Technological Innovation and Resources
Identification of Endogenous Kinase Substrates by Proximity Labeling Combined with Kinase Perturbation and Phosphorylation Motifs

https://doi.org/10.1016/j.mcpro.2021.100119Get rights and content
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Highlights

  • Identification of novel kinase interactors by BioID.

  • Applying two orthogonal filters, kinase perturbation and phosphorylation motif.

  • Identification of novel CK2 and PKA substrates.

  • A universal method for the identification of endogenous substrates for all kinases.

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.

Keywords

kinase substrate
BioID (proximity-dependent biotin identification)
phosphoproteomics

Abbreviations

ACN
acetonitrile
AP-MS
affinity-purification mass spectrometry
BioID
proximity-dependent biotin identification
BirA
biotin ligase
CK2
casein kinase 2
GO
gene ontology
HAMMOC
hydroxy-acid-modified metal oxide chromatography
LC/MS/MS
liquid chromatography/tandem mass spectrometry
PKA
protein kinase A
PPI
protein–protein interaction
PSM
peptide spectrum match
PWMs
position weight matrices
TMT
tandem Mass Tag

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