Kidney Week

Abstract: TH-PO336

Using CRISPR-Cas9/Phosphoproteomics to Identify Kinase Substrates: Calcium/Calmodulin-Dependent Protein Kinase 2δ

Session Information

• Fluid, Electrolyte, and Acid-Base Disorders: Basic
  November 03, 2022 | Location: Exhibit Hall, Orange County Convention Center‚ West Building
  Abstract Time: 10:00 AM - 12:00 PM

Category: Fluid‚ Electrolyte‚ and Acid-Base Disorders

• 1001 Fluid‚ Electrolyte‚ and Acid-Base Disorders: Basic

Authors

• Park, Euijung, National Heart Lung and Blood Institute, Bethesda, Maryland, United States

Euijung Park,

• Yang, Chin-Rang, National Heart Lung and Blood Institute, Bethesda, Maryland, United States

Chin-Rang Yang,

• Chen, Lihe, National Heart Lung and Blood Institute, Bethesda, Maryland, United States

Lihe Chen,

• Raghuram, Viswanathan, National Heart Lung and Blood Institute, Bethesda, Maryland, United States

Viswanathan Raghuram,

• Knepper, M. A., National Heart Lung and Blood Institute, Bethesda, Maryland, United States

M. A. Knepper,

Background

Arginine vasopressin (AVP) plays a critical role in the regulation of water permeability in renal collecting duct (CD) cells via the V2 vasopressin receptor, which triggers a rise of intracellular cAMP and Ca²⁺. Ca²⁺ stimulates a wide range of cellular processes including actin cytoskeleton rearrangement, in part through Ca²⁺/CaM-dependent signaling. One potential target, strongly expressed in CD cells, is Ca²⁺/CaM-dependent protein kinase 2d (Camk2d). Here, we deleted Camk2d in mpkCCD cells and carried out phosphoproteomics to identify Camk2d target proteins.

Methods

Using two different gRNAs which target the Camk2d kinase domain, CRISPR/Cas9-mediated Camk2d knock-out cell lines were created. We carried out large-scale proteomic and phosphoproteomic analysis in the presence of dDAVP, (V2 selective AVP analog, 0.1 nM, 30 min) using TMT quantification versus wild-type cells to identify direct targets of Camk2d.

Results

The quantitative phosphoproteomics revealed that, of the 11570 phosphopeptides quantified, 78 were significantly increased and 60 were decreased. Of the 60 downregulated sites, Tpd52 were previously identified as Camk2d targets in existing literature, but 59 of the identified sites are novel. Motif analysis of the decreased phosphorylation sites revealed a novel target preference of –(R/K)-X-X-p(S/T)-X-D- where R,K,S,T and D are standard single letter amino acid codes, X means “any amino acid” and p indicates the phosphorylated amino acid. Gene Ontology (GO) analysis identified that Cam2d-mediated protein phosphorylation is likely to be involved in regulation of actin polymerization as well as chromatin organization. In standard proteomics (n=5198), 23 proteins significantly increased and 24 were decreased. GO term analysis mapped the regulated proteins to chromatin organization and histone modification, consistent with the phosphoproteomic data.

Conclusion

We identified multiple novel Camk2d phosphorylation targets that have potential roles in vasopressin’s action to regulate water transport in the renal collecting duct, especially with regard to Ca²⁺/CaM-dependent regulation of the actin cytoskeleton and chromatin organization. The large increase in the number of recognized phosphorylation targets allowed identification of a new target sequence preference motif for Camk2d.