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 phosphositespecific 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.renin-angiotensin-aldosterone system | NCC | hypertension | renal electrolyte transport | distal convoluted tubule T he distal portion of the mammalian nephron plays a key role in water, electrolyte, and blood pressure homeostasis. Mutations that alter normal renal Na-Cl homeostasis in this nephron segment modulate blood pressure and result in diverse electrolyte abnormalities (1). One such rare Mendelian trait is Pseudohypoaldosteronism type II (PHAII, OMIM 145260), which is characterized by hypertension, hyperkalemia, and metabolic acidosis; these features can be corrected by low doses of thiazide diuretics, inhibitors of the Na-Cl cotransporter of the distal convoluted tubule (NCC).Genetic analysis of PHAII has revealed a previously unrecognized pathway that regulates blood pressure and electrolyte homeostasis in the distal nephron. Causative mutations have been found in four genes; two encode the serine-threonine kinases with-no-lysine 1 and 4 (WNK1 and WNK4) (2), and two encode Cullin 3 (CUL3) and Kelch-like 3 (KLHL3), components of an E3-RING ubiquitin ligase complex (3). At the time of the discovery of their causal relationship to this Mendelian disease, none of these proteins were known to play a role in electrolyte or blood pressure homeostasis. The biochemical mechanisms that link mutations to clinical phenoytpes are becoming understood. WNK4 is a Cl − -regulated kinase (4); when active, the kinase phosphorylates the kinases SPAK (Ste20-related proline alaninerich kinase) and OSR1 (oxidative stress responsive kinase), which in turn phosphorylate and activate the thiazide-sensitive Na-Cl cotransporter of the renal distal convoluted tubule (DCT). The phenotype of WNK4 knockout (WNK4-KO) mice recapitulates Gitelman syndrome (OMIM 263800), the mirror image of PHAII, sugg...