Identification of FAK substrate peptides via colorimetric screening of a one-bead one-peptide combinatorial library

Laurie A Witucki; Lauren Sanford Borowicz; Anthony M Pedley; Jaime Curtis-Fisk; Elizabeth Girnys Kuszpit

doi:10.1002/psc.2751

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Identification of FAK substrate peptides via colorimetric screening of a one-bead one-peptide combinatorial library

Journal article

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Identification of FAK substrate peptides via colorimetric screening of a one-bead one-peptide combinatorial library

Laurie A Witucki, Lauren Sanford Borowicz, Anthony M Pedley, Jaime Curtis-Fisk and Elizabeth Girnys Kuszpit

Journal of peptide science, Vol.21(4), pp.302-311

04/2015

DOI: 10.1002/psc.2751

PMID: 25728406

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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.

Focal Adhesion Protein-Tyrosine Kinases - chemistry

Peptide Library

Peptides - chemistry

Peptides - metabolism

Phosphorylation

Details

Title: Subtitle: Identification of FAK substrate peptides via colorimetric screening of a one-bead one-peptide combinatorial library
Creators: Laurie A Witucki - Grand Valley State University
Lauren Sanford Borowicz - Grand Valley State University
Anthony M Pedley - Grand Valley State University
Jaime Curtis-Fisk - Grand Valley State University
Elizabeth Girnys Kuszpit - Grand Valley State University
Resource Type: Journal article
Publication Details: Journal of peptide science, Vol.21(4), pp.302-311
DOI: 10.1002/psc.2751
PMID: 25728406
ISSN: 1075-2617
eISSN: 1099-1387
Language: English
Date published: 04/2015
Academic Unit: Biochemistry and Molecular Biology
Record Identifier: 9984772261502771

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