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PXD005938

PXD005938 is an original dataset announced via ProteomeXchange.

Dataset Summary
TitlePKA signaling network-quantitative proteomics and phosphoproteomics
DescriptionClassically, 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 Gs-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.
HostingRepositoryPRIDE
AnnounceDate2017-11-23
AnnouncementXMLSubmission_2017-11-23_01:10:44.xml
DigitalObjectIdentifier
ReviewLevelPeer-reviewed dataset
DatasetOriginOriginal dataset
RepositorySupportUnsupported dataset by repository
PrimarySubmitterCHIN-RANG YANG
SpeciesList scientific name: Mus musculus (Mouse); NCBI TaxID: 10090;
ModificationListphosphorylated residue; monohydroxylated residue; acetylated residue; deamidated residue; iodoacetamide derivatized residue
InstrumentOrbitrap Fusion ETD
Dataset History
RevisionDatetimeStatusChangeLog Entry
02017-02-20 03:10:30ID requested
12017-11-23 01:10:45announced
Publication List
Isobe K, Jung HJ, Yang CR, Claxton J, Sandoval P, Burg MB, Raghuram V, Knepper MA, Systems-level identification of PKA-dependent signaling in epithelial cells. Proc Natl Acad Sci U S A, 114(42):E8875-E8884(2017) [pubmed]
Keyword List
curator keyword: Biological
submitter keyword: Mouse, Kindey, PKA, Proteomics, Phosphorylation
Contact List
Mark A Knepper
contact affiliationEpithelial Systems Biology Laboratory National Heart, Lung, and Blood Institute National Institutes of Health U.S.A.
contact emailmark_knepper@nih.gov
lab head
CHIN-RANG YANG
contact affiliationNational Institutes of Health, USA
contact emailchin-rang.yang@nih.gov
dataset submitter
Full Dataset Link List
Dataset FTP location
PRIDE project URI
Repository Record List
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