Abstract: PO1394
Protein Kinase A Catalytic-α and Catalytic-β Proteins Have Non-Redundant Functions
Session Information
- Fluid, Electrolyte, and Acid-Base Disorders: Basic
October 22, 2020 | Location: On-Demand
Abstract Time: 10:00 AM - 12:00 PM
Category: Fluid, Electrolyte, and Acid-Base Disorders
- 901 Fluid, Electrolyte, and Acid-Base Disorders: Basic
Authors
- Raghuram, Viswanathan, National Institutes of Health, Bethesda, Maryland, United States
- Yang, Chin-Rang, National Institutes of Health, Bethesda, Maryland, United States
- Park, Euijung, National Institutes of Health, Bethesda, Maryland, United States
- Knepper, Mark A., National Institutes of Health, Bethesda, Maryland, United States
Background
Vasopressin regulates osmotic water transport in the renal collecting duct by PKA-mediated control of the water channel aquaporin-2 (AQP2). Collecting duct principal cells express two seemingly redundant PKA catalytic subunits, PKA catalytic α (PKA-Cα, Gene symbol: Prkaca) and PKA catalytic β (PKA-Cβ, Gene symbol: Prkacb). At an amino-acid level, the two are 91 percent identical and the catalytic domains are virtually identical. However, whether the two PKA catalytic subunits have redundant functions, as is implicitly assumed in many studies involving PKA-mediated regulation, has not been tested.
Methods
To identify the roles of these two protein kinases in in kidney collecting duct cells, either PKA-Cα or PKA-Cβ was deleted using CRISPR-Cas9-based genome editing. Controls were cells carried through the genome editing procedure, but without deletion of either PKA catalytic subunit. Protein mass spectrometry-based quantitative proteomics and phosphoproteomics was carried out in both PKA catalytic-α and PKA catalytic-β single knockouts cells. TMT mass tagging was used for protein mass spectrometric quantification.
Results
Of the 4635 phosphopeptides that were quantified 67 were significantly altered in abundance with PKA-Cα deletion, while 21 were significantly altered in abundance with PKA-Cβ deletion. However, only four sites were changed in both. The target proteins identified in PKA-Cα-null cells were largely associated with cell membranes and membrane vesicles, while target proteins in the PKA-Cβ-null cells were largely associated with the actin cytoskeleton and cell junctions. In contrast, in vitro incubation of mpkCCD proteins with recombinant PKA-Cα and PKA-Cβ resulted in virtually identical phosphorylation changes. In addition, analysis of total protein abundances in the in vivo samples showed that PKA-Cα deletion resulted in a near disappearance of AQP2 protein, while PKA-Cβ deletion did not decrease AQP2 abundance.
Conclusion
We conclude that PKA-Cα and PKA-Cβ serve substantially different functions in renal collecting duct cells and that differences in phosphorylation targets may be due to differences in protein interactions, e.g. mediated by AKAP, C-KAP or PDZ binding.
Funding
- Other NIH Support