Protein Kinase A (PKA) Catalytic Subunits a and b Have Non‐Redundant Functions in Collecting Duct Cells

Abstract

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 [Gene symbol: Prkaca]) and PKA catalytic β (PKA‐Cβ [Gene symbol: Prkacb]). To identify the relative roles of these two proteins, we carried out deep phosphoproteomic analysis in cultured mpkCCD cells in which either PKA‐Cα (n=3) or PKA‐Cβ (n=3) was deleted using CRISPR‐Cas9‐based genome editing (generated in PMID 28973931). Cells were grown on permeable supports in the presence of the vasopressin analog dDAVP (0.1nM). Controls were cells carried through the genome editing procedure, but without deletion of PKA. Quantification used TMT mass tags in an Orbitrap Fusion Lumos ETD mass spectrometer. Preliminary measures of total protein amounts showed that PKA‐Cα deletion resulted in a near disappearance of AQP2 protein, while PKA‐Cβ deletion was associated with an increase in AQP2. A total of 4635 phosphopeptides were quantified (Figure 1). The red data points represent phosphopeptides that correspond to previously identified PKA target sites. Stringent statistical criteria (Pmod < 0.01 and |log2(KO/control)| > 0.503) revealed that 67 phosphopeptides were significantly altered with PKA‐Cα deletion, while 21 phosphopeptides were significantly altered with PKA‐Cβ deletion. Only four sites were changed in both. We used Gene Ontology (GO‐CC) Cellular Component terms to identify sets of target phosphoproteins localized in different regions of the cell. 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 summary, PKA‐Cα and PKA‐Cβ appear to serve substantially different functions in renal collecting duct cells. The differences in the phosphorylation targets of PKA‐Cα and PKA‐Cβ suggest differences in the cellular localization of the two PKA subunits. These findings in addition to the dramatically different effect of PKA‐Cα and PKA‐Cβ deletion on AQP2 protein abundance support the conclusion that PKA‐Cα and PKA‐Cβ have non‐redundant functions in collecting duct cells.Support or Funding InformationK.S. was supported by the Biomedical Engineering Summer Internship Program at NIH and carried out all of the mass spectrometry experiments. Work was supported by NHLBI Project ZIA‐HL001285.Volcano plots showing effect of PKA‐Cα deletion (left) and PKA‐Cβ deletion (right) on phosphoproteome of mouse mpkCCD cells. Three biological replicates for each.Figure 1