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Novel vasopressin‐regulated phosphorylation site of the NaCl co‐transporter, NCC
Author(s) -
Rosenbaek Lena Lindtoft,
Knepper Mark A,
Fenton Robert A
Publication year - 2011
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.25.1_supplement.1038.27
Subject(s) - vasopressin , phosphorylation , transporter , chemistry , microbiology and biotechnology , endocrinology , biochemistry , biology , gene
Using large‐scale LC‐MS/MS‐based phosphoproteomic profiling of biochemically isolated membranes from the rat renal cortex we identified a novel phosphorylation site (S124) in NCC. Bioinformatic studies suggested the neighbouring S127 site as also highly likely to be phosphorylated. Immunoblotting with a novel pS124‐NCC antibody demonstrated a band of ~ 160 kDa in the kidney cortex, but not medulla, which was preabsorbed by a corresponding phosphorylated peptide. Confocal microscopy with segment‐specific markers localized pS124‐NCC to all DCT cells, with greater abundance in the early DCT. Double immunogold electron microscopy with total NCC revealed that pS124‐NCC was associated predominantly with the apical plasma membrane of DCT cells, although some labelling was associated with intracellular vesicles. Acute, but not long‐term, vasopressin treatment significantly increased pS124‐NCC abundance at the plasma membrane. Similar observations were apparent after rats were fed a low salt diet. Kinase profiling assays using a synthetic peptide corresponding to the region surrounding S124 against the protein kinases mTOR/FRAP, ERK5, NLK, p38d, and ERK2 showed either weak or no significant activity. Funded by Lundbeck.