Lysine Kinase Research Articles

Apoptosis-associated tyrosine kinase scaffolding of protein phosphatase 1 and SPAK reveals a novel pathway for Na-K-2C1 cotransporter regulation

Previous work from our laboratory and others has established that Ste-20-related proline-alanine-rich kinase (SPAK/PASK) is central to the regulation of NKCC1 function. With no lysine (K) kinase (WNK4) has also been implicated in the regulation of NKCC1 activity through upstream activation of SPAK. Because previous studies from our laboratory also demonstrated a protein-protein interaction between SPAK and apoptosis-associated tyrosine kinase (AATYK), we explore here the possibility that AATYK is another component of the regulation of NKCC1. Heterologous expression of AATYK1 in NKCC1-injected Xenopus laevis oocytes markedly inhibited cotransporter activity under isosmotic conditions. Interestingly, mutation of key residues in the catalytic domain of AATYK1 revealed that the kinase activity does not play a role in the suppression of NKCC1 function. However, mutagenesis of the two SPAK-binding motifs in AATYK1 completely abrogated this effect. As protein phosphatase 1 (PP1) also plays a central role in the dephosphorylation and inactivation of NKCC1, we investigated the possibility that AATYK1 interacts with the phosphatase. We identified a PP1 docking motif in AATYK1 and demonstrated interaction using yeast-2-hybrid analysis. Mutation of a key valine residue (V1175) within this motif prevented protein-protein interaction. Furthermore, the physical interaction between PP1 and AATYK was required for inhibition of NKCC1 activity in Xenopus laevis oocytes. Taken together, our data are consistent with AATYK1 indirectly inhibiting the SPAK/WNK4 activation of the cotransporter by scaffolding an inhibitory phosphatase in proximity to a stimulatory kinase.

Cotransporters, WNKs and hypertension: important leads from the study of monogenetic disorders of blood pressure regulation

Major advances are being made in identifying the structure and behaviour of regulatory cascades that control the activity of cation-Cl(-) cotransporters and certain Na(+), K(+) and Cl(-) channels. These transporters play key roles in regulating arterial blood pressure as they are not only responsible for NaCl reabsorption in the thick ascending limb and distal tubule of the kidney, but are also involved in regulating smooth muscle Ca(2+) levels. It is now apparent that defects in these transporters, and particularly in the regulatory cascades, cause some monogenetic forms of hypertension and may contribute to essential hypertension and problems with K(+) homoeostasis. Two families of kinases are prominent in these processes: the Ste-20-related kinases [OSR1 (oxidative stress-responsive kinase 1) and SPAK (Ste20/SPS1-related proline/alanine-rich kinase)] and the WNKs [with no lysine kinases]. These kinases affect the behaviour of their targets through both phosphorylation and by acting as scaffolding proteins, bringing together regulatory complexes. This review analyses how these kinases affect transport by activating or inhibiting individual transporters at the cell surface, or by changing the surface density of transporters by altering the rate of insertion or removal of transporters from the cell surface, and perhaps through controlling the rate of transporter degradation. This new knowledge should not only help us target antihypertensive therapy more appropriately, but could also provide the basis for developing new therapeutic approaches to essential hypertension.

WNK4 Kinase is a Negative Regulator of K + ‐Cl − Cotransporters

WNK kinases (with no lysine kinase(K)) are regulators of several membrane transport proteins in which these kinases act as a molecular switches that coordinate the activity of several players. Members of the Solute Carrier Family number 12 are one of the main targets. WNK3 activates the Na+-driven NCC, NKCC1, and NKCC2 and inhibits the K+-driven 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 upon 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 by 50 to 60% 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. WNK4 effect upon K+-Cl− cotransporters is not affected by co-injection with wild type SPAK cRNA or its catalytically inactive form (SPAK-K104R). In contrast, 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 KCC3 activation in isotonic conditions. This effect is enhanced by the presence of catalytically inactive SPAK and was not present in KCC1 and KCC4. Our results reveal that WNK4 regulates the activity of the K+-Cl− cotransporters.

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

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.

Regulation of the expression of the Na/Cl cotransporter by WNK4 and WNK1: evidence that accelerated dynamin-dependent endocytosis is not involved

The novel serine/threonine kinases (with no lysine kinases or WNKs), WNK1 and WNK4, are encoded by the disease genes for Gordon syndrome (PRKWNK1 and PRKWNK4), a rare monogenic syndrome of hypertension and hyperkalemia. These proteins alter the expression of the thiazide-sensitive Na/Cl cotransporter (NCCT) in Xenopus laevis oocytes, although the details are controversial. We describe here our own experience and confirm that kinase-dead WNK4 (318D>A) is unable to affect Na+ fluxes through the thiazide-sensitive Na/Cl transporter (NCCT) or its membrane expression as an ECFP-NCCT fusion protein. However, the kinase domain is not sufficient for a functional WNK4 since deletion of the acidic motif (a motif unique to WNK family members) completely abolishes functional activity. Indeed, the NH2 terminal of WNK4 (1-620) containing the kinase domain and acidic motif retains full activity, but does not interact directly with NCCT in pull-down assays. Coexpression of WNK1 antagonizes the action of WNK4, and kinase-dead WNK1 (368D>A) or WNK1 carrying a WNK4 disease mutation (565Q>E) behaves in the same way as wild-type WNK1. This suggests kinase activity and charge conservation within the acidic motif are not essential for the WNK1-WNK4 interaction. We also report that WNK4 probably reduces surface expression largely through an effect on forward trafficking. Hence, the effect of WNK4 on NCCT expression is mimicked by dynamin, but the dominant-negative K44A dynamin mutant does not block the action of WNK4 itself. These results further highlight important differences in the mechanism by which WNK kinases affect expression of NCCT vs. other membrane proteins such as ROMK.

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.

Autophosphorylation of SPAK and OSR1 are required for Na‐K‐2Cl cotransporter activation

Hormonal, oxidative, and osmotic stimuli activate the Na-K-2Cl cotransporter, NKCC1, through phosphorylation. Our recent in vivo studies demonstrate that co-expression of SPAK (Ste20p related Proline Alanine rich Kinase), and WNK4 (With No lysine (K) kinase)with the Na-K-2Cl cotransporter (NKCC1) in Xenopus laevis oocytes, phosphorylates and stimulates NKCC1, whereas catalytically-inactive SPAK (K104R) fails to activate the cotransporter. In this study, we identified four threonine residues (T231, T236, T243, and T247) within the activation loop between the well-conserved DFG and APE motifs, which we speculated might be sites of phosphorylation and kinase activation, therefore we mutated each residue into an alanine. Co-expression 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 a similar effect to that of the catalytically-inactive SPAK (K104R) mutant. In vitro phosphorylation experiments demonstrate that both intramolecular autophosphorylation of SPAK and phosphorylation of NKCC1 is significantly stronger in the presence of Mn2+, rather than Mg2+. Consistent with the in vivo heterologous expression studies, 32P incorporation was significantly decreased in the T243A mutant and completely absent in the T247A and K104R mutants. We also show that SPAK activity is markedly inhibited by staurosporine and K252a, partially inhibited by N-ethylmaleimide and diamide, and unaffected by arsenite or ML7. OSR1, a kinase closely related to SPAK, exhibited similar kinase properties and similar functional activation of NKCC1 when co-expressed with WNK4. Together, these in vivo and in vitro data confirm that SPAK and OSR1 are kinases that phosphorylate and activate NKCC1.

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.