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. 2004 Mar 30;101(13):4690-4.
doi: 10.1073/pnas.0306924101. Epub 2004 Mar 19.

Disease-causing mutant WNK4 increases paracellular chloride permeability and phosphorylates claudins

Kozue Yamauchi  1 , Tatemitsu Rai, Katsuki Kobayashi, Eisei Sohara, Tatsunori Suzuki, Tomohiro Itoh, Shin Suda, Atsushi Hayama, Sei Sasaki, Shinichi Uchida
Affiliations

Disease-causing mutant WNK4 increases paracellular chloride permeability and phosphorylates claudins

Kozue Yamauchi et al. Proc Natl Acad Sci U S A. .

Abstract

Mutations in the WNK4 gene cause pseudohypoaldosteronism type II (PHAII), an autosomal-dominant disorder of hyperkalemia and hypertension. The target molecules of this putative kinase and the molecular mechanisms by which the mutations cause the phenotypes are currently unknown. Although recent reports found that expression of WNK4 in Xenopus oocytes causes inhibition of the thiazide-sensitive NaCl cotransporter and the renal K channel ROMK, there may be additional targets of WNK4. For example, an increase in paracellular chloride permeability has been postulated to be a mediator of PHAII pathogenesis, a possibility supported by the localization of WNK4 at tight junctions in vivo. To determine the validity of this hypothesis, we measured transepithelial Na and Cl permeability in Madin-Darby canine kidney II cells stably expressing wild-type or a pathogenic mutant of WNK4. We found that transepithelial paracellular Cl permeability was increased in cells expressing a disease-causing mutant WNK4 (D564A) but that Na permeability was decreased slightly. Furthermore, WNK4 bound and phosphorylated claudins 1-4, major tight-junction membrane proteins known to be involved in the regulation of paracellular ion permeability. The increases in phosphorylation of claudins were greater in cells expressing the mutant WNK4 than in cells expressing wild-type protein. These results clearly indicate that the pathogenic WNK4 mutant possesses a gain-of-function activity and that the claudins may be important molecular targets of WNK4 kinase. The increased paracellular "chloride shunt" caused by the mutant WNK4 could be the pathogenic mechanism of PHAII.

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Figures

Fig. 1.
Fig. 1.
Generation of wild-type and mutant WNK4-expressing MDCK II cell lines. (a) Expression of wild-type and mutant WNK4 in MDCK II cells. HA-tagged WNK4 proteins were detected by using an anti-HA (9Y10) Ab. Expression of WNK4 was induced by removal of doxycycline from the medium for 4 days. W28 and W31 are wild-type WNK4-expressing cell lines, and M18 and M19 are mutant WNK4-expressing cell lines. (b) Immunofluorescence of wild-type and mutant WNK4 expressed in isolated stable cell lines. Cells were grown on a permeable support, and the expression of WNK4 was induced for 4 days. WNK4 was detected by using an anti-HA Ab in conjunction with an Alexa 546-conjugated anti-rat IgG Ab. Cells were stained for occludin by using an anti-occludin Ab (Zymed) in conjunction with an Alexa 488-conjugated anti-rabbit IgG Ab.
Fig. 2.
Fig. 2.
Transepithelial 22Na (a) and 36Cl (b) permeability in MDCKII cells expressing wild-type and mutant WNK4. Each bar represents experiments using at least three cell lines. The numbers of assays are given in parentheses. At least three assays were performed with each cell line.
Fig. 3.
Fig. 3.
Tight-junction proteins in WNK4-expressing cells. (a) Immunofluorescence of tight-junction proteins in the WNK4-expresing cells. Stable WNK4-expressing cell lines were grown on a permeable support. Cells were immunostained with anti-claudin 1-4 Abs or with an anti-occludin Ab. (b) Expression of claudins and occludin in Triton X-100-soluble and -insoluble fractions in WNK4-expressing cells. WNK4 expression was induced for 4 days, and cells were harvested in a buffer containing 1% Triton X-100. The Triton X-100-soluble and -insoluble fractions were resolved by SDS/PAGE, and the expression of claudins and occludin were determined by Western blot analysis.
Fig. 4.
Fig. 4.
Phosphorylation of claudins by WNK4. (a) Phosphorylation of Flag-tagged claudins by WNK4 in COS7 cells. COS7 cells were transfected with HA-tagged WNK4 and Flag-tagged claudins or occuludin. Claudin 4 delC lacks the entire C-terminal cytosolic region of claudin4. Cells were labeled with [32P]Pi (1 mCi/ml), and proteins were immunoprecipitated with an anti-Flag Ab (M2). The immunoprecipitated Flag-claudins were separated by SDS/PAGE, electrophoretically transferred to nitrocellulose, and analyzed by Western blotting with an anti-Flag polyclonal Ab. After detecting the immunoprecipitated claudins, claudin phosphorylation was detected by autoradiography of the nitrocellulose membrane. (b) Phosphorylation of Flag-tagged claudins in the WNK4-expressing MDCK II cells. MDCK II cells stably expressing HA-tagged WNK4 were transfected with Flag-tagged claudin 2 and 3. Phosphorylated claudin 2 and 3 were detected, as described in a.(c) Phosphorylation of endogenous claudins in the mutant WNK4-expressing MDCK II cells. The WNK4-expressing cells were labeled with [32P]Pi, and the endogenous claudins were immunoprecipitated by using anti-claudin 1 and 4 Abs (Zymed). Phosphorylation of claudins was visualized by autoradiography. (d) Phosphorylation of wild-type and mutant WNK4. MDCK II cells were transfected with HA-tagged wild-type and mutant WNK4. Cells were labeled with [32P]Pi, and WNK4 proteins were immunoprecipitated with an anti-HA Ab. Immunoprecipitated proteins were resolved on SDS/PAGE, electrophoretically transferred to nitrocellulose, and analyzed by Western blotting with an anti-HA Ab. WNK4 phosphorylation was detected by autoradiography. (e) In vitro kinase assay with GST-WNK4. GST-WNK4 (kinase domain) and GST-claudin 4 (C-terminal cytoplasmic domain) were incubated at 37°C for 15 min in kinase buffer, and the phosphorylation of GST-claudin 4 was visualized by SDS/PAGE, followed by autoradiography.
Fig. 5.
Fig. 5.
Analysis of WNK4-claudin interaction. (a) Coimmunoprecipitation of WNK4 and claudins. COS7 cells were transfected with HA-tagged WNK4 and Flag-tagged claudins. Proteins were immunoprecipitated with an anti-Flag M2 Ab, and the immunoprecipitates were analyzed by Western blot analysis by using anti-HA Ab. (b) Coimmunoprecipitation of WNK4 and endogenous tight-junction proteins. Proteins from MDCK II cells stably expressing the wild-type and mutant HA-WNK4 were immunoprecipitated with anti-HA Ab, and the immunoprecipitates were analyzed by Western blot analysis by using Abs to claudin 1, claudin 4, ZO-1, and occludin. (c) Binding of claudin 4 and its mutants by mutant WNK4. Claudin 4 binding was assessed by coimmunoprecipitation in lysates from MDCK II cells as described in a. Mutants examined include delC (the claudin 4 mutant lacking the entire C terminal cytoplasmic region) and del C plus YV (the del C mutant with Y and V added to the C terminus). Addition of YV residues restored the binding to WNK4.
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References

    1. Wilson, F. H., Disse-Nicodeme, S., Choate, K. A., Ishikawa, K., Nelson-Williams, C., Desitter, I., Gunel, M., Milford, D. V., Lipkin, G. W., Achard, J. M., et al. (2001) Science 293, 1107-1112. - PubMed
    1. Schambelan, M., Sebastian, A. & Rector, F. C., Jr. (1981) Kidney Int. 19, 716-727. - PubMed
    1. Farfel, Z., Iaina, A., Rosenthal, T., Waks, U., Shibolet, S. & Gafni, J. (1978) Arch. Intern. Med. (Moscow) 138, 1828-1832. - PubMed
    1. Wilson, F. H., Kahle, K. T., Sabath, E., Lalioti, M. D., Rapson, A. K., Hoover, R. S., Hebert, S. C., Gamba, G. & Lifton, R. P. (2003) Proc. Natl. Acad. Sci. USA 100, 680-684. - PMC - PubMed
    1. Yang, C. L., Angell, J., Mitchell, R. & Ellison, D. H. (2003) J. Clin. Invest. 111, 1039-1045. - PMC - PubMed

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