With-no-lysine kinase 4 (WNK4) regulates electrolyte homeostasis and blood pressure. WNK4 phosphorylates the kinases SPAK (Ste20-related proline alanine-rich kinase) and OSR1 (oxidative stress responsive kinase), which then phosphorylate and activate the renal Na-Cl cotransporter (NCC). WNK4 levels are regulated by binding to Kelch-like 3, targeting WNK4 for ubiquitylation and degradation. Phosphorylation of Kelch-like 3 by PKC or PKA downstream of AngII or vasopressin signaling, respectively, abrogates binding. We tested whether these pathways also affect WNK4 phosphorylation and activity. By tandem mass spectrometry and use of phosphosite-specific antibodies, we identified five WNK4 sites (S47, S64, S1169, S1180, S1196) that are phosphorylated downstream of AngII signaling in cultured cells and in vitro by PKC and PKA. Phosphorylation at S64 and S1196 promoted phosphorylation of the WNK4 kinase T-loop at S332, which is required for kinase activation, and increased phosphorylation of SPAK. Volume depletion induced phosphorylation of these sites in vivo, predominantly in the distal convoluted tubule. Thus, AngII, in addition to increasing WNK4 levels, also modulates WNK4 kinase activity via phosphorylation of sites outside the kinase domain.
Keywords: NCC; distal convoluted tubule; hypertension; renal electrolyte transport; renin-angiotensin-aldosterone system.