Identification of Phosphorylation‐Dependent Binding Partners of Aquaporin‐2 (AQP2) By Protein Mass Spectrometry

Abstract

Phosphorylation of Ser‐256 at AQP2's cytoplasmic COOH‐terminus is well‐accepted as a key step for regulated AQP2 trafficking. Here we identify binding partners to phosphorylated and nonphosphorylated forms of the AQP2 COOH‐terminus using mass spectrometry. Cytosol from inner medullary collecting ducts (IMCDs) isolated from rat kidneys was incubated with biotinylated "bait" peptides, representing the COOH‐terminal AQP2 tail in its nonphosphorylated and phosphorylated forms, to capture differentially‐bound proteins prior to LC‐MS/MS analysis and immunoblotting. We found and confirmed 7 proteins that bound preferentially to the nonphospho‐peptide (hsc70, hsp70‐1 and ‐2, annexin II, PP1c, GDI‐2, and RAN) and one protein that bound preferentially to the S256 phosphopeptide (hsp70‐5 or BiP). AQP2 interactions with hsc70, hsp70‐1 and ‐2, as well as annexin II were previously identified. Immunoprecipitation of AQP2 from native IMCD lysates confirmed interactions with annexin II, PP1c, and BiP. Immunogold EM studies demonstrated that BiP is present not only in the ER, but in the cytoplasm and apical plasma membrane of rat collecting duct cells. Furthermore, confocal immunofluorescence showed partial colocalization of BiP with AQP2 in non‐ER compartments. These results suggest that phosphorylation of AQP2 at Ser‐256 may regulate binding of hsp70 family proteins, including BiP, to the COOH‐terminal tail.