Paola de los Heros

Regulation of NKCC2 by a chloride-sensing mechanism involving the WNK3 and SPAK kinases

The Na+:K+:2Cl− cotransporter (NKCC2) is the target of loop diuretics and is mutated in Bartters syndrome, a heterogeneous autosomal recessive disease that impairs salt reabsorption in the kidneys thick ascending limb (TAL). Despite the importance of this cation/chloride cotransporter (CCC), the mechanisms that underlie its regulation are largely unknown. Here, we show that intracellular chloride depletion in Xenopus laevis oocytes, achieved by either coexpression of the K-Cl cotransporter KCC2 or low-chloride hypotonic stress, activates NKCC2 by promoting the phosphorylation of three highly conserved threonines (96, 101, and 111) in the amino terminus. Elimination of these residues renders NKCC2 unresponsive to reductions of [Cl−]i. The chloride-sensitive activation of NKCC2 requires the interaction of two serine-threonine kinases, WNK3 (related to WNK1 and WNK4, genes mutated in a Mendelian form of hypertension) and SPAK (a Ste20-type kinase known to interact with and phosphorylate other CCCs). WNK3 is positioned upstream of SPAK and appears to be the chloride-sensitive kinase. Elimination of WNK3s unique SPAK-binding motif prevents its activation of NKCC2, as does the mutation of threonines 96, 101, and 111. A catalytically inactive WNK3 mutant also completely prevents NKCC2 activation by intracellular chloride depletion. Together these data reveal a chloride-sensing mechanism that regulates NKCC2 and provide insight into how increases in the level of intracellular chloride in TAL cells, as seen in certain pathological states, could drastically impair renal salt reabsorption.

Regulation of the NKCC2 ion cotransporter by SPAK-OSR1-dependent and -independent pathways.

Ion cotransporters, such as the Na+/Cl− cotransporter (NCC), control renal salt re-absorption and are regulated by the WNK-signalling pathway, which is over-stimulated in patients suffering from Gordons hypertension syndrome. Here, we study the regulation of the NKCC2 (SLC12A1) ion cotransporter that contributes towards ~25% of renal salt re-absorption and is inhibited by loop-diuretic hypertensive drugs. We demonstrate that hypotonic low-chloride conditions that activate the WNK1-SPAK and OSR1 pathway promote phosphorylation of NKCC2 isoforms (A, B and F) at five residues (Ser91, Thr95, Thr100, Thr105 and Ser130). We establish that the SPAK and OSR1 kinases activated by WNK interact with an RFQV motif on NKCC2 and directly phosphorylate Thr95, Thr100, Thr105 and, possibly, Ser91. Our data indicate that a SPAK-OSR1-independent kinase, perhaps AMP-activated protein kinase (AMPK), phosphorylates Ser130 and that phosphorylation of Thr105 and Ser130 plays the most important roles in stimulating NKCC2 activity. In contrast with NCC, whose membrane translocation is triggered by SPAK-OSR1 phosphorylation, NKCC2 appears to be constitutively at the membrane. Our findings provide new insights into how NKCC2 is regulated and suggest that inhibitors of SPAK and/or OSR1 for the treatment of hypertension would be therapeutically distinct from thiazide or loop diuretics, as they would suppress the activity of both NCC and NKCC2.

MO25 is a master regulator of SPAK/OSR1 and MST3/MST4/YSK1 protein kinases

Mouse protein‐25 (MO25) isoforms bind to the STRAD pseudokinase and stabilise it in a conformation that can activate the LKB1 tumour suppressor kinase. We demonstrate that by binding to several STE20 family kinases, MO25 has roles beyond controlling LKB1. These new MO25 targets are SPAK/OSR1 kinases, regulators of ion homeostasis and blood pressure, and MST3/MST4/YSK1, involved in controlling development and morphogenesis. Our analyses suggest that MO25α and MO25β associate with these STE20 kinases in a similar manner to STRAD. MO25 isoforms induce approximately 100‐fold activation of SPAK/OSR1 dramatically enhancing their ability to phosphorylate the ion cotransporters NKCC1, NKCC2 and NCC, leading to the identification of several new phosphorylation sites. siRNA‐mediated reduction of expression of MO25 isoforms in mammalian cells inhibited phosphorylation of endogenous NKCC1 at residues phosphorylated by SPAK/OSR1, which is rescued by re‐expression of MO25α. MO25α/β binding to MST3/MST4/YSK1 also stimulated kinase activity three‐ to four‐fold. MO25 has evolved as a key regulator of a group of STE20 kinases and may represent an ancestral mechanism of regulating conformation of pseudokinases and activating catalytically competent protein kinases.

The WNK-regulated SPAK/OSR1 kinases directly phosphorylate and inhibit the K + -Cl − co-transporters

Precise homoeostasis of the intracellular concentration of Cl− is achieved via the co-ordinated activities of the Cl− influx and efflux. We demonstrate that the WNK (WNK lysine-deficient protein kinase)-activated SPAK (SPS1-related proline/alanine-rich kinase)/OSR1 (oxidative stress-responsive kinase 1) known to directly phosphorylate and stimulate the N[K]CCs (Na+–K+ ion co-transporters), also promote inhibition of the KCCs (K+–Cl− co-transporters) by directly phosphorylating a recently described C-terminal threonine residue conserved in all KCC isoforms [Site-2 (Thr1048)]. First, we demonstrate that SPAK and OSR1, in the presence of the MO25 regulatory subunit, robustly phosphorylates all KCC isoforms at Site-2 in vitro. Secondly, STOCK1S-50699, a WNK pathway inhibitor, suppresses SPAK/OSR1 activation and KCC3A Site-2 phosphorylation with similar efficiency. Thirdly, in ES (embryonic stem) cells lacking SPAK/OSR1 activity, endogenous phosphorylation of KCC isoforms at Site-2 is abolished and these cells display elevated basal activity of 86Rb+ uptake that was not markedly stimulated further by hypotonic high K+ conditions, consistent with KCC3A activation. Fourthly, a tight correlation exists between SPAK/OSR1 activity and the magnitude of KCC3A Site-2 phosphorylation. Lastly, a Site-2 alanine KCC3A mutant preventing SPAK/OSR1 phosphorylation exhibits increased activity. We also observe that KCCs are directly phosphorylated by SPAK/OSR1, at a novel Site-3 (Thr5 in KCC1/KCC3 and Thr6 in KCC2/KCC4), and a previously recognized KCC3-specific residue, Site-4 (Ser96). These data demonstrate that the WNK-regulated SPAK/OSR1 kinases directly phosphorylate the N[K]CCs and KCCs, promoting their stimulation and inhibition respectively. Given these reciprocal actions with anticipated net effects of increasing Cl− influx, we propose that the targeting of WNK–SPAK/OSR1 with kinase inhibitors might be a novel potent strategy to enhance cellular Cl− extrusion, with potential implications for the therapeutic modulation of epithelial and neuronal ion transport in human disease states.

WNK Kinases, Renal Ion Transport and Hypertension

Two members of a recently discovered family of protein kinases are the cause of an inherited disease known as pseudohypoaldosteronism type II (PHAII). These patients exhibit arterial hypertension together with hyperkalemia and metabolic acidosis. This is a mirror image of Gitelman disease that is due to inactivating mutations of the SLC12A3 gene that encodes the thiazide-sensitive Na+:Cl– cotransporter. The uncovered genes causing PHAII encode for serine/threonine kinases known as WNK1 and WNK4. Physiological and biochemical studies have revealed that WNK1 and WNK4 modulate the activity of several transport pathways of the aldosterone-sensitive distal nephron, thus increasing our understanding of how diverse renal ion transport proteins are coordinated to regulate normal blood pressure levels. Observations discussed in the present work place WNK1 and WNK4 as genes involved in the genesis of essential hypertension and as potential targets for the development of antihypertensive drugs.

N-terminal Serine Dephosphorylation Is Required for KCC3 Cotransporter Full Activation by Cell Swelling

Background: KCC3 lacking the two known phosphorylation sites is still regulated by cell swelling and WNK3. Results: Dephosphorylation of serine 96 is necessary for full activation of the cotransporter. Conclusion: Serine 96 is a third phospho-site involved in KCC3 regulation. Significance: The finding of new phosphorylation sites sheds light on an increasingly complex regulation of K+:Cl− cotransporters. The K+:Cl− cotransporter (KCC) activity is modulated by phosphorylation/dephosphorylation processes. In isotonic conditions, KCCs are inactive and phosphorylated, whereas hypotonicity promotes their dephosphorylation and activation. Two phosphorylation sites (Thr-991 and Thr-1048) in KCC3 have been found to be critical for its regulation. However, here we show that the double mutant KCC3-T991A/T1048A could be further activated by hypotonicity, suggesting that additional phosphorylation site(s) are involved. We observed that in vitro activated STE20/SPS1-related proline/alanine-rich kinase (SPAK) complexed to its regulatory MO25 subunit phosphorylated KCC3 at Ser-96 and that in Xenopus laevis oocytes Ser-96 of human KCC3 is phosphorylated in isotonic conditions and becomes dephosphorylated during incubation in hypotonicity, leading to a dramatic increase in KCC3 function. Additionally, WNK3, which inhibits the activity of KCC3, promoted phosphorylation of Ser-96 as well as Thr-991 and Thr-1048. These observations were corroborated in HEK293 cells stably transfected with WNK3. Mutation of Ser-96 alone (KCC3-S96A) had no effect on the activity of the cotransporter when compared with wild type KCC3. However, when compared with the double mutant KCC3-T991A/T1048A, the triple mutant KCC3-S96A/T991A/T1048A activity in isotonic conditions was significantly higher, and it was not further increased by hypotonicity or inhibited by WNK3. We conclude that serine residue 96 of human KCC3 is a third site that has to be dephosphorylated for full activation of the cotransporter during hypotonicity.

Functional kinomics establishes a critical node of volume-sensitive cation-Cl− cotransporter regulation in the mammalian brain

Cell volume homeostasis requires the dynamically regulated transport of ions across the plasmalemma. While the ensemble of ion transport proteins involved in cell volume regulation is well established, the molecular coordinators of their activities remain poorly characterized. We utilized a functional kinomics approach including a kinome-wide siRNA-phosphoproteomic screen, a high-content kinase inhibitor screen, and a kinase trapping-Orbitrap mass spectroscopy screen to systematically identify essential kinase regulators of KCC3 Thr991/Thr1048 phosphorylation – a key signaling event in cell swelling-induced regulatory volume decrease (RVD). In the mammalian brain, we found the Cl−-sensitive WNK3-SPAK kinase complex, required for cell shrinkage-induced regulatory volume decrease (RVI) via the stimulatory phosphorylation of NKCC1 (Thr203/Thr207/Thr212), is also essential for the inhibitory phosphorylation of KCC3 (Thr991/Thr1048). This is mediated in vivo by an interaction between the CCT domain in SPAK and RFXV/I domains in WNK3 and NKCC1/KCC3. Accordingly, genetic or pharmacologic WNK3-SPAK inhibition prevents cell swelling in response to osmotic stress and ameliorates post-ischemic brain swelling through a simultaneous inhibition of NKCC1-mediated Cl− uptake and stimulation of KCC3-mediated Cl− extrusion. We conclude that WNK3-SPAK is an integral component of the long-sought “Cl−/volume-sensitive kinase” of the cation-Cl− cotransporters, and functions as a molecular rheostat of cell volume in the mammalian brain.

Role of WNK Kinases in the Modulation of Cell Volume

Ion Transport across the cell membrane is required to maintain cell volume homeostasis. In response to changes in extracellular osmolarity, most cells activate specific metabolic or membrane-transport pathways to respond to cell swelling or shrinkage and return their volume to its normal resting state. This process involves the rapid adjustment of the activities of channels and transporters that mediate flux of K+, Na+, Cl-, and small organic osmolytes. Cation chloride cotransporters (CCCs) NKCCs and KCCs are a family of membrane proteins modulated by changes in cell volume and/or in the intracellular chloride concentration ([Cl-]i). Cell swelling triggers regulatory volume decrease (RVD), promoting solute and water efflux to restore normal cell volume. Swelling-activated KCCs mediate RVD in most cell types. In contrast, cell shrinkage triggers regulatory volume increase (RVI), which involves the activation of the NKCC1 cotransporter of the CCC family. Regulation of the CCCs during RVI and RVD by protein phosphorylation is a well-characterized mechanism, where WNK kinases and their downstream kinase substrates, SPAK and OSR1 constitute the essential phospho-regulators. WNKs-SPAK/OSR1-CCCs complex is required to regulate cell shrinkage-induced RVI or cell swelling-induced RVD via activating or inhibitory phosphorylation of NKCCs or KCCs, respectively. WNK1 and WNK4 kinases have been established as [Cl-]i sensors/regulators, while a role for WNK3 kinase as a cell volume-sensing kinase has emerged and is proposed in this chapter.

Mexneurin is a novel precursor of peptides in the central nervous system of rodents

Endomorphins (EMs) have been proposed as the endogenous ligand agonists of the μ‐opioid receptor; however, no propeptide precursor protein for EMs has been identified. Here, to identify the presumed precursor of EMs, we designed an immunoscreening assay using specific affinity‐purified rabbit antisera raised against synthetic EMs in a whole‐mouse brain cDNA library. Following this approach, we identify a DNA sequence encoding a protein precursor, which we name proMexneurin, that contains three different peptide sequences: Mexneurin‐1 (an EM‐like peptide), Mexneurin‐2, and Mexneurin‐3, a peptide which appears to be unrelated to EMs. RT‐PCR analysis and in situ hybridization reveal a widespread distribution of proMexneurin mRNA throughout the mouse brain. Both Mexneurin‐1 and Mexneurin‐3 peptides display biological activities in the mouse CNS.