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Vasopressin regulates phosphorylation of aquaporin‐2 (AQP2) at ser‐264 and ser‐269
Author(s) -
Hoffert Jason,
Pisitkun Trairak,
McDill Brad,
Chen Feng,
Fenton Rob,
Knepper Mark
Publication year - 2008
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.22.1_supplement.934.21
Subject(s) - phosphorylation , vasopressin , aquaporin 2 , protein phosphorylation , phosphoproteomics , phosphoserine , chemistry , phosphopeptide , microbiology and biotechnology , biology , protein kinase a , endocrinology , serine , water channel , mechanical engineering , engineering , inlet
Arginine vasopressin increases phosphorylation of the water channel AQP2 at Ser‐256 (S256). We recently discovered two novel AQP2 phosphorylation sites, S264 and S269, via shotgun phosphoproteomics. Here, we generated two distinct phospho‐specific antibodies to these sites to assess changes in AQP2 phosphorylation. Through immunoblotting we detected significant increases in pS256 (t 1/2 = 41 sec), pS269 (t 1/2 = 3.2 min), and pS264 (t 1/2 = 4.2 min) following short‐term incubation of rat inner medullary collecting duct suspensions with 1 nM dDAVP. The magnitude of increase at steady state was: S256 3.0‐fold; S264 8.5‐fold; S269 14.9‐fold. Short‐term incubation with cpt‐cAMP (0.1 mM) produced nearly identical responses. Preincubation of tubules with the PKA inhibitor H89 (10 μM) prevented cAMP‐dependent increases in AQP2 phosphorylation at all 3 sites, suggesting coordinate regulation through the cAMP‐PKA pathway. However, incubation of synthetic AQP2 peptides with PKA in vitro resulted in phosphorylation of S256, but not S264 or S269. Both S264 and S269 phosphorylation failed to occur in mice with an S256L mutation, suggesting that S256 phosphorylation may be necessary for downstream phosphorylation events. These results demonstrate that all 3 sites are regulated by vasopressin, via cAMP‐PKA, but do not identify the kinases responsible for S264 and S269 phosphorylation. Support: ZO1‐HL001285.