Identification of Phosphorylation-Dependent Binding Partners of Aquaporin-2 Using Protein Mass Spectrometry

Abstract

Vasopressin-mediated control of water permeability in the renal collecting duct occurs in part through regulation of the distribution of aquaporin-2 (AQP2) between the apical plasma membrane and intracellular membrane compartments. Phosphorylation of Ser-256 at AQP2's cytoplasmic COOHterminus is well-accepted as a critical step for translocation. The aim of this study was to identify binding partners to phosphorylated versus nonphosphorylated forms of the AQP2 COOH-terminus via a targeted comparative proteomic approach. Cytosol from inner medullary collecting ducts isolated from rat kidneys was incubated with "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. Mass spectrometric results were confirmed by immunoblotting. Immunoprecipitation was performed using an AQP2 COOH-terminal antibody combined with immunblotting against the proposed binding partners in order to demonstrate interactions with native AQP2. Our studies confirmed previously identified interactions between AQP2 and hsc70, hsp70−1 and −2, as well as annexin II. These proteins were found to bind less to the Ser-256-phosphorylated AQP2 than to the non-phosphorylated form. In contrast, another heat shock protein, hsp70−5 (BiP/ grp78), bound to phosphorylated AQP2 more avidly than to non-phosphorylated AQP2. 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 studies showed partial colocalization of BiP with AQP2 in non-ER compartments. These results suggest that phosphorylation of AQP2 at Ser-256 may regulate AQP2 trafficking in part by mediating differential binding of hsp70 family proteins to the COOH-terminal tail.

Phosphorylation and ubiquitylation are opposing players in regulating endocytosis of the water channel Aquaporin-2

“…Selectively inhibiting PP2A or PP2B using endothall, fostriecin, cantharidic acid or deltamethrin did not result in significant changes in pS269 levels, suggesting that PP1 is the most likely protein phosphatase targeting pS269-AQP2 in vivo. Our results are in line with other studies that suggest a role for PP1 in regulation of AQP2 phosphorylation (Moeller et al, 2010;Zwang et al, 2009). A functional role for protein phosphatases in regulation of AQP2 has previously been suggested, although the effects of okadaic acid observed were attributed to a depolymerizing effect (disruption) on the actin cytoskeleton (Valenti et al, 2000).…”

Vasopressin increases S261 phosphorylation in AQP2-P262L, a mutant in recessive nephrogenic diabetes insipidus

“…Similarly, the different running patterns of AQP2-E258K variants could also not be explained by differences in their masses [12,13]. Moreover, studies using circular dichroism showed that the secondary structure of non-phosphorylated peptides of the AQP2 C-terminus differs from the structure of peptides phosphorylated at S256 or S261 [29].…”

Phosphorylation of aquaporin-2 regulates its endocytosis and protein–protein interactions

“…Polyphosphorylation of AQP2 does not affect the relative unit water permeability of the channel (12), so it is unlikely to be involved in channel gating. Another hypothesis is that phosphorylation can influence AQP2 interactions with proteins involved in exocytosis or endocytosis (13), and several proteins have been identified that bind to AQP2, including Hsp70, Hsc70, clathrin, dynamin, AP2 (14), MAL (15), Spa-1 (16), lip 5 (17), annexin (18), Hsp50-5(BiP/ Grp78), and PP1 (13).…”