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Published on: December 6, 2017
Identification of FAK substrate peptides via colorimetric screening of a one-bead one-peptide combinatorial library
Laurie A Witucki1, Lauren Sanford Borowicz, Anthony M Pedley
1Department of Chemistry, Grand Valley State University, Allendale, MI, 49401, USA.
Abstract:
Focal adhesion kinase (FAK) is a protein tyrosine kinase that is associated with regulating cellular functions such as cell adhesion and migration and has emerged as an important target for cancer research. Short peptide substrates that are selectively and efficiently phosphorylated by FAK have not been previously identified and tested. Here we report the synthesis and screening of a one-bead one-peptide combinatorial library to identify novel substrates for FAK. Using a solid-phase colorimetric antibody tagging detection platform, the peptide beads phosphorylated by FAK were sequenced via Edman degradation and then validated through radioisotope kinetic studies with [γ-(32)P] ATP to derive Michaelis-Menton constants. The combination of results gathered from both colorimetric and radioisotope kinase assays led to the rational design of a second generation of FAK peptide substrates. Out of all the potential peptide substrates evaluated, the most active was GDYVEFKKK with a K(M) = 92 μM and a Vmax = 1920 nmol/min/mg. Peptide substrates discovered within this study may be useful diagnostic tools for future kinase investigations and may lead to novel therapeutic agents.
Insights
Researchers identified new peptide substrates for focal adhesion kinase (FAK), a key protein in cell migration and cancer. These novel FAK substrates may serve as diagnostic tools and lead to new cancer therapies.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Focal adhesion kinase (FAK) is a protein tyrosine kinase regulating cell adhesion and migration.
- FAK is a significant target in cancer research.
- Novel, selective peptide substrates for FAK have not been previously identified.
Purpose of the Study:
- To synthesize and screen a combinatorial library for novel FAK peptide substrates.
- To identify and validate peptides efficiently phosphorylated by FAK.
Main Methods:
- Utilized a one-bead one-peptide combinatorial library approach.
- Employed a solid-phase colorimetric antibody tagging detection platform.
- Sequenced phosphorylated peptides via Edman degradation and validated using radioisotope kinetic studies with [γ-(32)P] ATP.
Main Results:
- Identified and validated novel FAK peptide substrates.
- The most active peptide substrate, GDYVEFKKK, exhibited a K(M) of 92 μM and Vmax of 1920 nmol/min/mg.
- Results informed the rational design of a second generation of FAK peptide substrates.
Conclusions:
- Discovered novel peptide substrates for FAK.
- These substrates may serve as valuable diagnostic tools for kinase research.
- The identified peptides could potentially lead to the development of novel therapeutic agents for cancer.
