WNK3-SPAK Interaction is Required for the Modulation of NCC and other Members of the SLC12 Family

Abstract

The serine/threonine with no lysine kinase 3 (WNK3) modulates the activity of the electroneutral cation-coupled chloride cotransporters (CCC) to promote Cl^- influx and prevent Cl^- efflux, thus fitting the profile for a putative “Cl^--sensing kinase”. The Ste20-type kinases, SPAK/OSR1, become phosphorylated in response to reduction in intracellular chloride concentration and regulate the activity of NKCC1. Several studies have now shown that WNKs function upstream of SPAK/OSR1. This study was designed to analyze the role of WNK3-SPAK interaction in the regulation of CCCs with particular emphasis on NCC. In this study we used the functional expression system of Xenopus laevis oocytes to show that different SPAK binding sites in WNK3 (^{241, 872, 1336}RFxV) are required for the kinase to have effects on CCCs. WNK3-F1337A no longer activated NKCC2, but the effects on NCC, NKCC1, and KCC4 were preserved. In contrast, the effects of WNK3 on these cotransporters were prevented in WNK3-F242A. The elimination of F873 had no consequence on WNK3 effects. WNK3 promoted NCC phosphorylation at threonine 58, even in the absence of the unique SPAK binding site of NCC, but this effect was abolished in the mutant WNK3-F242A. Thus, our data support the hypothesis that the effects of WNK3 upon NCC and other CCCs require the interaction and activation of the SPAK kinase. The effect is dependent on one of the three binding sites for SPAK that are present in WNK3, but not on the SPAK binding sites on the CCCs, which suggests that WNK3 is capable of binding both SPAK and CCCs to promote their phosphorylation.

Hypotonicity Stimulates Potassium Flux through the WNK-SPAK/OSR1 Kinase Cascade and the Ncc69 Sodium-Potassium-2-Chloride Cotransporter in the Drosophila Renal Tubule

“…For example, Ponce-Coria et al (73) observed activation of NKCC2 by WNK3-SPAK signaling by either bathing Xenopus oocytes in low Cl Ϫ hypotonic medium or by decreasing intracellular Cl Ϫ via coexpression of the potassium-chloride cotransporter, KCC2. Similarly, WNK3 powerfully stimulates both NCC and NKCC1 in Xenopus oocytes even under hypotonic conditions, in which cotransporter activity is otherwise suppressed (70,74,75).…”

Revisiting the NaCl cotransporter regulation by with-no-lysine kinases

“…However, another study reported that all WNK3 variants displayed the same effect on all members of the SLC12 family, including NCC (13). The WNK3-mediated activation of NCC requires the interaction with SPAK/OSR1 and is associated with an increased phosphorylation level of the cotransporter (56). Although WNK3 induces a dramatic activation of NCC, the elimination of WNK3 by knockout in mice generates no apparent consequences or obvious phenotype.…”

With no lysine L-WNK1 isoforms are negative regulators of the K+-Cl− cotransporters

“…Among the KCCs, only the NH 2 -terminal sequence of KCC3a and KCC2a isoforms contains a true SPAK/OSR1 binding site. However, we previously showed that, although the unique SPAK/OSR1 binding site on NCC and NKCC2 seems to be required for basal activity of the cotransporters, it is not necessary for WNK-induced activation (44). We indeed observed that the WNK3-SPAK-NCC complex could be formed as long as WNK3 is able to interact with SPAK, which in turn activates NCC, even in the absence of the SPAK binding site on the cotransporter (44).…”

“…However, we previously showed that, although the unique SPAK/OSR1 binding site on NCC and NKCC2 seems to be required for basal activity of the cotransporters, it is not necessary for WNK-induced activation (44). We indeed observed that the WNK3-SPAK-NCC complex could be formed as long as WNK3 is able to interact with SPAK, which in turn activates NCC, even in the absence of the SPAK binding site on the cotransporter (44). As shown in the present work, L-WNK1 can inhibit the activity of all KCCs, even though only KCC3a and KCC2a isoforms contain a SPAK/OSR1 binding site.…”

“…Thus L-WNK1 kinase, which is ubiquitously expressed, is expected to be a modulator in the SLC12 family of cotransporters. Although previous studies have shown that other WNKs mediate the activity of all members of the SLC12 family (10,12,19,44,52), the precise effect of L-WNK1 on these cotransporters remains unknown. Rinehart et al (51) observed that knocking down L-WNK1 with two distinct siRNAs reduced the phosphorylation of the key residues T991 and T1048 of the KCC3a isoform, a condition that is associated with its activation, suggesting that by promoting KCC3 phosphorylation, L-WNK1 could also be an inhibitor of this cotransporter.…”