Using CRISPR-Cas9/Phosphoproteomics to Identify Kinase Substrates: Calmodulin Kinase 2 Delta

ABSTRACT: The Ca2+/CaM-dependent protein kinase 2 (CAMK2) family of protein kinases are key protein kinases involved in regulation of cellular processes in a variety of tissues including brain, smooth muscle and kidney. One member, CAMK2-delta (CAMK2D), has been proposed to be involved in vasopressin signaling in the renal collecting duct, which controls water and salt excretion by the kidney. To identify CAMK2D target proteins in renal collecting duct cells (mpkCCD), we deleted Camk2d in mpkCCD cells and carried out LC-MS/MS-based quantitative phoshoproteomics analysis. Using CRISPR/Cas9 with two different gRNAs targeting the CAMK2D catalytic domain, we created multiple CAMK2D knock-out cell lines. Using these cells and control cell lines, we carried out large-scale quantitative phosphoproteomic analysis (TMT-labeling) in the presence of the vasopressin analog dDAVP (0.1 nM, 30 min). Of the 11,570 phosphopeptides quantified, 206 were significantly increased and 169 were decreased. These data are available for browsing or download at https://esbl.nhlbi.nih.gov/Databases/CAMK2D-proteome/. Motif analysis of the decreased phosphorylation sites revealed a target preference of –(R/K)-X-X-p(S/T)-X-(D/E), matching the motif identified in previous in vitro studies using recombinant CAMK2D. Thirty-five of the significantly down-regulated phosphorylation sites in CAMK2D knock-out cells had exactly this motif and are judged to be sites that are most likely direct CAMK2D targets. These targets include Ser256 of aquaporin-2 (AQP2), a site known to regulate AQP2 trafficking to the plasma membrane, controlling water excretion by the kidney. The other changes, including phosphorylation sites that increased in response to CAMK2D deletion, are likely phosphorylated by other protein kinases that are regulated by CAMK2D in kinase cascades.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Epithelial Cell, Kidney

SUBMITTER: CHIN-RANG YANG  

LAB HEAD: Mark A. Knepper

PROVIDER: PXD040159 | Pride | 2023-11-06

REPOSITORIES: Pride