proline-alanine-rich kinase (SPAK), and OSR1/SPAK substrates, cation chloride cotransporters. The pathway regulates ion transport across cell membranes, among other functions, and is implicated in human diseases including hypertension, cancer and neurological diseases. However, the functions of WNK pathway beyond cotransporter regulation have not been extensively studied. The purpose of my work has been to understand novel functions of the WNK pathway. I demonstrated that WNK1, largest and ubiquitously expressed WNK isoform, is an inhibitor of autophagy, an intracellular degradation pathway. WNK1 inhibited the class III phosphatidylinositol 3-kinase (PI3KC3) complex which acts upstream in the autophagy pathway. In addition, WNK1 inhibited the unc-51-like kinase 1 (ULK1) complex that acts upstream of PI3KC3. WNK1 also inhibited AMP-activated protein kinase (AMPK), the upstream activator of ULK1. The actions of WNK1 on the AMPK-ULK1 axis only partially mediated its effects on autophagy. WNK1 directly bound UV radiation resistance-associated gene (UVRAG) in vitro and had an overlapping localization with it in cells, and autophagy induction led to a decrease in this property. OSR1 had no significant effect on autophagy while SPAK acted as an autophagy inhibitor. Therefore, WNK pathway most likely inhibits autophagy through multiple mechanisms. I also discovered that OSR1 regulates the cellular localization of inward-rectifier potassium channel (Kir) 2.3 that contains an OSR1/SPAK recognition motif, and is activated by WNK. OSR1 promoted Kir2.3 localization to shift towards the cell membrane in the presence of sodium chloride. Similar to OSR1, WNK kinase activity also promoted the change in localization of Kir2.3 elicited by NaCl. Therefore, I suggest that activated WNK induces Kir2.3 channel activity by driving it to the cell membrane.