WNK4 kinase is a negative regulator of K⁺-Cl⁻cotransporters

Abstract

WNK kinases [with no lysine (K) kinase] are emerging as regulators of several membrane transport proteins in which WNKs act as molecular switches that coordinate the activity of several players. Members of the cation-coupled chloride cotransporters family (solute carrier family number 12) are one of the main targets. WNK3 activates the Na(+)-driven cotransporters NCC, NKCC1, and NKCC2 and inhibits the K(+)-driven cotransporters KCC1 to KCC4. WNK4 inhibits the activity of NCC and NKCC1, while in the presence of the STE20-related proline-alanine-rich kinase SPAK activates NKCC1. Nothing is known, however, regarding the effect of WNK4 on the K(+)-Cl(-) cotransporters. Using the heterologous expression system of Xenopus laevis oocytes, here we show that WNK4 inhibits the activity of the K(+)-Cl(-) cotransporters KCC1, KCC3, and KCC4 under cell swelling, a condition in which these cotransporters are maximally active. The effect of WNK4 requires its catalytic activity because it was lost by the substitution of aspartate 318 for alanine (WNK4-D318A) that renders WNK4 catalytically inactive. In contrast, three different WNK4 missense mutations that cause pseudohypoaldosteronism type II do not affect the WNK4-induced inhibition of KCC4. Finally, we observed that catalytically inactive WNK4-D318A is able to bypass the tonicity requirements for KCC2 and KCC3 activation in isotonic conditions. This effect is enhanced by the presence of catalytically inactive SPAK, was prevented by the presence of protein phosphatase inhibitors, and was not present in KCC1 and KCC4. Our results reveal that WNK4 regulates the activity of the K(+)-Cl(-) cotransporters expressed in the kidney.

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

“…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. We have also previously observed that other WNKs inhibit the KCCs cotransporter that contains no SPAK/OSR1 binding sites (12,19,52). Our data thus suggest that L-WNK1 activates SPAK/OSR1, which in turn phosphorylates KCCs, decreasing their activity.…”

“…In their study, however, de los Heros et al (11) indicated that SPAK/OSR1 phosphorylates T1048 but may not itself phosphorylate the T991 directly. Regarding S96, which is only present in KCC3a, Melo et al (36) (4,46), and they are able to bypass tonicity requirements for the activation or inhibition of the SLC12 cotransporters (12,19 L-WNK1 is a ubiquitous kinase and its activity is regulated by hypertonicity or hypotonicity. It has been involved in cell volume regulation during changes in osmolality (31,49,68).…”

“…12 We previously reported that eliminating the catalytic activity of WNK3 and WNK4 by the D294A and D318A substitution, respectively (4,10,12,19,36,50), switched the way in which these kinases affect the SLC12 family members. Wild-type WNK3 activates NCC, NKCC1, and NKCC2, whereas the catalytically inactive version WNK3-D294A completely prevents the activity of these Na ϩ -driven cotransporters, even in the presence of cell shrinkage (26,50).…”

“…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.…”

Taurine Inhibits K+-Cl− Cotransporter KCC2 to Regulate Embryonic Cl− Homeostasis via With-no-lysine (WNK) Protein Kinase Signaling Pathway

“…Lifton and colleagues (54,61,62) propose that the WNK signaling pathway also activates KCCs, including KCC2. WNKs are activated by osmotic stress; however, the upstream signaling remains unknown (38,59,63).…”