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Abstract: FR-PO626

Intracellular Sites of AQP2 S256 Phosphorylation in the Plasma Membrane, Cytoplasmic Vesicles, and the Trans Golgi Network Identified Using Inhibitors of the AQP2 Recycling Itinerary

Session Information

Category: Fluid and Electrolytes

  • 901 Fluid and Electrolytes: Basic

Authors

  • Bouley, Richard, Massachusetts General Hospital, Boston, Massachusetts, United States
  • Cheung, Pui Susan Wen, Massachusetts General Hospital, Boston, Massachusetts, United States
  • Brown, Dennis, Massachusetts General Hospital, Boston, Massachusetts, United States

Group or Team Name

  • Program in Membrane Biology
Background

Vasopressin-regulated trafficking of aquaporin 2 between cytoplasmic vesicles and the plasma membrane of kidney principal cells is essential for body water homeostasis.VP-induced phosphorylation of AQP2 at serine residue S256 is required for its accumulation at the cell membrane, but the intracellular location(s) where this phosphorylation occurs remains poorly understood. Here, we used strategies to block AQP2 trafficking at different cellular locations in LLC-PK1 cells, and we monitored phosphorylation of AQP2 S256 at these sites using anti-phospho S256 antibodies after vasopressin/forskolin (VP/FK) stimulation.

Methods

Phosphorylation extent and location were assessed by western blotting and immunocytochemistry, respectively.

Results

Methyl-b-cyclodextrin (MBCD) treatment blocks endocytosis, and recycling AQP2 accumulates at the cell surface without an increase in S256 phosphorylation. However, VP/FK applied to MBCD treated cells resulted in a significant increase in S256 phosphorylation, indicating AQP2 can be phosphorylated when present in the plasma membrane. Taxol, an inhibitor of microtubule function, results in AQP2 containing vesicles being scattered throughout the cytoplasm, and inhibits VP-induced membrane accumulation of AQP2. Taxol alone did not affect AQP2 phosphorylation, but VP/FK treatment of taxol exposed cells caused a significant increase in S256 phosphorylation, indicating that AQP2 can be phosphorylated on scattered cytoplasmic vesicles. Finally, AQP2 trafficking is blocked in the peri-nuclear, trans-Golgi network both by incubating cells at 20°C for 2 h or by using the V-ATPase inhibitor bafilomycin. VP/FK stimulated AQP2 phosphorylation significantly under both conditions.

Conclusion

These findings suggest that the VP/FK induced phosphorylation of AQP2 at S256 can occur at various locations during its recycling itinerary to and from the cell surface; at the plasma membrane itself, on cytoplasmic vesicles, or in the trans-Golgi network. Whether protein kinase A is involved in AQP2 S256 phosphorylation in all these locations is unclear, but the ability to dissect different intracellular phosphorylation stations may help to uncover new strategies to regulate AQP2 trafficking in conditions such as nephrogenic diabetes insipidus and hyponatremia.

Funding

  • NIDDK Support