Abstract: TH-PO0060
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9/Phosphoproteomic Identification of Direct and Indirect Phosphorylation Targets of CAMKK2 in Renal Collecting Duct Cells
Session Information
- Fluid, Electrolyte, and Acid-Base Disorders: Basic Research
October 22, 2026 | Location: Exhibit Hall A, Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Fluid, Electrolytes, and Acid-Base Disorders
- 1101 Fluid, Electrolyte, and Acid-Base Disorders: Basic
Authors
- Khan, Shaza, National Institutes of Health, Bethesda, Maryland, United States
- Raghuram, Viswanathan, National Institutes of Health, Bethesda, Maryland, United States
- Yang, Chin-Rang, National Institutes of Health, Bethesda, Maryland, United States
- Nita-Lazar, Aleksandra, National Institutes of Health, Bethesda, Maryland, United States
- Knepper, Mark A., National Institutes of Health, Bethesda, Maryland, United States
Background
Vasopressin regulates water transport in the collecting duct through a PKA-dependent pathway involving hundreds of phosphoproteins. Beyond PKA, signaling involves downstream kinases like Calcium/Calmodulin-Dependent Protein Kinase Kinase 2 (CAMKK2), an activator of AMP-activated protein kinase (AMPK) that is regulated by calcium and phosphorylation. This study aims to elucidate the role of CAMKK2 in vasopressin-regulated signaling by identifying its direct and indirect substrates via CRISPR-Cas9 deletion and large-scale phosphoproteomics.
Methods
We used CRISPR-Cas9 to generate multiple CAMKK2 KO mpkCCD cell lines. Western blotting confirmed loss of CAMKK2 protein, and Sanger sequencing confirmed indels at target sites. KO and control cells treated with the V2R-selective vasopressin analog dDAVP were subjected to tandem mass tag TMT labeling-based quantitative proteomics and phosphoproteomics.
Results
Overall, 13,012 phosphopeptides were quantified in 3,296 phosphoproteins. Using stringent criteria (Pjoint <0.0025 with |log2[Camkk2-KO/Cont]| >0.5), 230 phosphopeptides (<2%) were identified as likely direct or indirect CAMKK2 targets, with 88 decreased and 142 increased in CAMKK2 KO clones. Total proteome analysis showed few abundance changes following CAMKK2 deletion, none of which overlapped with phosphoproteins containing altered phosphopeptides. PTM-Logo analysis revealed enrichment of leucine at +4 and methionine at −5, along with arginine or lysine at −2/−3 relative to the phosphosite, consistent with AMPK-related kinase motifs. General proteomics revealed that principal cell differentiation markers (AQP2, α-ENaC, FXYD4, GATA3, LGALS3) were statistically unchanged relative to control clones. While several transcription factors involved in epithelial development and polarity (TCF7L2, TEAD2, SOX4, FOXJ2, FOXA1) exhibited significant decreases in abundance.
Conclusion
Quantitative phosphoproteomics shows that CAMKK2 exerts a selective but functionally important control over cellular phosphorylation networks. Our findings suggest that AMPK is the primary CAMKK2 target in mpkCCD cells, though contributions from secondary AMPK-regulated kinases cannot be excluded. While CAMKK2 is not essential for collecting duct cell differentiation, we identified several transcription factors linked to epithelial development and polarity signaling that merit further study.
Funding
- Other NIH Support