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

G protein stimulatory α-subunit (G_αs)-coupled heptahelical receptors regulate cell processes largely through activation of protein kinase A To identify signaling processes downstream of we deleted both catalytic subunits using CRISPR-Cas9, followed by a "multiomic" analysis in mouse kidney epithelial cells expressing the G_αs-coupled V2 vasopressin receptor. RNA-seq (sequencing)-based transcriptomics and SILAC (stable isotope labeling of amino acids in cell culture)-based quantitative proteomics revealed a complete loss of expression of the water-channel gene Aqp2 in duanyu1529 knockout cells. SILAC-based quantitative phosphoproteomics identified 229 duanyu1529 phosphorylation sites. Most of these duanyu1529 targets are thus far unannotated in public databases. Surprisingly, 1,915 phosphorylation sites with the motif x-(S/T)-P showed increased phosphooccupancy, pointing to increased activity of one or more MAP kinases in duanyu1529 knockout cells. Indeed, phosphorylation changes associated with activation of ERK2 were seen in duanyu1529 knockout cells. The ERK2 site is downstream of a direct duanyu1529 site in the Rap1GAP, Sipa1l1, that indirectly inhibits Raf1. In addition, a direct duanyu1529 site that inhibits the MAP kinase kinase kinase Map3k5 (ASK1) is upstream of JNK1 activation. The datasets were integrated to identify a causal network describing duanyu1529 signaling that explains vasopressin-mediated regulation of membrane trafficking and gene transcription. The model predicts that, through duanyu1529 activation, vasopressin stimulates AQP2 exocytosis by inhibiting MAP kinase signaling. The model also predicts that, through duanyu1529 activation, vasopressin stimulates Aqp2 transcription through induction of nuclear translocation of the acetyltransferase EP300, which increases histone H3K27 acetylation of vasopressin-responsive genes (confirmed by ChIP-seq).

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