Kidney Week

Abstract: SA-PO1017

Systems-Level Identification of PKA-Independent Vasopressin Signaling in Renal Epithelial Cells

Session Information

• Fluid and Electrolytes: Basic - II
  October 27, 2018 | Location: Exhibit Hall, San Diego Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Fluid and Electrolytes

• 901 Fluid and Electrolytes: Basic

Authors

• Datta, Arnab, NHLBI, NIH, Bethesda, Maryland, United States

Arnab Datta,

• Yang, Chin-Rang, NHLBI, NIH, Bethesda, Maryland, United States

Chin-Rang Yang,

• Raghuram, Viswanathan, NHLBI, NIH, Bethesda, Maryland, United States

Viswanathan Raghuram,

• Knepper, Mark A., NHLBI, NIH, Bethesda, Maryland, United States

Mark A. Knepper,

Background

Vasopressin signaling in the renal collecting duct is believed to be mediated predominantly by the activation of protein kinase A (PKA). The recent generation of mouse cell lines (“PKA dKO” cells), derived from mpkCCD cells, in which both PKA catalytic subunit genes have been deleted via CRISPR-Cas9 affords us an opportunity to identify PKA-independent signaling.

Methods

We used protein mass spectrometry to profile proteome-wide phosphorylation changes in response to vasopressin (dDAVP, 0.1 nM, 30 min) in PKA dKO cells and in control mpkCCD cells. dDAVP-treated and vehicle-treated cells were metabolically labeled using the SILAC method to allow quantification of dDAVP-induced changes in tryptic phospho-peptide abundances in an Orbitrap Fusion Lumos Tribrid mass spectrometer. Experiments were done in triplicate.

Results

Overall, >13,000 unique phosphopeptides were quantified in both PKA dKO and control cells. In control cells, 691 distinct phosphopeptides were altered in abundance in response to dDAVP. In contrast, in PKA dKO cells, only 73 phosphopeptides were altered in abundance, indicating that a large component of the vasopressin response is PKA dependent. However, altered phosphorylation in response to dDAVP in PKA dKO cells is indicative of substantial PKA-independent signaling. Notably, phosphorylation of Ser256 of AQP2 (thought to be a PKA site) increased in response to dDAVP in PKA dKO cells, indicating that other kinases can phosphorylate this site. The upregulated phosphorylation sites in PKA dKO cells mapped to an X-R-(A/S/T)-X-S*-X motif, consistent with activation of other basophilic kinases such as SGK, PKG, CAMK2B or PAK2. Interestingly, cAMP measurements showed that baseline cAMP levels in PKA dKO cells, were ~10-fold higher than in control mpkCCD cells, although both PKA dKO and control cells showed significant increases in response to dDAVP. This indicates that there is likely to be a PKA-dependent feedback on some component of the Avpr2-G_αs-Adcy6 signaling pathway responsible for cAMP generation.

Conclusion

In cultured mouse principal cells: (1) the signaling response to vasopressin is largely PKA-dependent; (2) a smaller component of the vasopressin response is PKA-independent; (3) Ser256 of AQP2 can be phosphorylated by kinases other than PKA; (4) PKA is responsible for a feedback inhibition of cAMP generation.

Funding

• Other NIH Support