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Characterization of a novel phosphorylation site in the sodium–chloride cotransporter, NCC
Author(s) -
Rosenbaek L. L.,
Assentoft M.,
Pedersen N. B.,
MacAulay N.,
Fenton R. A.
Publication year - 2012
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1113/jphysiol.2012.240986
Subject(s) - distal convoluted tubule , phosphorylation , kinase , endocrinology , medicine , chemistry , microbiology and biotechnology , biology , biochemistry , kidney , nephron
Key points• The sodium–chloride cotransporter, NCC, is essential for renal electrolyte balance and its function can be regulated by protein phosphorylation • Here we report the role and regulation of a novel phosphorylation site in NCC at Ser124 • Ser124 phosphorylation plays a role in mediating full NCC transport activity, but does not seem to be involved in NCC trafficking • Various physiological stimuli such as vasopressin and aldosterone regulate the abundance of the Ser124 phosphorylation status and other phosphorylation sites in NCC • Unlike other known phosphorylation sites in NCC, the STE20/SPS1‐related proline–alanine‐rich kinase and oxidative stress‐response kinases (SPAK and OSR1) were not able to phosphorylate NCC at Ser124 • The results demonstrate that phosphorylation of NCC is a major factor in determining the function of NCC under various physiological conditionsAbstract The sodium–chloride cotransporter, NCC, is essential for renal electrolyte balance. NCC function can be modulated by protein phosphorylation. In this study, we characterized the role and physiological regulation of a novel phosphorylation site in NCC at Ser124 (S124). Novel phospho‐specific antibodies targeting pS124‐NCC demonstrated a band of 160 kDa in the kidney cortex, but not medulla, which was preabsorbed by a corresponding phosphorylated peptide. Confocal microscopy with kidney tubule segment‐specific markers localized pS124‐NCC to all distal convoluted tubule cells. Double immunogold electron microscopy demonstrated that pS124‐NCC co‐localized with total NCC in the apical plasma membrane of distal convoluted tubule cells and intracellular vesicles. Acute treatment of Munich–Wistar rats or vasopressin‐deficient Brattleboro rats with the vasopressin type 2 receptor‐specific agonist dDAVP significantly increased pS124‐NCC abundance, with no changes in total NCC plasma membrane abundance. pS124‐NCC levels also increased in abundance in rats after stimulation of the renin–angiotensin–aldosterone system by dietary low sodium intake. In contrast to other NCC phosphorylation sites, the STE20/SPS1‐related proline–alanine‐rich kinase and oxidative stress‐response kinases (SPAK and OSR1) were not able to phosphorylate NCC at S124. Protein kinase arrays identified multiple kinases that were able to bind to the region surrounding S124. Four of these kinases (IRAK2, CDK6/Cyclin D1, NLK and mTOR/FRAP) showed weak but significant phosphorylation activity at S124. In oocytes, 36 Cl uptake studies combined with biochemical analysis showed decreased activity of plasma membrane‐associated NCC when replacing S124 with alanine (A) or aspartic acid (D). In novel tetracycline‐inducible MDCKII‐NCC cell lines, S124A and S124D mutants were able to traffic to the plasma membrane similarly to wildtype NCC.