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

Abstract

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.

KCC2 activity is critical in limiting the onset and severity of status epilepticus

“…OKA limits NMDAR-dependent PP1-mediated dephosphorylation of S940, and our results demonstrated that the ability of OKA to potentiate KCC2 activity was directly mediated by this residue. Presumably, the inhibitory effects of OKA in the absence of S940 may reflect enhanced phosphorylation of other residues in KCC2 such as T906 and T1007, which have been postulated to cause transporter inactivation (28,29). Of note, S940 is not conserved in the other KCCs that are inhibited by OKA under basal conditions (30), suggesting a unique role for this residue in regulating neuronal Cl -homeostasis.…”

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

“…The total and phospho-specific KCC3a antibodies for KCC3a and SPAK/OSR1 used in this study were previously described and validated (11,36). Polyclonal anti-c-Myc antibody was used to identify L-WNK1 (Sigma).…”

“…Then, 25 g of protein were resolved in 7.5% SDS-PAGE and transferred to PVDF membranes. In both cases, membranes were blocked with TBST (Tris-buffered saline/Tween 20, in mM: 100 Tris·HCl, 150 NaCl, and 0.2% Tween 20, pH 7.5) containing 5% (wt/vol) of nonfat dry milk and incubated overnight at 4°C, with anti-KCC3a phospho-specific antibodies T991 and T1048 (2 g/ml plus 2 l/ml nonphosphopeptide) (11,36), polyclonal anti-cMyc (1:1,000; Sigma), anti-␤ actin (1:2,500; Santa Cruz Biotechnology), or anti-GAPDH (1:10,000; Sigma). After further being washed, blots were incubated with (horseradish peroxidase) conjugated anti-sheep secondary antibody (1:5,000; Sigma) for 1 h at room temperature.…”

“…Therefore, L-WNK1 could inhibit the cotransporter by inducing its phosphorylation. To test this hypothesis, we used previously generated and validated phospho-specific antibodies that recognize KCC3a at the phospho-residues Thr991 and Thr1048 (11,35,36) and SPAK/OSR1 at the phospho-residue Ser373/Ser325, respectively (11). These residues, as well as the corresponding to the endogenous SPAK/OSR1 kinase, are phosphorylated in Xenopus oocytes maintained in isotonic conditions, in which KCC3 is inactive.…”

From nephron to neuron: an exciting journey in search of a cure for epilepsy

“…SPAK/OSR1 act in combination with the WNK (With No lysine = K) type kinases (WNK1-4). SPAK/OSR1 and WNK act as intracellular Cl -sensors reducing their activity when Cl -concentration decreases, thus closing the regulatory loop [19][20]. The kinases involved in this complex regulation of Cl-intracellular concentration at the nephron level, may represent not only new therapeutic targets for kidney dysfunction, but also a new field for research in epilepsy.…”