Phosphopeptides in IMCD aquaporins (See below for narrative)
*Peptides in blue satisfied dual criteria: |Mean log2(dDAVP/Control)| > 0.342 and FDR < 0.005. The value 0.342 defines the 95% confidence interval for control:control comparisons.
§, Phosphorylated amino acid indicated by asterisk *; ?, phosphorylation site (ambiguous site assignment); ^, carbamidomethyl; @, oxidation; #, deamidation; †, False Discovery Rate
| Gene Symbol | UniProt no. | Residue(s) | Annotation§ | Peptide | Log2(dDAVP/Vehicle) | FDR† |
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Vasopressin-mediated changes in AQP2 and AQP4 phosphorylation. Vasopressin binding triggers changes in phosphorylation of the apical water channel AQP2 at four sites, causing increases in phosphorylation of Ser256 [(Christensen, 2000), (Hoffert, 2006)], Ser264 [(Hoffert, 2006), (Fenton, 2008)] and Ser269 [(Hoffert, 2006), (Hoffert, 2008), (Moeller, 2009)] and a decrease in phosphorylation of Ser261 [(Hoffert, 2006), (Hoffert, 2007)]. All of these sites were detected and the changes that were observed are consistent with prior data.
Multiple phosphorylation sites were detected in AQP4, present in the basolateral plasma membrane of IMCD cells (Terris, 1995). Genetic deletion of AQP4 in mice reduced transepithelial water permeability in isolated perfused IMCDs by 77%, establishing that AQP4 is the dominant pathway for basolateral water movement in the IMCD (Chou, 1998). Of interest, three AQP4 phosphopeptides in the COOH-terminal tail of AQP2 showed significant increases in response to dDAVP (double phosphopeptide at S285/T289, double phosphopeptide at S276/T289, and monophosphopeptide at S315). A prior study found protein kinase A-mediated phosphorylation of AQP4 in gastric parietal cells although the phosphorylation specific site was not identified (Carmosino, 2007). Mutations of S276, S285 or S315 to alanine did not affect water channel gating when expressed in Xenopus oocytes (Assentoft, 2014), although an indirect effect of phosphorylation changes on AQP4-mediated water transport, e.g. via membrane trafficking, cannot be ruled out.