PXD033523

PXD033523 is an original dataset announced via ProteomeXchange.

Title	Chemical genetic validation of CSNK2 substrates using an inhibitor-resistant mutant in combination with triple SILAC quantitative phosphoproteomics
Description	Casein Kinase 2 (CSNK2) is an extremely pleiotropic, ubiquitously expressed protein kinase involved in the regulation of numerous key biological processes. Mapping the CSNK2-dependent phosphoproteome is necessary for better characterization of its fundamental role in cellular signalling. While ATP-competitive inhibitors have enabled the identification of many putative kinase substrates, compounds targeting the highly conserved ATP-binding pocket often exhibit off-target effects limiting their utility for definitive kinase-substrate assignment. To overcome this limitation, we devised a strategy combining chemical genetics and quantitative phosphoproteomics to identify and validate physiological CSNK2 substrates. We engineered U2OS cells expressing exogenous wild type CSNK2A1 (WT) or a triple mutant (TM, V66A/H160D/I174A) with substitutions at residues important for inhibitor binding. These cells were treated with CX-4945, a clinical-stage inhibitor of CSNK2, and analyzed using large-scale triple SILAC (Stable Isotope Labelling of Amino Acids in Cell Culture) quantitative phosphoproteomics. In contrast to wild-type CSNK2A1, CSNK2A1-TM retained activity in the presence of CX-4945 enabling identification and validation of several physiological CSNK2 substrates on the basis of their increased phosphorylation in cells expressing CSNK2A1-TM. Based on high conservation within the kinase family, we expect that this strategy can be broadly adapted for identification of other kinase-substrate relationships.
HostingRepository	PRIDE
AnnounceDate	2023-11-14
AnnouncementXML	Submission_2023-11-14_08:56:51.012.xml
DigitalObjectIdentifier	https://dx.doi.org/10.6019/PXD033523
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Supported dataset by repository
PrimarySubmitter	Kristina Jurcic
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	phosphorylated residue; monohydroxylated residue; deamidated residue; iodoacetamide derivatized residue
Instrument	LTQ Orbitrap Elite

Revision	Datetime	Status	ChangeLog Entry
0	2022-04-28 01:23:42	ID requested
1	2022-07-14 09:07:48	announced
⏵ 2	2023-11-14 08:56:51	announced	2023-11-14: Updated project metadata.

10.6019/PXD033523;

submitter keyword: protein kinase CK2, U2OS cells, CSNK2, SILAC, CX-4945, phosphoproteomics, LC-MS/MS

David William Litchfield
contact affiliation	Department of Biochemistry, Department of Oncology, Schulich School of Medicine & Dentistry, Western University, London, Ontario, Canada
contact email	litchfi@uwo.ca
lab head
Kristina Jurcic
contact affiliation	University of Western Ontario
contact email	kjurcic@uwo.ca
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD033523
     1. Label: PRIDE project
     2. Name: Chemical genetic validation of CSNK2 substrates using an inhibitor-resistant mutant in combination with triple SILAC quantitative phosphoproteomics