WNK4 inhibits NCC protein expression through MAPK ERK1/2 signaling pathway

WNK [with no lysine (K)] kinase is a subfamily of serine/threonine kinases. Mutations in two members of this family (WNK1 and WNK4) cause pseudohypoaldosteronism type II featuring hypertension, hyperkalemia, and metabolic acidosis. WNK1 and WNK4 were shown to regulate sodium chloride cotransporter (NCC) activity through phosphorylating SPAK and OSR1. Previous studies including ours have also shown that WNK4 inhibits NCC function and its protein expression. A recent study reported that a phorbol ester inhibits NCC function via activation of extracellular signal-regulated kinase (ERK) 1/2 kinase. In the current study, we investigated whether WNK4 affects NCC via the MAPK ERK1/2 signaling pathway. We found that WNK4 increased ERK1/2 phosphorylation in a dose-dependent manner in mouse distal convoluted tubule (mDCT) cells, whereas WNK4 mutants with the PHA II mutations (E562K and R1185C) lost the ability to increase the ERK1/2 phosphorylation. Hypertonicity significantly increased ERK1/2 phosphorylation in mDCT cells. Knock-down of WNK4 expression by siRNA resulted in a decrease of ERK1/2 phosphorylation. We further showed that WNK4 knock-down significantly increases the cell surface and total NCC protein expressions and ERK1/2 knock-down also significantly increases cell surface and total NCC expression. These data suggest that WNK4 inhibits NCC through activating the MAPK ERK1/2 signaling pathway.     

WNK1 and WNK4 modulate CFTR activity

The cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-gated chloride channel. WNK kinases are widely expressed modulators of ion transport. WNK1 and WNK4, two WNK kinases that are mutated in familial hyperkalemic hypertension (FHHt), are co-expressed with CFTR in several organs, raising the possibility that WNK kinases might alter CFTR activity in vivo or that CFTR could be involved in the pathogenesis of FHHt. Here, we report that WNK1 co-localizes with CFTR protein in pulmonary epithelial cells. Co-expression of WNK1 or WNK4 with CFTR in Xenopus laevis oocytes suppresses chloride channel activity. The effect of WNK4 is dose dependent and occurs, at least in part, by reducing CFTR protein abundance at the plasma membrane. This effect is independent of WNK4 kinase activity. In contrast, the effect of WNK1 on CFTR activity requires intact WNK1 kinase activity. Moreover WNK1 and WNK4 exhibit additive CFTR inhibition. Previous reports suggest that patients with FHHt exhibit mild changes in nasal potential difference that resemble the more severe changes that occur in cystic fibrosis. We report that the FHHt-causing mutant WNK4 Q562E is a more potent inhibitor of CFTR activity than is the wild-type WNK4. Taken together, these results suggest that WNK1 and WNK4 may modulate CFTR activity; they further suggest that WNK kinases may be potential therapeutic targets for cystic fibrosis.     

WNK3 is a putative chloride-sensing kinase

The with-no-lysine kinase 3 (WNK3) is a serine/threonine kinase that modulates the activity of the electroneutral cation-coupled chloride cotransporters (CCC). Using the Xenopus laevis oocyte heterologous expression system, it has been shown that WNK3 activates the Na(+)-coupled chloride cotransporters NKCC1, NKCC2, and NCC and inhibits the K(+)-coupled chloride cotransporters KCC1 through KCC4. Interestingly, the effect of catalytically inactive WNK3 is opposite to that of wild type WNK3: inactive WNK3 inhibits NKCCs and activates KCCs. In doing so, wild type and catalytically inactive WNK3 bypass the tonicity requirement for activation/inhibition of the cotransporter. Thus, WNK3 modulation of the electroneutral cotransporters promotes Cl(-) influx and prevents Cl(-) efflux, thus fitting the profile for a putative "Cl(-)-sensing kinase". Other kinases that potentially have these properties are the Ste20-type kinases, SPAK/OSR1, which become phosphorylated in response to reductions in intracellular chloride concentration and regulate the activity of NKCC1. It has been demonstrated that WNKs lie upstream of SPAK/OSR1 and that the activity of these kinases is activated by phosphorylation of threonines in the T-loop by WNKs. It is possible that a protein phosphatase is also involved in the WNK3 effects on its associated cotransporters because activation of KCCs and inhibition of NKCCs by inactive WNK3 can be prevented by known inhibitors of protein phosphatases, such as calyculin A and cyclosporine, suggesting that a protein phosphatase is also involved in the protein complex.     

Comparison of WNK4 and WNK1 kinase and inhibiting activities

WNK kinases are novel serine/threonine protein kinases. Mutations in two members of the WNK family, WNK1 and WNK4, cause familial hyperkalemic hypertension. These kinases regulate ion transport across diverse epithelia; WNK4 reduces activity of the Na-Cl cotransporter activity and the potassium channel, ROMK, by reducing their appearance at the plasma membrane. We examined the kinase activity of WNK1 and WNK4 in vitro. A glutathione S-transferase (GST) fusion protein of the WNK1 kinse domain phosphorylated itself and a substrate protein, as reported previously. A longer construct, containing the autoinhibitory domain, did not. A GST WNK4 kinase domain construct demonstrated no kinase activity, in vitro or in HEK 293 cells. WNK4 constructs that included a region homologous to the autoinhibitory domain of WNK1 inhibited WNK1 kinase activity. Inhibition by a short WNK4 segment, WNK4 (444-518), was greater than inhibition by WNK4 (444-563). Together, these results suggest that WNK4 must be activated by currently unknown factors to exhibit kinase activity and that WNK4 contains an inhibitory domain that can inhibit the kinase activity of WNK1.     

WNK1, a novel mammalian serine/threonine protein kinase lacking the catalytic lysine in subdomain II

We have cloned and characterized a novel mammalian serine/threonine protein kinase WNK1 (with no lysine (K)) from a rat brain cDNA library. WNK1 has 2126 amino acids and can be detected as a protein of approximately 230 kDa in various cell lines and rat tissues. WNK1 contains a small N-terminal domain followed by the kinase domain and a long C-terminal tail. The WNK1 kinase domain has the greatest similarity to the MEKK protein kinase family. However, overexpression of WNK1 in HEK293 cells exerts no detectable effect on the activity of known, co-transfected mitogen-activated protein kinases, suggesting that it belongs to a distinct pathway. WNK1 phosphorylates the exogenous substrate myelin basic protein as well as itself mostly on serine residues, confirming that it is a serine/threonine protein kinase. The demonstration of activity was striking because WNK1, and its homologs in other organisms lack the invariant catalytic lysine in subdomain II of protein kinases that is crucial for binding to ATP. A model of WNK1 using the structure of cAMP-dependent protein kinase suggests that lysine 233 in kinase subdomain I may provide this function. Mutation of this lysine residue to methionine eliminates WNK1 activity, consistent with the conclusion that it is required for catalysis. This distinct organization of catalytic residues indicates that WNK1 belongs to a novel family of serine/threonine protein kinases.     

Protein phosphatase 1 modulates the inhibitory effect of With-no-Lysine kinase 4 on ROMK channels

With-no-Lysine kinase 4 (WNK4) inhibited ROMK (Kir1.1) channels and the inhibitory effect of WNK4 was abolished by serum-glucocorticoid-induced kinase 1 (SGK1) but restored by c-Src. The aim of the present study is to explore the mechanism by which Src-family tyrosine kinase (SFK) modulates the effect of SGK1 on WNK4 and to test the role of SFK-WNK4-SGK1 interaction in regulating ROMK channels in the kidney. Immunoprecipitation demonstrated that protein phosphatase 1 (PP1) binds to WNK4 at amino acid (aa) residues 695-699 (PP1(#1)) and at aa 1211-1215 (PP1(#2)). WNK4(-PP1#1) and WNK4(-PP1#2), in which the PP1(#1) or PP1(#2) binding site was deleted or mutated, inhibited ROMK channels as potently as WNK4. However, c-Src restored the inhibitory effect of WNK4 but not WNK4(-PP1#1) on ROMK channels in the presence of SGK1. Moreover, expression of c-Src inhibited SGK1-induced phosphorylation of WNK4 but not WNK4(-PP1#1) at serine residue 1196 (Ser(1196)). In contrast, coexpression of c-Src restored the inhibitory effect of WNK4(-PP1#2) on ROMK in the presence of SGK1 and diminished SGK1-induced WNK4 phosphorylation at Ser(1196) in cells transfected with WNK4(-PP1#2). This suggests the possibility that c-Src regulates the interaction between WNK4 and SGK1 through activating PP1 binding to aa 695-9 thereby decreasing WNK4 phosphorylation and restoring the inhibitory effect of WNK4. This mechanism plays a role in suppressing ROMK channel activity during the volume depletion because inhibition of SFK or serine/threonine phosphatases increases ROMK channel activity in the cortical collecting duct of rats on a low-Na diet. We conclude that regulation of phosphatase activity by SFK plays a role in determining the effect of aldosterone on ROMK channels and on renal K secretion.     

Thyroid hormones inhibit the Ca2+ calmodulin-induced activation of myosin light chain kinase

L-Thyroxine (T4) and L-triiodothyronine (T3) specifically, inhibited myosin light chain kinase (MLC-kinase) from various tissues whereas inhibitory effects of T4 and T3 on other protein kinases such as protein kinase C, cAMP-dependent protein kinase, casein kinase I, casein kinase II and calmodulin kinase II were much weaker. T4 was a more potent inhibitor of MLC-kinase than T3. Kinetic studies showed that T4 behaved as a competitive inhibitor of MLC-kinase toward calmodulin (CaM) and that Ki value was 2.5 microM. The activity of the catalytic fragment of MLC-kinase, which is active without CaM, was not inhibited by T4. 125I-T4 gel overlay revealed that CaM did not bind T4 but MLC-kinase had 125I-T4 binding activity. These observations suggest that T4 binds at or near CaM binding domain of MLC-kinase and inhibits CaM-induced activation of MLC-kinase.     

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.The with-no-Lysine (WNK)² kinases are a unique family of serine-threonine protein kinases that regulate ion transport in diverse epithelia (1). In the kidney the gene products of several members of the WNK family, including WNK1, WNK3, and WNK4, converge in a signaling network that coordinates distal nephron sodium chloride and potassium handling. WNK4 participates in this network by suppressing NaCl reabsorption via the thiazide-sensitive NaCl cotransporter (NCC, SLC12A3), and potassium secretion via the potassium channel Kir 1.1 (ROMK) (2, 3). The importance of this signaling pathway is underscored by a link to human disease; WNK4 mutations cause familial hyperkalemic hypertension (pseudohypoaldosteronism type II, Gordon''s syndrome), an autosomal dominant disorder featuring chloride-dependent thiazide-sensitive hypertension and hyperkalemia (4). These mutations release NCC from inhibition, leading to an increase in renal sodium chloride reabsorption and blood pressure (2, 5).Ample evidence demonstrates that WNK4 suppresses NCC activity, at least in part by modulating its cell surface expression. This effect has been observed at steady state in multiple heterologous overexpression systems, including Xenopus oocytes (2, 5, 6), COS-7 cells (7), and polarized Madin-Darby canine kidney cells epithelia (8). More recently, the inhibitory effect of wild type WNK4 on NCC has been verified in vivo. Mice overexpressing wild type WNK4 exhibit a reduced total amount of NCC expressed at the apical surface of the distal convoluted tubule (DCT), coincident with a reduction in DCT cell mass (9). Conversely, knock-in mice bearing a familial hyperkalemic hypertension-causing WNK4 mutation overexpress NCC at the apical surface, leading to chloride-dependent hypertension and hyperkalemia (10).Although the underlying mechanism by which WNK4 regulates NCC trafficking remains unresolved, some clues are available. Two groups have shown that, unlike the effect of WNK4 on ROMK channel activity (3), WNK4-mediated NCC inhibition is not attenuated by a dominant-negative dynamin mutant (6, 7). These observations strongly suggest that the kinase acts via independent mechanisms to modulate the cell surface expression of NCC and ROMK. Cai et al. (7) found that the suppressive effect of WNK4 on NCC was sensitive to vacuolar H+ ATPase inhibition, suggesting that the kinase might promote the trafficking of NCC to a low pH endosomal compartment. However, the precise identity of this compartment and the mechanism by which the cotransporter arrives there remains undefined.In this study we elucidated the mechanism by which WNK4 suppresses NCC surface expression by directly measuring the effect of WNK4 on NCC cell surface lifetime, forward trafficking, subcellular localization, and interactions with intracellular trafficking machinery. Our results show that WNK4 does not affect the net internalization rate of NCC expressed at the cell surface. Instead, WNK4 influences the biosynthetic trafficking of NCC, diverting itinerant cotransporters exiting the trans-Golgi network (TGN) away from the plasma membrane and to the lysosome for degradation. Consistent with this observation, WNK4 enhances the physical association between NCC and the AP-3 adaptor complex, which marks cargo for sorting to lysosomes. Thus, these findings reveal a novel mechanism by which cotransporters destined for the cell surface are instead bypassed directly into the endolysosomal pathway for degradation.     

WNK1 activates ERK5 by an MEKK2/3-dependent mechanism

WNK1 belongs to a unique protein kinase family that lacks the catalytic lysine in its normal position. Mutations in human WNK1 and WNK4 have been implicated in causing a familial form of hypertension. Here we report that overexpression of WNK1 led to increased activity of cotransfected ERK5 in HEK293 cells. ERK5 activation was blocked by the MEK5 inhibitor U0126 and expression of a dominant negative MEK5 mutant. Expression of dominant negative mutants of MEKK2 and MEKK3 also blocked activation of ERK5 by WNK1. Moreover, both MEKK2 and MEKK3 coimmunoprecipitated with endogenous WNK1 from cell lysates. WNK1 phosphorylated both MEKK2 and -3 in vitro, and MEKK3 was activated by WNK1 in 293 cells. Finally, ERK5 activation by epidermal growth factor was attenuated by suppression of WNK1 expression using small interfering RNA. Taken together, these results place WNK1 in the ERK5 MAP kinase pathway upstream of MEKK2/3.     

Regulation of apical localization of the thiazide-sensitive NaCl cotransporter by WNK4 in polarized epithelial cells

Missense mutations in the WNK4 gene have been postulated to cause pseudohypoaldosteronism type II (PHAII), an autosomal-dominant disorder characterized by hyperkalemia and hypertension. Previous reports using Xenopus oocytes showed that wild-type WNK4 expression inhibited surface expression of the thiazide-sensitive NaCl cotransporter (NCC), while a disease-causing mutant lost the inhibitory effect on NCC surface expression. To determine if these changes observed in oocytes really occur in polarized epithelial cells, we generated stable MDCK II cell lines expressing NCC alone or NCC plus wild-type WNK4 or a disease-causing (D564A) WNK4. In contrast to the apical localization of NCC without co-expression of WNK4, immunofluorescence microscopy and biotin surface labeling revealed that this apical localization was equally decreased by both the wild-type and the mutant WNK4 expression. Apical localizations of two PHAII-unrelated apical transporters, sodium-independent amino acid transporter, BAT1 and bile salt export pump, Bsep, were also found to be decreased by both wild-type and mutant WNK4 expression. These results indicate that the regulation of NCC was not related to the disease-causing mutation and not restricted to the PHAII-related specific transporters. The regulation of intracellular localization of NCC by WNK4 might not be involved in the pathogenesis of PHAII.     

WNK3-SPAK interaction is required for the modulation of NCC and other members of the SLC12 family

The serine/threonine with no lysine kinase 3 (WNK3) modulates the activity of the electroneutral cation-coupled chloride cotransporters (CCC) to promote Cl(-) influx and prevent Cl(-) efflux, thus fitting the profile for a putative "Cl(-)-sensing kinase". The Ste20-type kinases, SPAK/OSR1, become phosphorylated in response to reduction in intracellular chloride concentration and regulate the activity of NKCC1. Several studies have now shown that WNKs function upstream of SPAK/OSR1. This study was designed to analyze the role of WNK3-SPAK interaction in the regulation of CCCs with particular emphasis on NCC. In this study we used the functional expression system of Xenopus laevis oocytes to show that different SPAK binding sites in WNK3 ((241, 872, 1336)RFxV) are required for the kinase to have effects on CCCs. WNK3-F1337A no longer activated NKCC2, but the effects on NCC, NKCC1, and KCC4 were preserved. In contrast, the effects of WNK3 on these cotransporters were prevented in WNK3-F242A. The elimination of F873 had no consequence on WNK3 effects. WNK3 promoted NCC phosphorylation at threonine 58, even in the absence of the unique SPAK binding site of NCC, but this effect was abolished in the mutant WNK3-F242A. Thus, our data support the hypothesis that the effects of WNK3 upon NCC and other CCCs require the interaction and activation of the SPAK kinase. The effect is dependent on one of the three binding sites for SPAK that are present in WNK3, but not on the SPAK binding sites on the CCCs, which suggests that WNK3 is capable of binding both SPAK and CCCs to promote their phosphorylation.     

JAK3 inhibitor VI is a mutant specific inhibitor for epidermal growth factor receptor with the gatekeeper mutation T790M

AIM: To identify non-quinazoline kinase inhibitors effective against drug resistant mutants of epidermal growth factor receptor (EGFR).METHODS: A kinase inhibitor library was subjected to screening for specific inhibition pertaining to the in vitro kinase activation of EGFR with the gatekeeper mutation T790M, which is resistant to small molecular weight tyrosine kinase inhibitors (TKIs) for EGFR in non-small cell lung cancers (NSCLCs). This inhibitory effect was confirmed by measuring autophosphorylation of EGFR T790M/L858R in NCI-H1975 cells, an NSCLC cell line harboring the gatekeeper mutation. The effects of a candidate compound, Janus kinase 3 (JAK3) inhibitor VI, on cell proliferation were evaluated using the MTT assay and were compared between T790M-positive and -negative lung cancer cell lines. JAK3 inhibitor VI was modeled into the ATP-binding pocket of EGFR T790M/L858R. Potential physical interactions between the compound and kinase domains of wild-type (WT) or mutant EGFRs or JAK3 were estimated by calculating binding energy. The gatekeeper residues of EGFRs and JAKs were aligned to discuss the similarities among EGFR T790M and JAKs.RESULTS: We found that JAK3 inhibitor VI, a known inhibitor for JAK3 tyrosine kinase, selectively inhibits EGFR T790M/L858R, but has weaker inhibitory effects on the WT EGFR in vitro. JAK3 inhibitor VI also specifically reduced autophosphorylation of EGFR T790M/L858R in NCI-H1975 cells upon EGF stimulation, but did not show the inhibitory effect on WT EGFR in A431 cells. Furthermore, JAK3 inhibitor VI suppressed the proliferation of NCI-H1975 cells, but showed limited inhibitory effects on the WT EGFR-expressing cell lines A431 and A549. A docking simulation between JAK3 inhibitor VI and the ATP-binding pocket of EGFR T790M/L858R predicted a potential binding status with hydrogen bonds. Estimated binding energy of JAK3 inhibitor VI to EGFR T790M/L858R was more stable than its binding energy to the WT EGFR. Amino acid sequence alignments revealed that the gatekeeper residues of JAK family kinases are methionine in WT, similar to EGFR T790M, suggesting that TKIs for JAKs may also be effective for EGFR T790M.CONCLUSION: Our findings demonstrate that JAK3 inhibitor VI is a gatekeeper mutant selective TKI and offer a strategy to search for new EGFR T790M inhibitors.     

Taurine inhibits K+-Cl- cotransporter KCC2 to regulate embryonic Cl- homeostasis via with-no-lysine (WNK) protein kinase signaling pathway

GABA inhibits mature neurons and conversely excites immature neurons due to lower K(+)-Cl(-) cotransporter 2 (KCC2) expression. We observed that ectopically expressed KCC2 in embryonic cerebral cortices was not active; however, KCC2 functioned in newborns. In vitro studies revealed that taurine increased KCC2 inactivation in a phosphorylation-dependent manner. When Thr-906 and Thr-1007 residues in KCC2 were substituted with Ala (KCC2T906A/T1007A), KCC2 activity was facilitated, and the inhibitory effect of taurine was not observed. Exogenous taurine activated the with-no-lysine protein kinase 1 (WNK1) and downstream STE20/SPS1-related proline/alanine-rich kinase (SPAK)/oxidative stress response 1 (OSR1), and overexpression of active WNK1 resulted in KCC2 inhibition in the absence of taurine. Phosphorylation of SPAK was consistently higher in embryonic brains compared with that of neonatal brains and down-regulated by a taurine transporter inhibitor in vivo. Furthermore, cerebral radial migration was perturbed by a taurine-insensitive form of KCC2, KCC2T906A/T1007A, which may be regulated by WNK-SPAK/OSR1 signaling. Thus, taurine and WNK-SPAK/OSR1 signaling may contribute to embryonic neuronal Cl(-) homeostasis, which is required for normal brain development.     

Properties of WNK1 and implications for other family members

WNKs are large serine/threonine protein kinases structurally distinct from all other members of the protein kinase superfamily. Of the four human WNK family members, WNK1 and WNK4 have been linked to a hereditary form of hypertension, pseudohypoaldosteronism type II. We characterized the biochemical properties and regulation of WNK1 that may contribute to its physiological activities and abnormal function in disease. We showed that WNK1 is activated by hypertonic stress in kidney epithelial cells and in breast and colon cancer cell lines. In addition, hypotonic stress also led to a modest increase in WNK1 activity. Gel filtration suggested that WNK1 exists as a tetramer, and yeast two-hybrid data showed that the N terminus of WNK1 (residues 1-222) interacts with residues 481-660, which includes the WNK1 autoinhibitory domain and a C-terminal coiled-coil domain. Although cell biological studies have suggested a functional interaction between WNK1 and WNK4, we found no evidence of stable interactions between these kinases. However, WNK1 phosphorylated both WNK4 and WNK2. In addition, the WNK1 autoinhibitory domain inhibited the catalytic activity of these WNKs. These findings suggest potential mechanisms for interconnected regulation of WNK family members.     

The protein inhibitor of calcium-dependent proteases: purification from bovine heart and possible mechanisms of regulation

A bovine heart protein which specifically inhibits calcium-dependent proteases has been purified to near homogeneity. The purified inhibitor had a Stokes radius of 6.8 nm estimated by gel filtration and a molecular weight of 145,000 estimated by sodium dodecyl sulfate-gel electrophoresis. There is evidence that it may be a glycoprotein. The inhibitor could be phosphorylated by bovine heart cyclic AMP-dependent protein kinase, and its inhibitory effect on Peak II (high-calcium-requiring) protease was modestly increased. However, no other phosphorylating or dephosphorylating conditions significantly influenced its activity. The inhibitor was not hydrolyzed by calcium-dependent proteases, but it was very sensitive to proteolytic inactivation by trypsin or proteases present in a lysosomal fraction from rat heart. Thus, proteolysis may represent a mechanism for decreasing the activity of the inhibitor in different physiologic or pathologic conditions.     

Protein kinase WNK1 promotes cell surface expression of glucose transporter GLUT1 by regulating a Tre-2/USP6-BUB2-Cdc16 domain family member 4 (TBC1D4)-Rab8A complex

One mechanism by which mammalian cells regulate the uptake of glucose is the number of glucose transporter proteins (GLUT) present at the plasma membrane. In insulin-responsive cells types, GLUT4 is released from intracellular stores through inactivation of the Rab GTPase activating protein Tre-2/USP6-BUB2-Cdc16 domain family member 4 (TBC1D4) (also known as AS160). Here we describe that TBC1D4 forms a protein complex with protein kinase WNK1 in human embryonic kidney (HEK293) cells. We show that WNK1 phosphorylates TBC1D4 in vitro and that the expression levels of WNK1 in these cells regulate surface expression of the constitutive glucose transporter GLUT1. WNK1 was found to increase the binding of TBC1D4 to regulatory 14-3-3 proteins while reducing its interaction with the exocytic small GTPase Rab8A. These effects were dependent on the catalytic activity because expression of a kinase-dead WNK1 mutant had no effect on binding of 14-3-3 and Rab8A, or on surface GLUT1 levels. Together, the data describe a pathway regulating constitutive glucose uptake via GLUT1, the expression level of which is related to several human diseases.     

Antagonistic regulation of cystic fibrosis transmembrane conductance regulator cell surface expression by protein kinases WNK4 and spleen tyrosine kinase

Members of the WNK (with-no-lysine [K]) subfamily of protein kinases regulate various ion channels involved in sodium, potassium, and chloride homeostasis by either inducing their phosphorylation or regulating the number of channel proteins expressed at the cell surface. Here, we describe findings demonstrating that the cell surface expression of the cystic fibrosis transmembrane conductance regulator (CFTR) is also regulated by WNK4 in mammalian cells. This effect of WNK4 is independent of the presence of kinase and involves interaction with and inhibition of spleen tyrosine kinase (Syk), which phosphorylates Tyr512 in the first nucleotide-binding domain 1 (NBD1) of CFTR. Transfection of catalytically active Syk into CFTR-expressing baby hamster kidney cells reduces the cell surface expression of CFTR, whereas that of WNK4 promotes it. This is shown by biotinylation of cell surface proteins, immunofluorescence microscopy, and functional efflux assays. Mutation of Tyr512 to either glutamic acid or phenylalanine is sufficient to alter CFTR surface levels. In human airway epithelial cells, downregulation of endogenous Syk and WNK4 confirms their roles as physiologic regulators of CFTR surface expression. Together, our results show that Tyr512 phosphorylation is a novel signal regulating the prevalence of CFTR at the cell surface and that WNK4 and Syk perform an antagonistic role in this process.     

Map2k4 functions as a tumor suppressor in lung adenocarcinoma and inhibits tumor cell invasion by decreasing peroxisome proliferator-activated receptor γ2 expression

MAP2K4 encodes a dual-specificity kinase (mitogen-activated protein kinase kinase 4, or MKK4) that is mutated in a variety of human malignancies, but the biochemical properties of the mutant kinases and their roles in tumorigenesis have not been fully elucidated. Here we showed that 8 out of 11 cancer-associated MAP2K4 mutations reduce MKK4 protein stability or impair its kinase activity. On the basis of findings from bioinformatic studies on human cancer cell lines with homozygous MAP2K4 loss, we posited that MKK4 functions as a tumor suppressor in lung adenocarcinomas that develop in mice owing to expression of mutant Kras and Tp53. Conditional Map2k4 inactivation in the bronchial epithelium of mice had no discernible effect alone but increased the multiplicity and accelerated the growth of incipient lung neoplasias induced by oncogenic Kras. MKK4 suppressed the invasion and metastasis of Kras-Tp53-mutant lung adenocarcinoma cells. MKK4 deficiency increased peroxisomal proliferator-activated receptor γ2 (PPARγ2) expression through noncanonical MKK4 substrates, and PPARγ2 enhanced tumor cell invasion. We conclude that Map2k4 functions as a tumor suppressor in lung adenocarcinoma and inhibits tumor cell invasion by decreasing PPARγ2 levels.     

WNK1 activates SGK1 by a phosphatidylinositol 3-kinase-dependent and non-catalytic mechanism

WNK1 (with no lysine (K) 1) is a protein-serine/threonine kinase with a unique catalytic site organization. Deletions in the first intron of the WNK1 gene were found in a group of hypertensive patients with pseudohypoaldosteronism type II. No changes in coding sequence of WNK1 were found, but its expression was increased severalfold. We have been investigating actions of WNK1 and have found that WNK1 activates the serum- and glucocorticoid-induced protein kinase SGK1, which impacts membrane expression of the epithelial sodium channel. Here we explore the role of WNK1 in SGK1 regulation. Activation of SGK1 by WNK1 is blocked by phosphatidylinositol 3-kinase inhibitors. Neither the catalytic activity nor the kinase domain of WNK1 is required; rather the N-terminal 220 residues of WNK1 are necessary and sufficient to activate SGK1. Phosphorylation of WNK1 on Thr-58 contributes to SGK1 activation. Finally, we show that WNK1 is required for the activation of SGK1 by insulin-like growth factor 1.