Arginine vasopressin regulates the renal Na+-Cl- and Na+-K+-Cl- cotransporters through with-no-lysine kinase 4 and inhibitor 1 phosphorylation.

Vasopressin regulates water homeostasis via the V2 receptor in the kidney at least in part through protein kinase A (PKA) activation. Vasopressin, through an unknown pathway, upregulates the activity and phosphorylation of Na+-Cl- cotransporter (NCC) and Na+-K+-2Cl- cotransporter 2 (NKCC2) by Ste20-related proline/alanine-rich kinase (SPAK) and oxidative stress-responsive kinase 1 (OSR1), which are regulated by the with-no-lysine kinase (WNK) family. Phosphorylation of WNK4 at PKA consensus motifs may be involved. Inhibitor 1 (I1), a protein phosphatase 1 (PP1) inhibitor, may also play a role. In human embryonic kidney (HEK)-293 cells, we assessed the phosphorylation of WNK4, SPAK, NCC, or NKCC2 in response to forskolin or desmopressin. WNK4 and cotransporter phosphorylation were studied in desmopressin-infused WNK4-/- mice and in tubule suspensions. In HEK-293 cells, only wild-type WNK4 but not WNK1, WNK3, or a WNK4 mutant lacking PKA phosphorylation motifs could upregulate SPAK or cotransporter phosphorylation in response to forskolin or desmopressin. I1 transfection maximized SPAK phosphorylation in response to forskolin in the presence of WNK4 but not of mutant WNK4 lacking PP1 regulation. We observed direct PP1 regulation of NKCC2 dephosphorylation but not of NCC or SPAK in the absence of WNK4. WNK4-/- mice with desmopressin treatment did not increase SPAK/OSR1, NCC, or NKCC2 phosphorylation. In stimulated tubule suspensions from WNK4-/- mice, upregulation of pNKCC2 was reduced, whereas upregulation of SPAK phosphorylation was absent. These findings suggest that WNK4 is a central node in which kinase and phosphatase signaling converge to connect cAMP signaling to the SPAK/OSR1-NCC/NKCC2 pathway.NEW & NOTEWORTHY With-no-lysine kinases regulate the phosphorylation and activity of the Na+-Cl- and Na+-K+-2Cl- cotransporters. This pathway is modulated by arginine vasopressin (AVP). However, the link between AVP and WNK signaling remains unknown. Here, we show that AVP activates WNK4 through increased phosphorylation at putative protein kinase A-regulated sites and decreases its dephosphorylation by protein phosphatase 1. This work increases our understanding of the signaling pathways mediating AVP actions in the kidney.

C-terminally truncated, kidney-specific variants of the WNK4 kinase lack several sites that regulate its activity.

WNK lysine-deficient protein kinase 4 (WNK4) is an important regulator of renal salt handling. Mutations in its gene cause pseudohypoaldosteronism type II, mainly arising from overactivation of the renal Na+/Cl- cotransporter (NCC). In addition to full-length WNK4, we have observed faster migrating bands (between 95 and 130 kDa) in Western blots of kidney lysates. Therefore, we hypothesized that these could correspond to uncharacterized WNK4 variants. Here, using several WNK4 antibodies and WNK4-/- mice as controls, we showed that these bands indeed correspond to short WNK4 variants that are not observed in other tissue lysates. LC-MS/MS confirmed these bands as WNK4 variants that lack C-terminal segments. In HEK293 cells, truncation of WNK4's C terminus at several positions increased its kinase activity toward Ste20-related proline/alanine-rich kinase (SPAK), unless the truncated segment included the SPAK-binding site. Of note, this gain-of-function effect was due to the loss of a protein phosphatase 1 (PP1)-binding site in WNK4. Cotransfection with PP1 resulted in WNK4 dephosphorylation, an activity that was abrogated in the PP1-binding site WNK4 mutant. The electrophoretic mobility of the in vivo short variants of renal WNK4 suggested that they lack the SPAK-binding site and thus may not behave as constitutively active kinases toward SPAK. Finally, we show that at least one of the WNK4 short variants may be produced by proteolysis involving a Zn2+-dependent metalloprotease, as recombinant full-length WNK4 was cleaved when incubated with kidney lysate.