CRISPR-Cas9/Phosphoproteomic Analysis of CAMKK2 Signaling in Collecting Duct

Background: Vasopressin regulates multiple physiological processes in collecting duct principal cells, including short- and long-term regulation of aquaporin-2 (AQP2). Prior phosphoproteomic studies have identified 45 proteins that undergo changes in phosphorylation in response to vasopressin, including Calcium/ Calmodulin-Dependent Protein Kinase Kinase 2 (CAMKK2). This protein kinase is known to be regulated both through changes in intracellular calcium and by phosphorylation. CAMKK2 is known to regulate AMP-activated protein kinases (AMPKs) and other SNF1-family kinases. In this study, we aim to elucidate the role of CAMKK2 in vasopressin signaling by identifying its substrates, determining its activity regulation, and identifying potential functional roles of CAMKK2 in collecting duct cells. Methods: We used CRISPR-Cas9 to generate multiple CAMKK2 knockout (KO) mpkCCD cell lines. Western blot analysis confirmed the absence of detectable CAMKK2 protein in KO clones, while Sanger sequencing confirmed indels at target sites. Control clones with intact CAMKK2 expression were included for comparative analysis. Both KO and control cell lines, treated with the V2R selective-vasopressin analog 1-desamino-8-D-arginine-vasopressin (dDAVP), were subjected to tandem mass tag (TMT) labeling-based quantitative proteomics and phosphoproteomics. A Thermo Orbitrap Eclipse™ mass spectrometer was used in data-dependent acquisition (DDA) mode. Results: General proteomics revealed that principal cell differentiation markers (AQP2, a-ENaC, FXYD4, GATA3, TMEM45B, LGALS3) showed no significant change in total abundance in CAMKK2 KO cells. However, several key transcription factors exhibited significant decreases in abundance (TCF7L2, TEAD2, SOX4, FOXJ2, FOXA1). The phosphoproteomic analysis identified 10,319 quantifiable phosphorylation sites of which 119 were significantly changed according to stringent criteria (29 increased; 90 decreased). The analysis demonstrated no significant changes in AQP2 phosphorylation. Analysis using PTM-Logo shows a preference for lysine or arginine in position -3 (relative to the phosphorylation site) and leucine in +4 in the downregulated phosphorylation sites. Although this sequence does not match the expected target sequence for CAMKK2 based on prior studies, it is a strong match for the sequence preference for SNF1-family kinases including AMPKs and Salt-Inducible Kinases. This suggests that CAMKK2 plays an important indirect role in collecting duct cells to activate SNF1-family kinases. Analysis of Gene Ontology Biological Process terms enriched among all regulated phosphoproteins indicated a significant association with the following processes: "regulation of cell adhesion", "regulation of cell differentiation", "regulation of transmembrane transport", "regulation of transcription by RNA polymerase II", and several terms related to cell-cycle regulation. Conclusion: We conclude that CAMKK2-mediated phosphorylation results in relatively few direct phosphorylation events that directly match the known CAMKK2 sequence preference. Instead, the phosphoproteomic results identify a larger number of indirect effects on phosphorylation, including targets of AMP-Regulated Kinases and Salt-Inducible Kinases. CAMKK2 is not essential for collecting duct cell differentiation. This work was funded by NHLBI Division of Intramural Research projects ZIA-HL001285 and ZIA-HL006129 (to M. Knepper). This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.