The Experts below are selected from a list of 1563 Experts worldwide ranked by ideXlab platform
Melanie H Cobb - One of the best experts on this subject based on the ideXlab platform.
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WNK1 is an unexpected autophagy inhibitor
Autophagy, 2017Co-Authors: Sachith Gallolu Kankanamalage, Steve Stippec, Chonlarat Wichaidit, Andres Lorenterodriguez, Akansha M Shah, Angelique W Whitehurst, Melanie H CobbAbstract:ABSTRACTAutophagy is a cellular degradation pathway that is essential to maintain cellular physiology, and deregulation of autophagy leads to multiple diseases in humans. In a recent study, we discovered that the protein kinase WNK1 (WNK lysine deficient protein kinase 1) is an inhibitor of autophagy. The loss of WNK1 increases both basal and starvation-induced autophagy. In addition, the depletion of WNK1 increases the activation of the class III phosphatidylinositol 3-kinase (PtdIns3K) complex, which is required to induce autophagy. Moreover, the loss of WNK1 increases the expression of ULK1 (unc-51 like kinase 1), which is upstream of the PtdIns3K complex. It also increases the pro-autophagic phosphorylation of ULK1 at Ser555 and the activation of AMPK (AMP-activated protein kinase), which is responsible for that phosphorylation. The inhibition of AMPK by compound C decreases the magnitude of autophagy induction following WNK1 loss; however, it does not prevent autophagy induction. We found that the UV...
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multistep regulation of autophagy by WNK1
Proceedings of the National Academy of Sciences of the United States of America, 2016Co-Authors: Sachith Gallolu Kankanamalage, Steve Stippec, Chonlarat Wichaidit, Andres Lorenterodriguez, Akansha M Shah, Angelique W Whitehurst, Melanie H CobbAbstract:The with-no-lysine (K) (WNK) kinases are an atypical family of protein kinases that regulate ion transport across cell membranes. Mutations that result in their overexpression cause hypertension-related disorders in humans. Of the four mammalian WNKs, only WNK1 is expressed throughout the body. We report that WNK1 inhibits autophagy, an intracellular degradation pathway implicated in several human diseases. Using small-interfering RNA-mediated WNK1 knockdown, we show autophagosome formation and autophagic flux are accelerated. In cells with reduced WNK1, basal and starvation-induced autophagy is increased. We also show that depletion of WNK1 stimulates focal class III phosphatidylinositol 3-kinase complex (PI3KC3) activity, which is required to induce autophagy. Depletion of WNK1 increases the expression of the PI3KC3 upstream regulator unc-51–like kinase 1 (ULK1), its phosphorylation, and activation of the kinase upstream of ULK1, the AMP-activated protein kinase. In addition, we show that the N-terminal region of WNK1 binds to the UV radiation resistance-associated gene (UVRAG) in vitro and WNK1 partially colocalizes with UVRAG, a component of a PI3KC3 complex. This colocalization decreases upon starvation of cells. Depletion of the SPS/STE20-related proline-alanine–rich kinase, a WNK1-activated enzyme, also induces autophagy in nutrient-replete or -starved conditions, but depletion of the related kinase and WNK1 substrate, oxidative stress responsive 1, does not. These results indicate that WNK1 inhibits autophagy by multiple mechanisms.
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actions of the protein kinase WNK1 on endothelial cells are differentially mediated by its substrate kinases osr1 and spak
Proceedings of the National Academy of Sciences of the United States of America, 2014Co-Authors: Hashem A Dbouk, Lauren M Weil, G Sachith K Perera, Michael T Dellinger, Gray W Pearson, Rolf A Brekken, Melanie H CobbAbstract:The with no lysine (K) (WNK) family of enzymes is best known for control of blood pressure through regulation of the function and membrane localization of ion cotransporters. In mice, global as well as endothelial-specific WNK1 gene disruption results in embryonic lethality due to angiogenic and cardiovascular defects. WNK1−/− embryos can be rescued by endothelial-specific expression of a constitutively active form of the WNK1 substrate protein kinase OSR1 (oxidative stress responsive 1). Using human umbilical vein endothelial cells (HUVECs), we explored mechanisms underlying the requirement of WNK1–OSR1 signaling for vascular development. WNK1 is required for cord formation in HUVECs, but the actions of the two major WNK1 effectors, OSR1 and its close relative SPAK (STE20/SPS1-related proline-, alanine-rich kinase), are distinct. SPAK is important for endothelial cell proliferation, whereas OSR1 is required for HUVEC chemotaxis and invasion. We also identified the zinc-finger transcription factor Slug in WNK1-mediated control of endothelial functions. Our study identifies a separation of functions for the WNK1-activated protein kinases OSR1 and SPAK in mediating proliferation, invasion, and gene expression in endothelial cells and an unanticipated link between WNK1 and Slug that is important for angiogenesis.
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WNK1 is required for mitosis and abscission
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Szu Wei Tu, Abhijit Bugde, Katherine Lubyphelps, Melanie H CobbAbstract:WNK [with no lysine (K)] protein kinases are found in all sequenced multicellular and many unicellular organisms. WNKs influence ion balance. Two WNK family members are associated with a single gene form of hypertension. RNA interference screens have implicated WNKs in survival and growth, and WNK1 is essential for viability of mice. We found that the majority of WNK1 is localized on cytoplasmic puncta in resting cells. During cell division, WNK1 localizes to mitotic spindles. Therefore, we analyzed mitotic phenotypes in WNK1 knockdown cells. A large percentage of WNK1 knockdown cells fail to complete cell division, displaying defects in mitotic spindles and also in abscission and cell survival. One of the best-characterized WNK1 targets is the protein kinase OSR1 (oxidative stress responsive 1). OSR1 regulates ion cotransporters, is activated in response to osmotic stress by WNK family members, and is largely associated with WNK1. In resting cells, the majority of OSR1, like WNK1, is on cytoplasmic puncta. OSR1 is also in nuclei. In contrast to WNK1, however, OSR1 does not concentrate around spindles during mitosis and does not show a WNK1-like localization pattern in mitotic cells. Knockdown of OSR1 has only a modest effect on cell survival and does not lead to spindle defects. We conclude that decreased cell survival associated with loss of WNK1 is attributable to defects in chromosome segregation and abscission and is independent of the effector kinase OSR1.
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WNK1 is a novel regulator of munc18c syntaxin 4 complex formation in soluble nsf attachment protein receptor snare mediated vesicle exocytosis
Journal of Biological Chemistry, 2007Co-Authors: Eunjin Oh, Melanie H Cobb, Jessie M English, Charles J Heise, Debbie C ThurmondAbstract:Abstract Defects in soluble NSF attachment protein receptor (SNARE)-mediated granule exocytosis occur in islet beta cells, adipocytes, and/or skeletal muscle cells correlate with increased susceptibility to insulin resistance and diabetes. The serine/threonine kinase WNK1 (with no K (lysine)) has recently been implicated in exocytosis and is expressed in all three of these cell types. To search for WNK1 substrates related to exocytosis, we conducted a WNK1 two-hybrid screen, which yielded Munc18c. Munc18c is known to be a key regulator of accessibility of the target membrane (t-SNARE) protein syntaxin 4 to participate in SNARE core complex assembly, although a paucity of Munc18c-binding factors has precluded discovery of its precise functions. To validate WNK1 as a new Munc18c-interacting partner, the direct interaction between WNK1 and Munc18c was confirmed using in vitro binding analysis, and endogenous WNK1-Munc18c complexes were detected in the cytosolic and plasma membrane compartments of the islet beta cell line MIN6. This binding interaction is mediated through the N-terminal 172 residues of Munc18c and the kinase domain residues of WNK1 (residues 159–491). Expression of either of these two minimal interaction domains resulted in inhibition of glucose-stimulated insulin secretion, consistent with a functional importance for the endogenous WNK1-Munc18c complex in exocytosis. Interestingly, Munc18c failed to serve as a WNK1 substrate in kinase activity assays, suggesting that WNK1 functions in SNARE complex assembly outside its role as a kinase. Taken together, these data support a novel role for WNK1 and a new mechanism for the regulation of SNARE complex assembly by WNK1-Munc18c complexes.
Chou-long Huang - One of the best experts on this subject based on the ideXlab platform.
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identification of a novel pp2a regulator WNK1 as critical for uterine function
bioRxiv, 2020Co-Authors: Tianyuan Wang, Chou-long Huang, Sanpin Wu, Steven L Young, John P Lydon, Francesco J DemayoAbstract:WNK1 is critical for uterine function as a mediator of stromal cell decidualization in vitro. Here, we employed a mouse model with conditional WNK1 ablation from the female reproductive tract to define its in vivo role in uterine biology. Loss of WNK1 altered uterine morphology, causing endometrial epithelial hyperplasia, adenomyosis and a delay in embryo implantation, ultimately resulting in compromised fertility. Mechanistic investigations through transcriptomic and proteomic approaches uncovered the regulatory role of WNK1 in controlling the PP2A-AKT-FOXO1 signaling axis. We show that WNK1 interacts directly with PPP2R1A, which is crucial for PP2A phosphatase activity. PP2A phosphatase in turn dephosphorylates AKT, thereby reducing its inhibitory effect on FOXO1. This permits the nuclear entry of FOXO1 to transcriptionally regulate implantation-associated genes. Our findings revealed a novel function of WNK1 in regulating AKT-FOXO1 post-translational modification, and demonstrated that this signaling pathway is critical in normal uterine physiology and pregnancy.
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WNK1 kinase stimulates angiogenesis to promote tumor growth and metastasis
Cancers, 2020Co-Authors: Rueiyang Li, Horngdar Wang, Bonifasius Putera Sampurna, Chou-long HuangAbstract:With-no-lysine (K)-1 (WNK1) is the founding member of family of four protein kinases with atypical placement of catalytic lysine that play important roles in regulating epithelial ion transport. Gain-of-function mutations of WNK1 and WNK4 cause a mendelian hypertension and hyperkalemic disease. WNK1 is ubiquitously expressed and essential for embryonic angiogenesis in mice. Increasing evidence indicates the role of WNK kinases in tumorigenesis at least partly by stimulating tumor cell proliferation. Here, we show that human hepatoma cells xenotransplanted into zebrafish produced high levels of vascular endothelial growth factor (VEGF) and WNK1, and induced expression of zebrafish WNK1. Knockdown of WNK1 in zebrafish decreased tumor-induced ectopic vessel formation and inhibited tumor proliferation. Inhibition of WNK1 or its downstream kinases OSR1 (oxidative stress responsive kinase 1)/SPAK (Ste20-related proline alanine rich kinase) using chemical inhibitors decreased ectopic vessel formation as well as proliferation of xenotransplanted hepatoma cells. The effect of WNK and OSR1 inhibitors is greater than that achieved by inhibitor of VEGF signaling cascade. These inhibitors also effectively inhibited tumorigenesis in two separate transgenic zebrafish models of intestinal and hepatocellular carcinomas. Endothelial-specific overexpression of WNK1 enhanced tumorigenesis in transgenic carcinogenic fish, supporting endothelial cell-autonomous effect of WNK1 in tumor promotion. Thus, WNK1 can promote tumorigenesis by multiple effects that include stimulating tumor angiogenesis. Inhibition of WNK1 may be a potent anti-cancer therapy.
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A unifying mechanism for WNK kinase regulation of sodium-chloride cotransporter
Pflügers Archiv - European Journal of Physiology, 2015Co-Authors: Chou-long Huang, Chih-jen ChengAbstract:Mammalian with-no-lysine [K] (WNK) kinases are a family of four serine-threonine protein kinases, WNK1-4. Mutations of WNK1 and WNK4 in humans cause pseudohypoaldosteronism type II (PHA2), an autosomal-dominant disease characterized by hypertension and hyperkalemia. Increased Na^+ reabsorption through Na^+–Cl^− cotransporter (NCC) in the distal convoluted tubule plays an important role in the pathogenesis of hypertension in patients with PHA2. However, how WNK1 and WNK4 regulate NCC and how mutations of WNKs cause activation of NCC have been controversial. Here, we review current state of literature supporting a compelling model that WNK1 and WNK4 both contribute to stimulation of NCC. The precise combined effects of WNK1 and WNK4 on NCC remain unclear but likely are positive rather than antagonistic. The recent discovery that WNK kinases may function as an intracellular chloride sensor adds a new dimension to the physiological role of WNK kinases. Intracellular chloride-dependent regulation of WNK’s may underlie the mechanism of regulation of NCC by extracellular K^+. Definite answer yet will require future investigation by tubular perfusion in mice with altered WNK kinase expression.
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zebrafish wnk lysine deficient protein kinase 1 WNK1 affects angiogenesis associated with vegf signaling
PLOS ONE, 2014Co-Authors: Su Mei Tsai, Hsiao Chen Tu, Wen Chuan Chen, Jeng Wei Lu, Horngdar Wang, Chou-long HuangAbstract:The WNK1 (WNK lysine deficient protein kinase 1) protein is a serine/threonine protein kinase with emerging roles in cancer. WNK1 causes hypertension and hyperkalemia when overexpressed and cardiovascular defects when ablated in mice. In this study, the role of WNK1 in angiogenesis was explored using the zebrafish model. There are two zebrafish WNK1 isoforms, WNK1a and WNK1b, and both contain all the functional domains found in the human WNK1 protein. Both isoforms are expressed in the embryo at the initiation of angiogenesis and in the posterior cardinal vein (PCV), similar to fms-related tyrosine kinase 4 (flt4). Using morpholino antisense oligonucleotides against WNK1a and WNK1b, we observed that WNK1 morphants have defects in angiogenesis in the head and trunk, similar to flk1/vegfr2 morphants. Furthermore, both WNK1a and WNK1b mRNA can partially rescue the defects in vascular formation caused by flk1/vegfr2 knockdown. Mutation of the kinase domain or the Akt/PI3K phosphorylation site within WNK1 destroys this rescue capability. The rescue experiments provide evidence that WNK1 is a downstream target for Vegfr2 (vascular endothelial growth factor receptor-2) and Akt/PI3K signaling and thereby affects angiogenesis in zebrafish embryos. Furthermore, we found that knockdown of vascular endothelial growth factor receptor-2 (flk1/vegfr2) or vascular endothelial growth factor receptor-3 (flt4/vegfr3) results in a decrease in WNK1a expression, as assessed by in situ hybridization and q-RT-PCR analysis. Thus, the Vegf/Vegfr signaling pathway controls angiogenesis in zebrafish via Akt kinase-mediated phosphorylation and activation of WNK1 as well as transcriptional regulation of WNK1 expression.
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WNK1 protein kinase regulates embryonic cardiovascular development through the osr1 signaling cascade
Journal of Biological Chemistry, 2013Co-Authors: Joonho Yoon, Sung-sen Yang, Chou-long HuangAbstract:Abstract WNK1 is a widely expressed serine/threonine protein kinase that regulates multiple cellular and organ functions via diverse mechanisms. We previously reported that endothelial-specific deletion of WNK1 in mice results in embryonic lethality, with angiogenesis and cardiac defects beginning at embryonic day ∼10.5. Here, we further investigated the signaling mechanism by which WNK1 regulates embryonic cardiovascular development. We found that mice with a global deletion of Osr1, which encodes oxidative stress-responsive kinase-1, a protein kinase activated by WNK1, died in utero beginning at embryonic day ∼11. The defects in Osr1-null yolk sacs and embryos were virtually identical to those observed in WNK1-knock-out mice: no mature large vessels in yolk sacs, defective angiogenesis in the brain and intersomitic vessels, and smaller chambers and reduced myocardial trabeculation in mutant hearts. Endothelial-specific deletion of Osr1 generated by crossing Osr1flox/flox mice with Tie2-Cre mice phenocopied defects caused by global Osr1 deletion. To investigate whether OSR1 acts downstream of WNK1 in embryonic angiogenesis, we generated a mouse line that carries a catalytically and constitutively active human OSR1 transgene in the ROSA26 locus under the control of a cassette of floxed transcription stop codons. We found that endothelial-specific expression of the constitutively active mutant OSR1, generated by Tie2-Cre-mediated excision of floxed stop codons in the mutated ROSA26 locus, rescued angiogenesis and cardiac defects in global WNK1-null embryos. These results indicate that WNK1 activation of the OSR1 signaling cascade is an essential pathway that regulates angiogenesis and cardiac formation during mouse embryo development.
Shinichi Uchida - One of the best experts on this subject based on the ideXlab platform.
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WNK1 regulates skeletal muscle cell hypertrophy by modulating the nuclear localization and transcriptional activity of foxo4
Scientific Reports, 2018Co-Authors: Shintaro Mandai, Eisei Sohara, Takayasu Mori, Naohiro Nomura, Emi Sasaki, Taisuke Furusho, Yohei Arai, Hiroaki Kikuchi, Shinichi UchidaAbstract:With-no-lysine (K) (WNK) kinases, which are mutated in the inherited form of hypertension pseudohypoaldosteronism type II, are essential regulators of membrane ion transporters. Here, we report that WNK1 positively regulates skeletal muscle cell hypertrophy via mediating the function of the pro-longevity transcription factor forkhead box protein O4 (FOXO4) independent of the conventional WNK signaling pathway linking SPS/STE20-related proline-alanine–rich kinase (SPAK)/oxidative stress response kinase 1 (OSR1) to downstream effector ion transporters. Small interfering RNA (siRNA)-mediated silencing of WNK1, but not SPAK/OSR1 kinases, induced myotube atrophy and remarkable increases in the mRNA expression of the muscle atrophy ubiquitin ligases MAFbx and MuRF1 in C2C12 mouse skeletal muscle cells. WNK1 silencing also increased FOXO4 nuclear localization, and co-transfection of Foxo4-targeted siRNA completely reversed the myotube atrophy and upregulation of atrogene transcription induced by WNK1 silencing. We further illustrated that WNK1 protein abundance in skeletal muscle was increased by chronic voluntary wheel running exercise (hypertrophic stimulus) and markedly decreased by adenine-induced chronic kidney disease (atrophic stimulus) in mice. These findings suggest that WNK1 is involved in the physiological regulation of mammalian skeletal muscle hypertrophy and atrophy via interactions with FOXO4. The WNK1-FOXO4 axis may be a potential therapeutic target in human diseases causing sarcopenia.
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Effect of heterozygous deletion of WNK1 on the WNK-OSR1/SPAK-NCC/NKCC1/NKCC2 signal cascade in the kidney and blood vessels
Clinical and Experimental Nephrology, 2012Co-Authors: Koichiro Susa, Satomi Kita, Takahiro Iwamoto, Sung-sen Yang, Akihito Ohta, Eisei Sohara, Sei Sasaki, Dario R. Alessi, Shinichi UchidaAbstract:Background We found that a mechanism of hypertension in pseudohypoaldosteronism type II (PHAII) caused by a WNK4 missense mutation (D561A) was activation of the WNK-OSR1/SPAK-NCC signal cascade. However, the pathogenic effect of intronic deletions in WNK1 genes also observed in PHAII patients remains unclear. To understand the pathophysiological roles of WNK1 in vivo, WNK1^+/− mice have been analyzed, because homozygous WNK1 knockout is embryonic lethal. Although WNK1^+/− mice have been reported to have hypotension, detailed analyses of the WNK signal cascade in the kidney and other organs of WNK1^+/− mice have not been performed. Method We assess the effect of heterozygous deletion of WNK1 on the WNK-OSR1/SPAK-NCC/NKCC1/NKCC2 signal cascade in the kidney and blood vessels. Results Contrary to the previous report, the blood pressure of WNK1^+/− mice was not decreased, even under a low-salt diet. Under a WNK4^D561A/+ background, the heterozygous deletion of the WNK1 gene did not reduce the high blood pressure either. We then evaluated the phosphorylation status of OSR1, SPAK, NCC, NKCC1, and NKCC2 in the kidney, but no significant decrease in the phosphorylation was observed in WNK1^+/− mice or WNK1^+/−WNK4^D561A/+ mice. In contrast, a significant decrease in NKCC1 phosphorylation in the aorta and a decreased pressure-induced myogenic response in the mesenteric arteries were observed in WNK1^+/− mice. Conclusion The contribution of WNK1 to total WNK kinase activity in the kidney may be small, but that WNK1 may play a substantial role in the regulation of blood pressure in the arteries.
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effect of heterozygous deletion of WNK1 on the wnk osr1 spak ncc nkcc1 nkcc2 signal cascade in the kidney and blood vessels
Clinical and Experimental Nephrology, 2012Co-Authors: Koichiro Susa, Satomi Kita, Takahiro Iwamoto, Sung-sen Yang, Akihito Ohta, Eisei Sohara, Sei Sasaki, Dario R. Alessi, Shinichi UchidaAbstract:Background We found that a mechanism of hypertension in pseudohypoaldosteronism type II (PHAII) caused by a WNK4 missense mutation (D561A) was activation of the WNK-OSR1/SPAK-NCC signal cascade. However, the pathogenic effect of intronic deletions in WNK1 genes also observed in PHAII patients remains unclear. To understand the pathophysiological roles of WNK1 in vivo, WNK1+/− mice have been analyzed, because homozygous WNK1 knockout is embryonic lethal. Although WNK1+/− mice have been reported to have hypotension, detailed analyses of the WNK signal cascade in the kidney and other organs of WNK1+/− mice have not been performed.
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Regulation of WNK1 kinase by extracellular potassium
Clinical and Experimental Nephrology, 2011Co-Authors: Shotaro Naito, Akihito Ohta, Eisei Sohara, Sei Sasaki, Eriko Ohta, Shinichi UchidaAbstract:Background Mutations of WNK kinase genes were identified as the cause of a hereditary hypertensive disease, pseudohypoaldosteronism type II; however, little is known about the regulation of WNK kinases. In the present study, we focused on anisosmotic conditions as the initial clues for clarifying a stimulating factor for WNK kinase activity. Methods Endogenous WNK kinase activity in COS7 cells was monitored by the phosphorylation of its substrate, OSR1. Results Knockdown experiments revealed that WNK1 was a major WNK kinase in COS7 cells. In contrast to the transient increase in WNK1 activity caused by hypertonic medium, hypotonic medium increased the phosphorylation of OSR1 for 24 h, suggesting that the hypotonic medium included a signal for continuously stimulating WNK1 kinase activity. To identify the signal, ion substitution experiments were performed. Surprisingly, even isotonic media with low Cl^− or low K^+ was found to increase OSR1 phosphorylation as well as the hypotonic medium. Furthermore, WNK1 activation by the hypotonic medium was completely blocked by quinine (500 μM) but not by 5-nitro-2-(3-phenylpropylamino) benzoic acid (100 μM), and this inhibition was closely correlated with the inhibition of ^86Rb^+ (=K^+) efflux but not with the inhibition of ^125I^− (=Cl^−) efflux. These results suggest that K^+, rather than hypotonicity or low Cl^−, may be an important regulator for WNK1 activation. Finally, we confirmed that high K^+ and low K^+ media under the physiological range decreased and increased WNK1 activity, respectively. Conclusion Extracellular K^+ is an important regulator of WNK1 kinase activity.
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Pathophysiological roles of WNK kinases in the kidney
Pflügers Archiv - European Journal of Physiology, 2010Co-Authors: Shinichi UchidaAbstract:Since the discovery of mutations in the WNK1 and WNK4 genes in pseudohypoaldosteronism type II (PHAII), the pathophysiological role of WNK kinases in hypertension and renal ion transport has been a hot topic for investigation. Analyses from a mouse model carrying the same mutation as seen in PHAII patients, reveal a new signal cascade in the kidney that regulates NaCl and K balance in the body. WNK kinases phosphorylate and activate oxidative stress responsive kinase 1 (OSR1) and STE20-like proline and alanine-rich kinase (SPAK), and OSR1 and SPAK phosphorylate and activate the thiazide-sensitive Na-Cl cotransporter (NCC). Furthermore, this cascade is regulated by aldosterone, indicating that WNK-OSR1/SPAK-NCC cooperates with this system including the epithelial Na channel (ENaC) to conserve NaCl. With regard to K excretion, however, both systems work in opposite directions whereby PHAII and Liddle syndrome show hyperkalemia and hypokalemia, respectively. Thus, the identification of such aldosterone effecters other than ENaC, will reveal a novel regulatory mechanism of K excretion in the distal nephron, and also provides basic evidence for the therapeutic use of thiazide in various clinical situations.
Bing E Xu - One of the best experts on this subject based on the ideXlab platform.
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WNK1 activates sgk1 by a phosphatidylinositol 3 kinase dependent and non catalytic mechanism
Journal of Biological Chemistry, 2005Co-Authors: Bing E Xu, Chou-long Huang, Steve Stippec, Ahmed Lazrak, Melanie H CobbAbstract:Abstract 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.
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properties of WNK1 and implications for other family members
Journal of Biological Chemistry, 2005Co-Authors: Lisa Y Lenertz, Bing E Xu, Kyle Wedin, Svetlana Earnest, Elizabeth J Goldsmith, Melanie H CobbAbstract: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.
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WNK1 activates sgk1 to regulate the epithelial sodium channel
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Bing E Xu, Chou-long Huang, Steve Stippec, Ahmed Lazrak, Xin Ji Li, Jessie M English, Bernardo Ortega, Melanie H CobbAbstract:WNK (with no lysine [K]) kinases are serine-threonine protein kinases with an atypical placement of the catalytic lysine. Intronic deletions increase the expression of WNK1 in humans and cause pseudohypoaldosteronism type II, a form of hypertension. WNKs have been linked to ion carriers, but the underlying regulatory mechanisms are unknown. Here, we report a mechanism for the control of ion permeability by WNK1. We show that WNK1 activates the serum- and glucocorticoid-inducible protein kinase SGK1, leading to activation of the epithelial sodium channel. Increased channel activity induced by WNK1 depends on SGK1 and the E3 ubiquitin ligase Nedd4-2. This finding provides compelling evidence that this molecular mechanism contributes to the pathogenesis of hypertension in pseudohypoaldosteronism type II caused by WNK1 and, possibly, in other forms of hypertension.
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WNK1: analysis of protein kinase structure, downstream targets, and potential roles in hypertension
Cell Research, 2005Co-Authors: Bing E Xu, Elizabeth J Goldsmith, Steve Stippec, Charles J Heise, Lisa Lenertz, Melanie H CobbAbstract:The WNK kinases are a recently discovered family of serine-threonine kinases that have been shown to play an essential role in the regulation of electrolyte homeostasis. Intronic deletions in the WNK1 gene result in its overexpression and lead to pseudohypoaldosteronism type II, a disease with salt-sensitive hypertension and hyperkalemia. This review focuses on the recent evidence elucidating the structure of the kinase domain of WNK1 and functions of these kinases in normal and disease physiology. Their functions have implications for understanding the biochemical mechanism that could lead to the retention or insertion of proteins in the plasma membrane. The WNK kinases may be able to influence ion homeostasis through its effects on synaptotagmin function.
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WNK1 phosphorylates synaptotagmin 2 and modulates its membrane binding
Molecular Cell, 2004Co-Authors: Charles J Heise, Bing E Xu, Elizabeth J Goldsmith, Katherine Lubyphelps, She Chen, Melanie H CobbAbstract:Abstract WNK ( w ith n o lysine [ K ]) protein kinases were named for their unique active site organization. Mutations in WNK1 and WNK4 cause a familial form of hypertension by undefined mechanisms. Here, we report that WNK1 selectively binds to and phosphorylates synaptotagmin 2 (Syt2) within its calcium binding C2 domains. Endogenous WNK1 and Syt2 coimmunoprecipitate and colocalize on a subset of secretory granules in INS-1 cells. Phosphorylation by WNK1 increases the amount of Ca 2+ required for Syt2 binding to phospholipid vesicles; mutation of threonine 202, a WNK1 phosphorylation site, partially prevents this change. These findings suggest that phosphorylation of Syts by WNK1 can regulate Ca 2+ sensing and the subsequent Ca 2+ -dependent interactions mediated by Syt C2 domains. These findings provide a biochemical mechanism that could lead to the retention or insertion of proteins in the plasma membrane. Interruption of this regulatory pathway may disturb membrane events that regulate ion balance.
Dario R. Alessi - One of the best experts on this subject based on the ideXlab platform.
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Effect of heterozygous deletion of WNK1 on the WNK-OSR1/SPAK-NCC/NKCC1/NKCC2 signal cascade in the kidney and blood vessels
Clinical and Experimental Nephrology, 2012Co-Authors: Koichiro Susa, Satomi Kita, Takahiro Iwamoto, Sung-sen Yang, Akihito Ohta, Eisei Sohara, Sei Sasaki, Dario R. Alessi, Shinichi UchidaAbstract:Background We found that a mechanism of hypertension in pseudohypoaldosteronism type II (PHAII) caused by a WNK4 missense mutation (D561A) was activation of the WNK-OSR1/SPAK-NCC signal cascade. However, the pathogenic effect of intronic deletions in WNK1 genes also observed in PHAII patients remains unclear. To understand the pathophysiological roles of WNK1 in vivo, WNK1^+/− mice have been analyzed, because homozygous WNK1 knockout is embryonic lethal. Although WNK1^+/− mice have been reported to have hypotension, detailed analyses of the WNK signal cascade in the kidney and other organs of WNK1^+/− mice have not been performed. Method We assess the effect of heterozygous deletion of WNK1 on the WNK-OSR1/SPAK-NCC/NKCC1/NKCC2 signal cascade in the kidney and blood vessels. Results Contrary to the previous report, the blood pressure of WNK1^+/− mice was not decreased, even under a low-salt diet. Under a WNK4^D561A/+ background, the heterozygous deletion of the WNK1 gene did not reduce the high blood pressure either. We then evaluated the phosphorylation status of OSR1, SPAK, NCC, NKCC1, and NKCC2 in the kidney, but no significant decrease in the phosphorylation was observed in WNK1^+/− mice or WNK1^+/−WNK4^D561A/+ mice. In contrast, a significant decrease in NKCC1 phosphorylation in the aorta and a decreased pressure-induced myogenic response in the mesenteric arteries were observed in WNK1^+/− mice. Conclusion The contribution of WNK1 to total WNK kinase activity in the kidney may be small, but that WNK1 may play a substantial role in the regulation of blood pressure in the arteries.
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effect of heterozygous deletion of WNK1 on the wnk osr1 spak ncc nkcc1 nkcc2 signal cascade in the kidney and blood vessels
Clinical and Experimental Nephrology, 2012Co-Authors: Koichiro Susa, Satomi Kita, Takahiro Iwamoto, Sung-sen Yang, Akihito Ohta, Eisei Sohara, Sei Sasaki, Dario R. Alessi, Shinichi UchidaAbstract:Background We found that a mechanism of hypertension in pseudohypoaldosteronism type II (PHAII) caused by a WNK4 missense mutation (D561A) was activation of the WNK-OSR1/SPAK-NCC signal cascade. However, the pathogenic effect of intronic deletions in WNK1 genes also observed in PHAII patients remains unclear. To understand the pathophysiological roles of WNK1 in vivo, WNK1+/− mice have been analyzed, because homozygous WNK1 knockout is embryonic lethal. Although WNK1+/− mice have been reported to have hypotension, detailed analyses of the WNK signal cascade in the kidney and other organs of WNK1+/− mice have not been performed.
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Effect of heterozygous deletion of WNK1 on the WNK-OSR1/SPAK-NCC/NKCC1/NKCC2 signal cascade in the kidney and blood vessels
Clinical and Experimental Nephrology, 2012Co-Authors: Koichiro Susa, Satomi Kita, Takahiro Iwamoto, Sung-sen Yang, Akihito Ohta, Eisei Sohara, Sei Sasaki, Dario R. AlessiAbstract:Background We found that a mechanism of hypertension in pseudohypoaldosteronism type II (PHAII) caused by a WNK4 missense mutation (D561A) was activation of the WNK-OSR1/SPAK-NCC signal cascade. However, the pathogenic effect of intronic deletions in WNK1 genes also observed in PHAII patients remains unclear. To understand the pathophysiological roles of WNK1 in vivo, WNK1+/− mice have been analyzed, because homozygous WNK1 knockout is embryonic lethal. Although WNK1+/− mice have been reported to have hypotension, detailed analyses of the WNK signal cascade in the kidney and other organs of WNK1+/− mice have not been performed.
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functional interactions of the spak osr1 kinases with their upstream activator WNK1 and downstream substrate nkcc1
Biochemical Journal, 2006Co-Authors: Alberto C Vitari, Fatema H Rafiqi, Jacob Thastrup, Maria Deak, Nick A Morrice, Hakan K R Karlsson, Dario R. AlessiAbstract:The SPAK (STE20/SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase-1) kinases interact and phosphorylate NKCC1 (Na + –K + –2Cl − co-transporter-1), leading to its activation. Recent studies indicated that SPAK and OSR1 are phosphorylated and activated by the WNK1 [with no K (lysine) protein kinase-1] and WNK4, genes mutated in humans affected by Gordon9s hypertension syndrome. In the present study, we have identified three residues in NKCC1 (Thr 175 /Thr 179 /Thr 184 in shark or Thr 203 /Thr 207 /Thr 212 in human) that are phosphorylated by SPAK and OSR1, and have developed a peptide substrate, CATCHtide (cation chloride co-transporter peptide substrate), to assess SPAK and OSR1 activity. Exposure of HEK-293 (human embryonic kidney) cells to osmotic stress, which leads to phosphorylation and activation of NKCC1, increased phosphorylation of NKCC1 at the sites targeted by SPAK/OSR1. The residues on NKCC1, phosphorylated by SPAK/OSR1, are conserved in other cation co-transporters, such as the Na + –Cl − co-transporter, the target of thiazide drugs that lower blood pressure in humans with Gordon9s syndrome. Furthermore, we characterize the properties of a 92-residue CCT (conserved C-terminal) domain on SPAK and OSR1 that interacts with an RFXV (Arg-Phe-Xaa-Val) motif present in the substrate NKCC1 and its activators WNK1/WNK4. A peptide containing the RFXV motif interacts with nanomolar affinity with the CCT domains of SPAK/OSR1 and can be utilized to affinity-purify SPAK and OSR1 from cell extracts. Mutation of the arginine, phenylalanine or valine residue within this peptide abolishes binding to SPAK/OSR1. We have identified specific residues within the CCT domain that are required for interaction with the RFXV motif and have demonstrated that mutation of these in OSR1 inhibited phosphorylation of NKCC1, but not of CATCHtide which does not possess an RFXV motif. We establish that an intact CCT domain is required for WNK1 to efficiently phosphorylate and activate OSR1. These data establish that the CCT domain functions as a multipurpose docking site, enabling SPAK/OSR1 to interact with substrates (NKCC1) and activators (WNK1/WNK4).
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the WNK1 and wnk4 protein kinases that are mutated in gordon s hypertension syndrome phosphorylate and activate spak and osr1 protein kinases
Biochemical Journal, 2005Co-Authors: Alberto C Vitari, Maria Deak, Nick A Morrice, Dario R. AlessiAbstract:Mutations in the human genes encoding WNK1 [with no K (lysine) protein kinase-1] and the related protein kinase WNK4 are the cause of Gordon9s hypertension syndrome. Little is known about the molecular mechanism by which WNK isoforms regulate cellular processes. We immunoprecipitated WNK1 from extracts of rat testis and found that it was specifically associated with a protein kinase of the STE20 family termed ‘STE20/SPS1-related proline/alanine-rich kinase’ (SPAK). We demonstrated that WNK1 and WNK4 both interacted with SPAK as well as a closely related kinase, termed ‘oxidative stress response kinase-1’ (OSR1). Wildtype (wt) but not catalytically inactive WNK1 and WNK4 phosphorylated SPAK and OSR1 to a much greater extent than with other substrates utilized previously, such as myelin basic protein and claudin-4. Phosphorylation by WNK1 or WNK4 markedly increased SPAK and OSR1 activity. Phosphopeptide mapping studies demonstrated that WNK1 phosphorylated kinase-inactive SPAK and OSR1 at an equivalent residue located within the T-loop of the catalytic domain (Thr 233 in SPAK, Thr 185 in OSR1) and a serine residue located within a C-terminal non-catalytic region (Ser 373 in SPAK, Ser 325 in OSR1). Mutation of Thr 185 to alanine prevented the activation of OSR1 by WNK1, whereas mutation of Thr 185 to glutamic acid (to mimic phosphorylation) increased the basal activity of OSR1 over 20-fold and prevented further activation by WNK1. Mutation of Ser 325 in OSR1 to alanine or glutamic acid did not affect the basal activity of OSR1 or its ability to be activated by WNK1. These findings suggest that WNK isoforms operate as protein kinases that activate SPAK and OSR1 by phosphorylating the T-loops of these enzymes, resulting in their activation. Our analysis also describes the first facile assay that can be employed to quantitatively assess WNK1 and WNK4 activity.
