Salhadar, K. | bioRxiv Science

bioRxiv2020

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

Background 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.

systems biology↗

bioRxiv2020

Protein kinase A catalytic-α and catalytic-β proteins have non-redundant functions

Vasopressin regulates osmotic water transport in the renal collecting duct by PKA-mediated control of the water channel aquaporin-2 (AQP2). Collecting duct principal cells express two seemingly redundant PKA catalytic subunits, PKA catalytic (PKA-C) and PKA catalytic {beta} (PKA-C{beta}). To identify the roles of these two protein kinases, we carried out deep phosphoproteomic analysis in cultured mpkCCD cells in which either PKA-C or PKA-C{beta} was deleted using CRISPR-Cas9-based genome editing. Controls were cells carried through the genome editing procedure, but without deletion of PKA. TMT mass tagging was used for protein mass spectrometric quantification. Of the 4635 phosphopeptides that were quantified 67 were significantly altered in abundance with PKA-C deletion, while 21 were significantly altered in abundance with PKA-C{beta} deletion. However, only four sites were changed in both. The target proteins identified in PKA-C-null cells were largely associated with cell membranes and membrane vesicles, while target proteins in the PKA-C{beta}-null cells were largely associated with the actin cytoskeleton and cell junctions. In contrast, in vitro incubation of mpkCCD proteins with recombinant PKA-C and PKA-C{beta} resulted in virtually identical phosphorylation changes. In addition, analysis of total protein abundances in the in vivo samples showed that PKA-C deletion resulted in a near disappearance of AQP2 protein, while PKA-C{beta} deletion did not decrease AQP2 abundance. We conclude that PKA-C and PKA-C{beta} serve substantially different functions in renal collecting duct cells and that differences in phosphorylation targets may be due to differences in protein interactions, e.g. mediated by AKAP, C-KAP or PDZ binding.

systems biology↗