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PP1 and PP2a phosphatases can modulate AQP2 endocytosis
Author(s) -
Moeller Hanne B.,
Aroankins Takwa S.,
Fenton Robert A.
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.885.17
Subject(s) - aquaporin 2 , internalization , forskolin , phosphorylation , microbiology and biotechnology , endocytosis , okadaic acid , intracellular , aquaporin , phosphatase , chemistry , biology , water channel , biochemistry , cell , receptor , mechanical engineering , engineering , inlet
Membrane accumulation of aquaporin‐2 (AQP2) is regulated by phosphorylation at S256 and S269. Our previous studies showed that mimicking phosphorylation at S256 and S269 (256D‐ and 269D‐AQP2) decreased internalization rates of AQP2 in MDCK cells. In cell lines expressing double mutated AQP2, 256D/269A‐AQP2 and 256D/269D‐AQP2 were predominantly located in the plasma membrane, whereas 256A/269A‐AQP2 and 256A/269D‐AQP2 had an intracellular localization. This suggests that pS256 is dominant for determining AQP2 localization. Cells expressing 256D/269A‐ or 256D/269D‐AQP2 had decreased AQP2 internalization compared to wt‐AQP2; with 256D/269D‐AQP2 being internalized slowest. To examine pS269 and pS256 in a dynamic system, wt‐AQP2 cells were treated with the phosphatase inhibitors Okadaic Acid and Calyculin A. Treatment decreased the rate of AQP2 endocytosis in response to forskolin removal. In parallel, pS269‐AQP2 levels were greatly increased and prolonged compared to control, whereas only minor changes in pS256‐AQP2 was observed. To examine if phosphorylation at S269 regulates AQP2‐ ubiquitination at K270, immunoprecipitations of N‐terminal flag‐tagged wt‐AQP2, 269A‐AQP2, and 269D‐AQP2 were assessed for ubiquitination. All AQP2 forms were ubiquitinated following either forskolin or combined forskolin and TPA treatment.