In vitro phosphorylation of synthetic AQP2 peptide by candidate kinases

Vasopressin-regulated water channel protein aquaporin-2 (AQP2) plays an essential role in water transport across kidney collecting duct. Vasopressin regulates AQP2 by triggering phosphorylation changes in four serine residues at its carboxyl-terminal end (S256, S261, S264, S269). Phosphorylation of AQP2 at S269 is known to potentiate AQP2 localization at the apical plasma membrane of collecting duct cells, rendering cell membrane permeable to water.  However, the protein kinase(s) responsible for S269 phosphorylation following vasopression stimulation remain unknown.  To address this question, we first performed Bayes' analysis to rank all mammalian protein kinases with regard to their likelihood of phosphorylating AQP2 at S269 using multi -omics datasets integration methods. We then used the list of prioritized kinases to choose a set of commerically available recombinant kinases to test their ability to phosphorylate synthetic AQP2 peptide in vitro.

Synthetic AQP2 peptide (QSVELHSPQSLPRGSKA) corresponding to the previously identified phosphorylation sites with or without pre-phosphorylation at S256 were used in in vitro phosphorylation experiments. In addition, as positive controls, a mixture of peptides consisting of all commercially available control peptide substrates for the kinases tested was also included. Synthetic peptides, 2 nmol each, were incubated with ATP and reaction buffer in the presence or absence of 0.5 μM of active recombinant kinases in a total reaction volume of 30 μl and incubated at 30°C for 1 h. Reactions were stopped by adding 70 μl water to reaction tube and filtering through a 30K molecular weight cut-off Amicon centrifuge filter at 14,000 x g for 30 min. The filtrate was dried and subjected to Zip-Tip cleaning for desalting before mass spectrometry analysis (LC-MS/MS).  

LC-MS/MS was carried out in an Orbitrap Fusion Lumos mass spectrometer (Thermo Scientific) using data-dependent proteomic analysis (DDA) method. MS spectrum raw files were searched for AQP2 phosphorylation at S256, S261, S264, S269 sites using MaxQuant 1.6.4.0.