Dataset Information


PKA signaling network-quantitative proteomics and phosphoproteomics

ABSTRACT: Classically, connections within a complex network can be identified through systematically removing its components one at a time and observing the resulting functional changes throughout the network. We applied this concept to identify the signaling network downstream from protein kinase A (PKA) in epithelial cells expressing the Gs-coupled V2 vasopressin receptor. Both genes coding for PKA catalytic subunits (Prkaca and Prkacb) were modified using CRISPR-Cas9 to introduce indels resulting in the absence of detectable PKA catalytic subunit protein (immunoblotting and SILAC-based quantitative protein mass spectrometry). Analysis of multiple PKA double knockout (dKO) lines using SILAC-based quantitative phosphoproteomics showed that phosphorylation sites with decreased phospho-occupancy were dominated by the basophilic motif (R/K)-(R/K)-x-S, consistent with that seen for previously identified PKA targets. Overall, 233 PKA target sites were identified, the majority of which are not annotated as PKA sites in public databases. In addition, we identified a large number of sites with increased phospho-occupancy and the motif x-(S/T)-P, consistent with activation of one or more CMGC family kinases in response to PKA deletion. An unexpected finding was a complete, selective loss of expression of the Aqp2 gene (coding for a kidney-specific water channel) with PKA deletion observed both with quantitative proteomics and RNA-Seq based transcriptomics. Using large-scale data integration techniques, the quantitative proteomic, phosphoproteomic, and RNA-Seq datasets were integrated with prior data from the literature to identify a PKA signaling network that explains most of the cellular physiological responses to vasopressin in the target cells, including the regulation of Aqp2 gene transcription.


INSTRUMENT(S): Orbitrap Fusion ETD

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Epithelial Cell, Cell Culture


LAB HEAD: Mark A Knepper

PROVIDER: PXD005938 | Pride | 2017-11-23


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Systems-level identification of PKA-dependent signaling in epithelial cells.

Isobe Kiyoshi K   Jung Hyun Jun HJ   Yang Chin-Rang CR   Claxton J'Neka J   Sandoval Pablo P   Burg Maurice B MB   Raghuram Viswanathan V   Knepper Mark A MA  

Proceedings of the National Academy of Sciences of the United States of America 20171002 42

G protein stimulatory α-subunit (G<sub>αs</sub>)-coupled heptahelical receptors regulate cell processes largely through activation of protein kinase A (PKA). To identify signaling processes downstream of PKA, we deleted both PKA catalytic subunits using CRISPR-Cas9, followed by a "multiomic" analysis in mouse kidney epithelial cells expressing the G<sub>αs</sub>-coupled V2 vasopressin receptor. RNA-seq (sequencing)-based transcriptomics and SILAC (stable isotope labeling of amino acids in cell c  ...[more]

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