RRC ID 67867
Author Niinae T, Imami K, Sugiyama N, Ishihama Y.
Title Identification of Endogenous Kinase Substrates by Proximity Labeling Combined with Kinase Perturbation and Phosphorylation Motifs.
Journal Mol Cell Proteomics
Abstract Mass-spectrometry-based phosphoproteomics can identify more than 10,000 phosphorylated sites in a single experiment. But, despite the fact that enormous phosphosite information has been accumulated in public repositories, protein kinase-substrate relationships remain largely unknown. Here, we describe a method to identify endogenous substrates of kinases by using a combination of a proximity-dependent biotin identification method, called BioID, with two other independent methods, kinase-perturbed phosphoproteomics and phosphorylation motif matching. For proof of concept, this approach was applied to casein kinase 2 (CK2) and protein kinase A (PKA), and we identified 24 and 35 putative substrates, respectively. We also show that known cancer-associated missense mutations near phosphosites of substrates affect phosphorylation by CK2 or PKA and thus might alter downstream signaling in cancer cells bearing these mutations. This approach extends our ability to probe physiological kinase-substrate networks by providing new methodology for large-scale identification of endogenous substrates of kinases.
Volume 20
Pages 100119
Published 2021-1-1
DOI 10.1016/j.mcpro.2021.100119
PII S1535-9476(21)00091-8
PMID 34186244
PMC PMC8325102
MeSH Casein Kinase II / genetics Casein Kinase II / metabolism* Cyclic AMP-Dependent Protein Kinases / genetics Cyclic AMP-Dependent Protein Kinases / metabolism* Dimethyl Sulfoxide / pharmacology HEK293 Cells HeLa Cells Humans Mass Spectrometry Mutation, Missense Phosphoproteins / genetics Phosphoproteins / metabolism* Phosphorylation Workflow
IF 4.87
DNA material Genome Network Project Human cDNA Clone W01A015A12 (HGE006012)
Human and Animal Cells 293T(RCB2202)