Glover M et al. (2010), A Single Amino Acid Substitution Makes WNK4 Sus...

XB-ART-42675

Open Med Chem J 2010 Sep 03;4:57-61. doi: 10.2174/1874104501004010057.

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A Single Amino Acid Substitution Makes WNK4 Susceptible to SB 203580 and SB 202190.

Glover M , Sweeny C , Davis B , O'Shaughnessy KM .

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Regulation of the SLC12 family of membrane transporters including NCCT involves a scaffold of interacting proteins including the STE 20 kinase SPAK and the WNK kinases, WNK 1 and WNK 4, which are mutated in the hypertensive syndrome of pseudohypoaldosteronism type 2 (PHAII). WNK4 regulates NCCT by affecting forward trafficking to the surface membrane. Studies in Xenopus using kinase dead WNK4 site mutants have produced inconsistent results with regard to the necessity of kinase function for NCCT regulation. Dynamic inhibition of WNK4 by small molecules may bring clarity to this issue however WNK4 is naturally resistant to commercial MAP kinase inhibitors owing to steric constraints prohibiting entry of small molecules to the active site. Using an approach similar to that used in p38 and ERK, we show that a single substitution in WNK4 (T261G) dramatically enhances its susceptibility to the inhibitors SB 202190 and SB 203580.

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Species referenced: Xenopus
Genes referenced: grap2 mapk1 mapk14 slc12a3 stk39 sult1e1 wnk4

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References [+] :

Asano, Role of phosphatidylinositol 3-kinase activation on insulin action and its alteration in diabetic conditions. 2007, Pubmed

	Fig. (1). Chemical structure of the p38 kinase inhibitors SB202190 and SB 203580.
	Fig. (2). The T261G WNK4 mutant behaves like wild type WNK4. A. Oocytes were injected with NCCT wt alone or in combination with WNK4 wt or WNK4 T261G. Mutant WNK4 reduced 22Na+ flux equivalent to the same extent as WNK4 wt. * P< 0.0001vs NCCT alone. B. Confocal microscopy of the fluorescent signal from eCFP-NCCT showed that changes in WNK4 induced 22Na+ flux were accompanied by similar changes in surface membrane expression of the cotransporter. * P< 0.0001 vs NCCT alone.
	Fig. (3). The T261G mutation of WNK4 confers kinase inhibitor sensitivity. A. Oocytes were injected with NCCT wt alone or in combination with either WNK4 wt or WNK4 T261G. The 22Na+ flux assay was then performed with or without 0.5 or 5 µM of each SB compound. * P<0.0001 vs NCCT alone. B. Confocal microscopy of the fluorescent signal from eCFP-NCCT showed that changes WNK4-induced 22Na+ flux were accompanied by parallel changes in surface membrane expression of the cotransporter. * P< 0.05 vs assay without an SB inhibitor.
	Fig. (4). Concentration response curves showing reversal of WNK4 T261G inhibition of NCCT 22Na+ flux in Xenopus oocytes by (A) SB 202190 and (B) SB203580. The fitted EC50 values (concentration of inhibitor producing 50% reversal) were respectively 147 and 109 nM.