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Identification of Novel CK2 Kinase Substrates Using a Versatile Biochemical Approach
Published on: February 21, 2019
A novel proteomic approach for specific identification of tyrosine kinase substrates using [13C]tyrosine
Nieves Ibarrola1, Henrik Molina, Akiko Iwahori
1McKusick-Nathans Institute for Genetic Medicine and the Department of Biological Chemistry, The Johns Hopkins University, Baltimore, Maryland 21205, USA.
Abstract:
Proteomic studies to find substrates of tyrosine kinases generally rely on identification of protein bands that are "pulled down" by antiphosphotyrosine antibodies from ligand-stimulated samples. One can obtain erroneous results from such experiments because of two major reasons. First, some proteins might be basally phosphorylated on tyrosine residues in the absence of ligand stimulation. Second, proteins can bind non-specifically to the antibodies or the affinity matrix. Induction of phosphorylation of proteins by ligand must therefore be confirmed by a different approach, which is not always feasible. We have developed a novel proteomic approach to identify substrates of tyrosine kinases in signaling pathways studies based on in vivo labeling of proteins with "light" (12C-labeled) or "heavy" (13C-labeled) tyrosine. This stable isotope labeling in cell culture method enables the unequivocal identification of tyrosine kinase substrates, as peptides derived from true substrates give rise to a unique signature in a mass spectrometry experiment. By using this approach, from a single experiment, we have successfully identified several known substrates of insulin signaling pathway and a novel substrate, polymerase I and transcript release factor, a protein that is implicated in the control of RNA metabolism and regulation of type I collagen promoters. This approach is amenable to high throughput global studies as it simplifies the specific identification of substrates of tyrosine kinases as well as serine/threonine kinases using mass spectrometry.
Insights
This study introduces a new proteomic method using stable isotope labeling to accurately identify tyrosine kinase substrates. This approach overcomes limitations of traditional antibody-based methods, enabling reliable discovery of signaling pathway components.
Area of Science:
- Biochemistry
- Molecular Biology
- Proteomics
Background:
- Traditional proteomic studies for tyrosine kinase substrates often yield erroneous results.
- Basal phosphorylation and non-specific antibody binding are major limitations of current methods.
- Confirmation of ligand-induced phosphorylation requires alternative, often infeasible, approaches.
Purpose of the Study:
- To develop a novel proteomic approach for the unequivocal identification of tyrosine kinase substrates.
- To overcome the limitations of conventional antiphosphotyrosine antibody-based pull-down assays.
- To enable high-throughput global studies of kinase signaling pathways.
Main Methods:
- Developed a stable isotope labeling in cell culture (SILC) method using light (12C) and heavy (13C) labeled tyrosine.
- Utilized mass spectrometry to analyze peptides derived from labeled proteins.
- Applied the method to identify substrates in insulin signaling pathways.
Main Results:
- Successfully identified known substrates of the insulin signaling pathway.
- Discovered a novel substrate: polymerase I and transcript release factor (PTRF).
- PTRF is implicated in RNA metabolism and regulation of type I collagen promoters.
Conclusions:
- The novel stable isotope labeling approach provides unequivocal identification of tyrosine kinase substrates.
- This method simplifies specific substrate identification for tyrosine kinases and serine/threonine kinases.
- The approach is suitable for high-throughput global proteomic studies.
