Dataset Information

Chemical genetic validation of CSNK2 substrates using an inhibitor-resistant mutant in combination with triple SILAC quantitative phosphoproteomics

ABSTRACT: 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.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

SUBMITTER: Kristina Jurcic

LAB HEAD: David William Litchfield

PROVIDER: PXD033523 | Pride | 2022-07-14

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

Items per page:

1 - 5 of 18

Similar Datasets

Project description:Ascending bacterial infections of the male genitourinary tract by Escherichia coli are an important cause of male infertility. Thus understanding mechanisms by which immunocompetent cells such as testicular macrophages (TM) respond to infection and how bacterial pathogens manipulate defense pathways is of great relevance. Following infection with uropathogenic E. coli (UPEC) whole genome expression profiling revealed induction of the calcium-dependent nuclear factor of activated T cells (NFAT) signaling pathway in TM as well as in peritoneal macrophages (PM) that were used in comparison. UPEC-dependent NFAT activation was confirmed in cultured TM and in TM using an in vivo infection model. Elevated expression of NFATC2-regulated anti-inflammatory cytokines was found in TM (IL-4, IL-13) and PM (IL-3, IL-4, IL-13). NFATC2 is activated by a rapid influx of calcium caused by the UPEC pore forming toxin alpha-hemolysin. Mutant analysis identified alpha-hemolysin as the main virulent factor responsible for UPEC-dependant suppression of IL-6 and TNF-a cytokine release from PM. Alpha-hemolysin caused differential activation of MAP kinase and AP-1 signaling pathways in TM and PM leading to differential expression profiles of key pro-inflammatory cytokines in PM (IL-1a, IL-1b and IL-6 downregulated) and TM (IL-1b and IL-6 upregulated). In contrast to PM, treatment with lipopolysaccharide did not result in activation of the NFkB pathway in TM as shown by lack of degradation of IkBa and lack of pro-inflammatory cytokine secretion (IL-6, TNF-a). In conclusion, these results propose a mechanism how TM can defend against microbes, while at the same time they are able to maintain the immune privileged status of the testis. 2 Macrophage populations ( peritoneal, testicular) infected with UPEC CFT073; timepoints of mrna harvest at 0 min (control), 30 min and 60 min; of each point 2 biological replicates; of each repliccate 2 technical replicates: summa summarum: 2*((1+1+1)*2*2)=24 samples

			Action	DRS
	aplfiles	Other
	checksum.txt	Txt
	combined_zipped.zip	Other
	kj_ec_17042017_phos2.raw	Raw
	kj_ec_17042017_phos3.raw	Raw

Dataset Information

Chemical genetic validation of CSNK2 substrates using an inhibitor-resistant mutant in combination with triple SILAC quantitative phosphoproteomics

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets