Phosphoproteomic Identification of Vasopressin-Regulated Protein Kinases in Collecting Duct Cells

Abstract

ABSTRACTBackground and PurposeThe peptide hormone vasopressin regulates water transport in the renal collecting duct largely via the V2 receptor, which triggers a cAMP-mediated activation of a protein kinase A (PKA)-dependent signaling network. The protein kinases downstream from PKA have not been fully identified or mapped to regulated phosphoproteins.Experimental ApproachWe carried out systems-level analysis of large-scale phosphoproteomic data quantifying vasopressin-induced changes in phosphorylation in aquaporin-2-expressing cultured collecting duct cells (mpkCCD). Quantification was done using stable isotope labeling (SILAC method).Key Results9640 phosphopeptides were quantified. Stringent statistical analysis identified significant changes in response to vasopressin in 429 of these phosphopeptides. The corresponding phosphoproteins were mapped to known vasopressin-regulated cellular processes. The vasopressin-regulated sites were classified according to the sequences surrounding the phosphorylated amino acids giving 11 groups distinguished predominantly by the amino acids at positions +1, −3, −2 and −5 relative to the phosphorylated amino acid. Among the vasopressin-regulated phosphoproteins were 25 distinct protein kinases. Among these, six of them plus PKA appeared to account for phosphorylation of more than 80% of the 313 vasopressin-regulated phosphorylation sites. The six downstream kinases were salt-inducible kinase 2 (Sik2), cyclin-dependent kinase 18 (PCTAIRE-3, Cdk18), calmodulin-dependent kinase kinase 2 (Camkk2), protein kinase D2 (Prkd2), mitogen-activated kinase 3 (ERK1; Mapk3), and myosin light chain kinase (Mylk).Conclusion and ImplicationsIn V2 receptor-mediated signaling, PKA is at the head of a complex network that includes at least 6 downstream vasopressin-regulated protein kinases that are prime targets for future study. The extensive phosphoproteomic data generated in this study is provided as a web-based data resource for future studies of G-protein coupled receptors.