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Proc Natl Acad Sci U S A. 2017 Jan 31;114(5):E879-E886. doi: 10.1073/pnas.1620315114. Epub 2017 Jan 17.

Phosphorylation by PKC and PKA regulate the kinase activity and downstream signaling of WNK4.

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Department of Genetics, Yale University School of Medicine, New Haven, CT 06510.
Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06510.
Department of Nephrology and Mineral Metabolism, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, 14080 Mexico City, Mexico.
Molecular Physiology Unit, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, 14080 Mexico City, Mexico.
Department of Cellular and Molecular Physiology and Systems Biology Institute, Yale University School of Medicine, New Haven, CT 06510.
Laboratory of Human Genetics and Genomics, The Rockefeller University, New York, NY 10065.


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.


NCC; distal convoluted tubule; hypertension; renal electrolyte transport; renin-angiotensin-aldosterone system

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