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 α (PKA-Cα) and PKA catalytic β (PKA-Cβ). To identify the roles of these two protein kinases, we carried out deep phosphoproteomic analysis in cultured mpkCCD cells in which either PKA-Cα or PKA-Cβ 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-Cα deletion, while 21 were significantly altered in abundance with PKA-Cβ deletion. However, only four sites were changed in both. The target proteins identified in PKA-Cα-null cells were largely associated with cell membranes and membrane vesicles, while target proteins in the PKA-Cβ-null cells were largely associated with the actin cytoskeleton and cell junctions. In contrast, in vitro incubation of mpkCCD proteins with recombinant PKA-Cα and PKA-Cβ resulted in virtually identical phosphorylation changes. In addition, analysis of total protein abundances in the in vivo samples showed that PKA-Cα deletion resulted in a near disappearance of AQP2 protein, while PKA-Cβ deletion did not decrease AQP2 abundance. We conclude that PKA-Cα and PKA-Cβ 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. Competing Interest Statement The authors have declared no competing interest. Footnotes * https://hpcwebapps.cit.nih.gov/ESBL/Database/Supplemental_Data_PKA_sKO/...

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

10.1101/2020.07.01.182691