TY - JOUR
T1 - Systems-level identification of PKA-dependent signaling in epithelial cells
AU - Isobe, Kiyoshi
AU - Jung, Hyun Jun
AU - Yang, Chin Rang
AU - Claxton, J’Neka
AU - Sandoval, Pablo
AU - Burg, Maurice B.
AU - Raghuram, Viswanathan
AU - Knepper, Mark A.
N1 - Funding Information:
ACKNOWLEDGMENTS. This work was primarily funded by the Division of Intramural Research, National Heart, Lung, and Blood Institute (NHLBI) (Projects ZIA-HL001285 and ZIA-HL006129; to M.A.K.). The NHLBI Proteomics Core Facility (M. Gucek, Director), NHLBI DNA Sequencing Core Facility (Y. Li, Director), NHLBI Light Microscopy Core Facility (C. Combs, Director), and NHLBI Flow Cytometry Core Facility (P. McCoy, Director) were used. K.I. was supported by a Japan Society for the Promotion of Science Research Fellowship.
Publisher Copyright:
© 2017, National Academy of Sciences. All rights reserved.
PY - 2017/10/17
Y1 - 2017/10/17
N2 - G protein stimulatory α-subunit (Gαs)-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 Gαs-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 PKA knockout cells. SILAC-based quantitative phosphoproteomics identified 229 PKA phosphorylation sites. Most of these PKA 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 PKA knockout cells. Indeed, phosphorylation changes associated with activation of ERK2 were seen in PKA knockout cells. The ERK2 site is downstream of a direct PKA site in the Rap1GAP, Sipa1l1, that indirectly inhibits Raf1. In addition, a direct PKA 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 PKA signaling that explains vasopressin-mediated regulation of membrane trafficking and gene transcription. The model predicts that, through PKA activation, vasopressin stimulates AQP2 exocytosis by inhibiting MAP kinase signaling. The model also predicts that, through PKA 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).
AB - G protein stimulatory α-subunit (Gαs)-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 Gαs-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 PKA knockout cells. SILAC-based quantitative phosphoproteomics identified 229 PKA phosphorylation sites. Most of these PKA 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 PKA knockout cells. Indeed, phosphorylation changes associated with activation of ERK2 were seen in PKA knockout cells. The ERK2 site is downstream of a direct PKA site in the Rap1GAP, Sipa1l1, that indirectly inhibits Raf1. In addition, a direct PKA 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 PKA signaling that explains vasopressin-mediated regulation of membrane trafficking and gene transcription. The model predicts that, through PKA activation, vasopressin stimulates AQP2 exocytosis by inhibiting MAP kinase signaling. The model also predicts that, through PKA 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).
KW - CRISPR-Cas9
KW - Next-generation sequencing
KW - Phosphoproteomics
KW - Protein mass spectrometry
KW - Vasopressin
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U2 - 10.1073/pnas.1709123114
DO - 10.1073/pnas.1709123114
M3 - Article
C2 - 28973931
AN - SCOPUS:85031768756
SN - 0027-8424
VL - 114
SP - E8875-E8884
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 42
ER -