PXD040159-3

PXD040159 is an original dataset announced via ProteomeXchange.

Title	Using CRISPR-Cas9/Phosphoproteomics to Identify Kinase Substrates: Calmodulin Kinase 2 Delta
Description	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.
HostingRepository	PRIDE
AnnounceDate	2024-10-22
AnnouncementXML	Submission_2024-10-22_06:13:40.619.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	CHIN-RANG YANG
SpeciesList	scientific name: Mus musculus (Mouse); NCBI TaxID: 10090;
ModificationList	phosphorylated residue; monohydroxylated residue; iodoacetamide derivatized residue
Instrument	Orbitrap Fusion Lumos

Revision	Datetime	Status	ChangeLog Entry
0	2023-02-16 09:49:54	ID requested
1	2023-11-06 12:03:10	announced
2	2023-11-14 08:26:34	announced	2023-11-14: Updated project metadata.
⏵ 3	2024-10-22 06:13:41	announced	2024-10-22: Updated project metadata.

10.1016/J.JBC.2023.105371;

submitter keyword: mass spectrometry, Camk2d, vasopressin,AQP2

Mark A. Knepper
contact affiliation	Epithelial Systems Biology Laboratory Systems Biology Center Division of Intramural Research National Heart, Lung and Blood Institute National Institutes of Health
contact email	knepperm@nhlbi.nih.gov
lab head
CHIN-RANG YANG
contact affiliation	National Institutes of Health, USA
contact email	chin-rang.yang@nih.gov
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD040159
     1. Label: PRIDE project
     2. Name: Using CRISPR-Cas9/Phosphoproteomics to Identify Kinase Substrates: Calmodulin Kinase 2 Delta