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Identification of Kinase-substrate Pairs Using High Throughput Screening
11:13

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Published on: August 29, 2015

A semisynthetic epitope for kinase substrates.

Jasmina J Allen1, Manqing Li, Craig S Brinkworth

  • 1Graduate Program in Chemistry and Chemical Biology, University of California, San Francisco, San Francisco, California 94143, USA.

Nature Methods
|May 9, 2007
PubMed
Summary

Researchers developed a novel semisynthetic method to tag and identify direct kinase substrates. This technique maps protein phosphorylation and reveals signaling network architecture by creating unique epitopes for antibody detection.

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Last Updated: Jul 15, 2026

Identification of Kinase-substrate Pairs Using High Throughput Screening
11:13

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Published on: August 29, 2015

Oligopeptide Competition Assay for Phosphorylation Site Determination
09:16

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Identification of Novel CK2 Kinase Substrates Using a Versatile Biochemical Approach
11:11

Identification of Novel CK2 Kinase Substrates Using a Versatile Biochemical Approach

Published on: February 21, 2019

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cell Signaling

Background:

  • Protein phosphorylation is crucial for cellular signaling, but mapping kinase-substrate interactions is complex.
  • Understanding kinase-substrate relationships is essential for deciphering signaling network architecture.

Purpose of the Study:

  • To develop a versatile method for tracing individual kinase activity.
  • To enable the affinity tagging and identification of direct kinase substrates.

Main Methods:

  • A semisynthetic reaction scheme was developed involving bio-orthogonal ATPgammaS analog incorporation by engineered kinases.
  • Thiophosphorylated residues were subsequently alkylated to create a specific epitope.
  • This epitope allows for detection using thiophosphate ester-specific antibodies.

Main Results:

  • The method's generality was demonstrated across 13 diverse kinases, including MAPK and Akt family members.
  • The approach successfully identified a direct substrate for Erk2 in mouse cells expressing endogenous levels of an analog-specific kinase.

Conclusions:

  • This semisynthetic epitope construction is a powerful tool for mapping kinase-substrate relationships.
  • The technique facilitates the elucidation of complex cellular signaling networks.