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Phosphorylation of Serine‐256 is essential for downstream polyphosphorylation of AQP2
Author(s) -
Møller Hanne B,
Hoffert Jason D,
Rützler Michael,
Praetorius Jeppe,
Knepper Mark A,
Fenton Robert A
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.934.20
Subject(s) - phosphorylation , aquaporin 2 , microbiology and biotechnology , serine , transfection , phosphorylation cascade , forskolin , chemistry , biology , protein phosphorylation , biochemistry , protein kinase a , in vitro , water channel , gene , mechanical engineering , engineering , inlet
Phosphorylation of AQP2 at S256 is essential for its membrane targeting. Recently, we determined that S261, S264 and S269 residues are also phosphorylated by AVP and have begun to examine their role in AQP2 trafficking. MDCK cells stably expressing constitutively non‐phosphorylated forms of AQP2 demonstrated that single mutations (S261A, S264A and S269A) had no significant effect on apical membrane targeting following either AVP or forskolin treatment. A mutation mimicking constitutively phosphorylated AQP2 at position S269 (S269D) resulted in predominantly apical membrane localization of AQP2 even in the absence of stimulation. Osmotically induced swelling assays determined that rates of swelling in S269D‐AQP2 mutants were increased relative to wt‐AQP2 or S269A‐AQP2. Time course studies determined that S256‐AQP2 phosphorylation occurs first, raising the possibility that downstream phosphorylation is dependent on S256 phosphorylation. MDCK cells stably transfected with S256A‐AQP2 showed an elimination of S264 or S269 phosphorylation, whereas WT‐AQP2 or S256D‐AQP2 transfected cells exbihited a high level of phosphorylation at these sites. Thus, these data are compatible with the view that S256 phosphorylation is required for downstream phosphorylation.