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WNK4 phosphorylates mouse thiazide‐sensitive Na‐Cl cotransporter NCC at threonine 48
Author(s) -
Na Tao,
Wu Guojin,
Peng Ji-Bin
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.867.38
Subject(s) - phosphorylation , threonine , kinase , protein kinase domain , serine , protein kinase a , cotransporter , chemistry , microbiology and biotechnology , biology , biochemistry , mutant , sodium , gene , organic chemistry
With‐No‐lysine (K) 4 (WNK4) is a serine/threonine kinase and mutations in WNK4 are associated with a genetic form of hypertension. WNK4 was shown to regulate the thiazide sensitive sodium chloride cotransporter NCC via SPAK and OSR1 kinases, which phosphorylate NCC and increase its transport activity. It is unclear whether WNK4 phosphorylates NCC directly. In this study, we purified human WNK4 kinase domain (amino‐acids 160–440) produced in E. coli. The WNK4 kinase domain was capable of phosphorylating GST‐fusion OSR1 D164A mutant. We then tested the ability of the WNK4 kinase domain in phosphorylating GST‐fusion mouse NCC cytoplasmic terminal regions using in vitro kinase assay. The NCC N‐terminal region (amino‐acids 1–135) was significantly phosphorylated by the WNK4 kinase domain whereas no strong phosphorylation was detected in the C‐terminal region (amino‐acids 604–1002). By truncation and site directed mutagenesis in the NCC N‐terminal region, threonine 48 was identified to be the main site of WNK4 phosphorylation in mouse NCC. Phospho‐mimicking mutation at this site (T48D) increased NCC‐mediated sodium uptake and NCC protein abundance in Xenopus oocytes by 84.2% and 106.5%, respectively. Furthermore, the phosphorylation of NCC 1–135 by OSR1 T185E was enhanced by the phospho‐mimicking mutation. In conclusion, direct phosphorylation of NCC by WNK4 may be involved in WNK4‐mediated regulation of NCC.