PXD005938-1

PXD005938 is an original dataset announced via ProteomeXchange.

Title	PKA signaling network-quantitative proteomics and phosphoproteomics
Description	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 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.
HostingRepository	PRIDE
AnnounceDate	2017-11-23
AnnouncementXML	Submission_2017-11-23_01:10:44.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	CHIN-RANG YANG
SpeciesList	scientific name: Mus musculus (Mouse); NCBI TaxID: 10090;
ModificationList	phosphorylated residue; monohydroxylated residue; acetylated residue; deamidated residue; iodoacetamide derivatized residue
Instrument	Orbitrap Fusion ETD

Revision	Datetime	Status	ChangeLog Entry
0	2017-02-20 03:10:30	ID requested
⏵ 1	2017-11-23 01:10:45	announced

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]

curator keyword: Biological

submitter keyword: Mouse, Kindey, PKA, Proteomics, Phosphorylation

Mark A Knepper
contact affiliation	Epithelial Systems Biology Laboratory National Heart, Lung, and Blood Institute National Institutes of Health U.S.A.
contact email	mark_knepper@nih.gov
lab head
CHIN-RANG YANG
contact affiliation	National Institutes of Health, USA
contact email	chin-rang.yang@nih.gov
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD005938
     1. Label: PRIDE project
     2. Name: PKA signaling network-quantitative proteomics and phosphoproteomics