WNK1 Regulates Phosphorylation of Cation-Chloride-coupled Cotransporters via the STE20-related Kinases, SPAK and OSR1

Abstract

The WNK1 and WNK4 genes have been found to be mutated in some patients with hyperkalemia and hypertension caused by pseudohypoaldosteronism type II. The clue to the pathophysiology of pseudohypoaldosteronism type II was its striking therapeutic response to thiazide diuretics, which are known to block the sodium chloride cotransporter (NCC). Although this suggests a role for WNK1 in hypertension, the precise molecular mechanisms are largely unknown. Here we have shown that WNK1 phosphorylates and regulates the STE20-related kinases, Ste20-related proline-alanine-rich kinase (SPAK) and oxidative stress response 1 (OSR1). WNK1 was observed to phosphorylate the evolutionary conserved serine residue located outside the kinase domains of SPAK and OSR1, and mutation of the OSR1 serine residue caused enhanced OSR1 kinase activity. In addition, hypotonic stress was shown to activate SPAK and OSR1 and induce phosphorylation of the conserved OSR1 serine residue, suggesting that WNK1 may be an activator of the SPAK and OSR1 kinases. Moreover, SPAK and OSR1 were found to directly phosphorylate the N-terminal regulatory regions of cation-chloride-coupled cotransporters including NKCC1, NKCC2, and NCC. Phosphorylation of NCC was induced by hypotonic stress in cells. These results suggested that WNK1 and SPAK/OSR1 mediate the hypotonic stress signaling pathway to the transporters and may provide insights into the mechanisms by which WNK1 regulates ion balance. WNK2 kinases (with no lysine (K)) comprise a family of novel serine/ threonine protein kinases conserved among multicellular organisms (1, 2). The kinase domain of this family is unique in that it lacks the conserved lysine residue previously known to be important for ATP binding in the catalytic site. A conserved lysine in subdomain I of the WNK kinases is thought to be essential for their catalytic activity (1, 3). There are four human WNK family members. WNK1 and WNK4 were identified as genes mutated in families of patients with pseudohypoaldosteronism type 2 (PHA II) human hypertension (4). The WNK1 gene mutation consists of a deletion within its first intron, leading to increased expression, whereas mutations in the WNK4 gene are found in the coding sequence near the coiled-coil domains.PHA II patients are treated by thiazide diuretics, which function as antagonists of the Na-Cl cotransporter (NCC, also known as thiazidesensitive cotransporter (TSC) or Na-Cl transporter (NCCT)), suggesting that the activity of NCC could be potentially involved in the development of PHA II. Previous studies using Xenopus oocytes have showed that wild-type WNK4 inhibits the surface expression and the activity of NCC, whereas one of the disease-causing mutants of WNK4 attenuated this inhibitory effect (5, 6). However, comparison of wild-type and mutant WNK4 revealed no differences in NCC surface expression in polarized epithelial cells (MDCK II cells), suggesting that the regulation of intracellular NCC localization by WNK4 might be unrelated to the pathogenesis of PHA II (7). WNK4 has ...

WNK1 and OSR1 regulate the Na + , K + , 2Cl − cotransporter in HeLa cells

“…Overexpression of kinase-dead SPAK and shark NKCC1 in HEK 293 cells reduced NKCC1 activation and its phosphorylation in response to low ionic strength (9), consistent with the view that phosphorylation of these sites activates cotransporter function. Phosphorylation sites identified in the N-terminal cytoplasmic domain of NKCC1, NKCC2, and sodium chloride cotransporter are phosphorylated by SPAK and OSR1 in vitro (36). Our findings suggest that WNK4 has little or no ability to activate OSR1 and SPAK.…”

Whole-genome association study identifies STK39 as a hypertension susceptibility gene

“…Consistent with this idea, recent in vitro studies have demonstrated that SPAK binds to and phosphorylates NKCC2 and NCC, 2 Na ϩ -dependent cation chloride cotransporters that mediate renal NaCl reabsorption (6). To explore whether SPAK has the capacity to regulate these transporters in vivo, we determined the renal expression pattern of SPAK through immunofluorescence microscopy of rat kidney sections.…”

Germinal center kinases in immune regulation

“…111 OSR1 has recently been identified as a key regulator of ion homeostasis and cell volume downstream of the WNK signaling pathway. [112][113][114][115][116] OSR1 and SPAK target the Na-K-Cl cotransporter. MO25 is a potent regulator of OSR1 kinase activity.…”