With-no-lysine Kinase Research Articles

WNK4 Diverts the Thiazide-sensitive NaCl Cotransporter to the Lysosome and Stimulates AP-3 Interaction

With-no-lysine kinase 4 (WNK4) inhibits electroneutral sodium chloride reabsorption by attenuating the cell surface expression of the thiazide-sensitive NaCl cotransporter (NCC). The underlying mechanism for this effect remains poorly understood. Here, we explore how WNK4 affects the trafficking of NCC through its interactions with intracellular sorting machinery. An analysis of NCC cell surface lifetime showed that WNK4 did not alter the net rate of cotransporter internalization. In contrast, direct measurements of forward trafficking revealed that WNK4 attenuated the rate of NCC surface delivery, inhibiting the anterograde movement of cotransporters traveling to the plasma membrane from the trans-Golgi network. The response was paralleled by a dramatic reduction in NCC protein abundance, an effect that was sensitive to the lysosomal protease inhibitor leupeptin, insensitive to proteasome inhibition, and attenuated by endogenous WNK4 knockdown. Subcellular localization studies performed in the presence of leupeptin revealed that WNK4 enhanced the accumulation of NCC in lysosomes. Moreover, NCC immunoprecipitated with endogenous AP-3 complexes, and WNK4 increased the fraction of cotransporters that associate with this adaptor, which facilitates cargo transport to lysosomes. WNK4 expression also increased LAMP-2-positive lysosomal content, indicating that the kinase may act by a general AP-3-dependent mechanism to promote cargo delivery into the lysosomal pathway. Taken together, these findings indicate that WNK4 inhibits NCC activity by diverting the cotransporter to the lysosome for degradation by way of an AP-3 transport carrier.

Gitelman Syndrome

Gitelman Syndrome

Domain swapping in the kinase superfamily: OSR1 joins the mix

Domain swapping in the kinase superfamily: OSR1 joins the mix

Regulation of potassium (K) handling in the renal collecting duct.

This review provides an overview of the molecular mechanisms of K transport in the mammalian connecting tubule (CNT) and cortical collecting duct (CCD), both nephron segments responsible for the regulation of renal K secretion. Aldosterone and dietary K intake are two of the most important factors regulating K secretion in the CNT and CCD. Recently, angiotensin II (AngII) has also been shown to play a role in the regulation of K secretion. In addition, genetic and molecular biological approaches have further identified new mechanisms by which aldosterone and dietary K intake regulate K transport. Thus, the interaction between serum-glucocorticoid-induced kinase 1 (SGK1) and with-no-lysine kinase 4 (WNK4) plays a significant role in mediating the effect of aldosterone on ROMK (Kir1.1), an important apical K channel modulating K secretion. Recent evidence suggests that WNK1, mitogen-activated protein kinases such as P38, ERK, and Src family protein tyrosine kinase are involved in mediating the effect of low K intake on apical K secretory channels.

Upregulation of human with-no-lysine kinase-4 gene expression by GATA-1 acetylation

With-no-lysine kinase-4 (WNK4), a member of the serine–threonine protein kinase family, acts as a multifunctional regulator of diverse ion transporters. Therefore, it is interesting to investigate the mechanisms that control its expression. We have previously demonstrated that glucocorticoid downregulates human WNK4 (hWNK4) expression through the negative glucocorticoid responsive element. Here, using real-time PCR and Western blot assays, we show that trichostatin A (TSA), a histone deacetylase inhibitor, upregulated hWNK4 mRNA and protein expression in human embryo kidney 293 cells. Analysis of the transcriptional activity of a series of the truncated hWNK4 promoters by luciferase assay indicated that the region −484 to −337 of the hWNK4 promoter was sensitive to TSA, and a GATA-1 binding motif was identified at position −426 using TRANSFAC-TESS program. Moreover, using electrophoresis mobility shift assay and chromatin immunoprecipitation assay, the GATA-1 binding affinity to the hWNK4 promoter was shown to increase with TSA under in vitro and in vivo conditions. Immunoprecipitation and Western blot analyses showed that the levels of acetylated GATA-1 were increased with TSA, in agreement with changes in its DNA-binding affinity. These findings indicate that TSA induces hWNK4 expression, at least in part, by increasing GATA-1 acetylation, and thereby its binding to the GATA-1 responsive element, within the hWNK4 promoter.

The plant WNK gene family and regulation of flowering time in Arabidopsis

The WNK (with no lysine kinase) protein kinase gene family may be involved in regulating ion homeostasis and other physiological processes in mammals. WNK-like genes have also been identified in plants, including nine in Arabidopsis, designated AtWNK1-AtWNK9. However, it is not clear if there are further plant WNK genes, and what the evolutionary relationships are among these genes, nor if these genes have functions other than their roles in regulating circadian rhythms and vacuolar H(+)-ATPase. In the present study, we found a tenth Arabidopsis WNK gene, designated AtWNK10. Further phylogenetic analysis of Arabidopsis and rice WNK genes suggests that the most recent common ancestor of monocots and eudicots had 4-8 WNK genes, and that the WNK gene family has experienced duplication events and possible gene losses. Semi-quantitative RT-PCR revealed that all Arabidopsis WNK genes except AtWNK6 are expressed in organs from the seedling to the flowering plant. T-DNA knockout mutations in the AtWNK2, AtWNK5 and AtWNK8 genes in different phylogenetic clades caused early flowering. In contrast, a T-DNA knockout wnk1 mutant had a much delayed flowering time. In addition, the transcript levels of several genes in the photoperiod pathway for flowering, such as ELF4, TOC1, CO and FT, were altered in atwnk mutants. Taken together, our results suggest that the Arabidopsis WNK gene family regulates flowering time by modulating the photoperiod pathway.

Cotransporters, WNKs and hypertension: an update

Studies of inherited conditions characterized by high or low blood pressure reveal the importance of a new signalling cascade, With no Lysine kinases (WNK) --> ste20/SPS1-related proline/alanine-rich kinase (SPAK)/oxidative stress-responsive kinase-1 (OSR1) --> Cation-Chloride Cotransporters (CCC), in regulating blood pressure and in the pathogenesis of essential hypertension. This review explores how these molecules interact to co-ordinate sodium homeostasis and how errors in these interactions may result in hypertension. Studies using transgenic animals and gene knockins have clarified the role of mutant WNK4 in hypertension, by revealing its main action to be increasing the expression and activity of sodium-chloride cotransporter (NCC) in the kidney. Functional studies show how phosphorylation of WNK1 regulates both its activity and ability to interact with SPAK/OSR1, and clearly place it upstream of SPAK/OSR1 in the cascade. The structural basis for the interactions between SPAK/OSR1 and targets has been identified. WNKs, activated by upstream kinases or autophosphorylation, bind and phosphorylate SPAK/OSR1, which in turn phosphorylate and activate NCCs and Na-K-Cl cotransporters (NKCCs). This increases sodium retention in the kidney (NKCC2, NCC) and vascular resistance (NKCC1), but decreases renin release (NKCC1). Hypertension-associated mutant WNKs increase surface expression and activation of renal tubular NKCC2 and NCC. Whether this adequately explains the hypertension awaits studies of these mutants in other tissues.

WNK1 Affects Surface Expression of the ROMK Potassium Channel Independent of WNK4

The WNK (with no lysine kinase) kinases are a novel class of serine/threonine kinases that lack a characteristic lysine residue for ATP docking. Both WNK1 and WNK4 are expressed in the mammalian kidney, and mutations in either can cause the rare familial syndrome of hypertension and hyperkalemia (Gordon syndrome, or pseudohypoaldosteronism type 2). The molecular basis for the action of WNK4 is through alteration in the membrane expression of the NaCl co-transporter (NCCT) and the renal outer-medullary K channel KCNJ1 (ROMK). The actions of WNK1 are less well defined, and evidence to date suggests that it can affect NCCT expression but only in the presence of WNK4. The results of co-expressing WNK1 with ROMK in Xenopus oocytes are reported for the first time. These studies show that WNK1 is able to suppress total current directly through ROMK by causing a marked reduction in its surface expression. The effect is mimicked by a kinase-dead mutant of WNK1 (368D > A), suggesting that it is not dependent on its catalytic activity. Study of the time course of ROMK expression further suggests that WNK1 accelerates trafficking of ROMK from the membrane, and this effect seems to be dynamin dependent. Using fragments of full-length WNK1, it also is shown that the effect depends on residues in the middle section of the protein (502 to 1100 WNK1) that contains the acidic motif. Together, these findings emphasize that the molecular mechanisms that underpin WNK1 regulation of ROMK expression are distinct from those that affect NCCT expression.

Characterization of SPAK and OSR1, Regulatory Kinases of the Na-K-2Cl Cotransporter

Our recent studies demonstrate that SPAK (Ste20p-related Proline Alanine-rich Kinase), in combination with WNK4 [With No lysine (K) kinase], phosphorylates and stimulates the Na-K-2Cl cotransporter (NKCC1), whereas catalytically inactive SPAK (K104R) fails to activate the cotransporter. The catalytic domain of SPAK contains an activation loop between the well-conserved DFG and APE motifs. We speculated that four threonine residues (T231, T236, T243, and T247) in the activation loop might be sites of phosphorylation and kinase activation; therefore, we mutated each residue into an alanine. In this report, we demonstrate that coexpression of SPAK (T243A) or SPAK (T247A) with WNK4 not only prevented, but robustly inhibited, cotransporter activity in NKCC1-injected Xenopus laevis oocytes. These activation loop mutations produced an effect similar to that of the SPAK (K104R) mutant. In vitro phosphorylation experiments demonstrate that both intramolecular autophosphorylation of SPAK and phosphorylation of NKCC1 are significantly stronger in the presence of Mn2+ rather than Mg2+. We also show that SPAK activity is markedly inhibited by staurosporine and K252a, partially inhibited by N-ethylmaleimide and diamide, and unaffected by arsenite. OSR1, a kinase closely related to SPAK, exhibited similar kinase properties and similar functional activation of NKCC1 when coexpressed with WNK4.