Abstract: FR-OR128
Global Identification of Protein Phosphorylation Changes Following CRISPR/Cas9-Deletion of cAMP-Dependent Protein Kinase (PKA) in Collecting Duct Cells
Session Information
- Urinary Concentration and Acidification
November 03, 2017 | Location: Room 290, Morial Convention Center
Abstract Time: 05:06 PM - 05:18 PM
Category: Fluid, Electrolytes, and Acid-Base
- 702 Water/Urea/Vasopressin, Organic Solutes
Authors
- Isobe, Kiyoshi, NHLBI/NIH, Bethesda, Maryland, United States
- Jung, Hyun Jun, NHLBI/NIH, Bethesda, Maryland, United States
- Yang, Chin-Rang, NHLBI/NIH, Bethesda, Maryland, United States
- Burg, Maurice B., NHLBI/NIH, Bethesda, Maryland, United States
- Raghuram, Viswanathan, NHLBI/NIH, Bethesda, Maryland, United States
- Knepper, Mark A., NHLBI/NIH, Bethesda, Maryland, United States
Background
Vasopressin regulates water and sodium transport in collecting duct principal cells by binding to the V2 receptor and increasing cAMP, thereby activating cAMP-regulated protein kinase PKA catalytic subunits PKA-Cα and/or PKA-Cβ. Signaling downstream from PKA is poorly understood.
Methods
To identify PKA-dependent phosphorylation changes, we deleted both PKA catalytic subunits using CRISPR-Cas9 in vasopressin-sensitive mpkCCD cells. Indel mutations were confirmed by Sanger sequencing. We carried out large-scale quantitative phosphoproteomic analysis using protein mass spectrometry in three pairs of PKA-knockout (KO) vs. control clones. The cells were grown on permeable supports in the presence of 0.1 nM dDAVP.
Results
The PKA-KO cells maintained viability and polarity. Phosphoproteomics identified 229 PKA substrate sites. These sites contained the motif R-(R/K)-X-pS and were significantly decreased in PKA-KO. Most of these PKA targets are not annotated in public databases. Surprisingly, a large number of phosphorylation sites with the motif X-(pS/pT)-P showed increased phospho-occupancy, pointing to increased activity of one or more MAP kinases in PKA-KO cells. Indeed, a marked increase in phosphorylation of ERK2 at T183 and Y185 (which activates ERK2) was seen in PKA-KO cells. The ERK2 site is downstream from a direct PKA site in Sipa1l1, which indirectly inhibits Raf1 through Rap1 inactivation. Aquaporin-2 phosphorylation at S256 was not decreased in PKA-KO cells. The datasets were integrated to identify a causal network describing PKA signaling that explains vasopressin’s actions to regulate membrane trafficking and gene transcription. The model predicts that, through PKA activation and inhibition of MAP kinase signaling, vasopressin stimulates AQP2 exocytosis (confirmed by immunofluorescence microscopy), induces nuclear translocation of the transcriptional co-activator/acetyltransferase EP300 (confirmed by immunoblotting of nuclear fractions) and increases histone H3K27 acetylation of vasopressin-responsive genes (confirmed by ChIP-Seq).
Conclusion
We conclude that PKA-dependent signaling is more complex than previously believed with both primary and secondary effects on phosphorylation that explain vasopressin responses.
Funding
- Other NIH Support