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Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein
Published on: June 30, 2019
Cellular characterization of a novel focal adhesion kinase inhibitor
Jill K Slack-Davis1, Karen H Martin, Robert W Tilghman
1Department of Microbiology and Cancer Center, Health Sciences System, University of Virginia, Charlottesville, Virginia 22908, USA.
Abstract:
Focal adhesion kinase (FAK) is a member of a family of non-receptor protein-tyrosine kinases that regulates integrin and growth factor signaling pathways involved in cell migration, proliferation, and survival. FAK expression is increased in many cancers, including breast and prostate cancer. Here we describe perturbation of adhesion-mediated signaling with a FAK inhibitor, PF-573,228. In vitro, this compound inhibited purified recombinant catalytic fragment of FAK with an IC(50) of 4 nM. In cultured cells, PF-573,228 inhibited FAK phosphorylation on Tyr(397) with an IC(50) of 30-100 nM. Treatment of cells with concentrations of PF-573,228 that significantly decreased FAK Tyr(397) phosphorylation failed to inhibit cell growth or induce apoptosis. In contrast, treatment with PF-573,228 inhibited both chemotactic and haptotactic migration concomitant with the inhibition of focal adhesion turnover. These studies show that PF-573,228 serves as a useful tool to dissect the functions of FAK in integrin-dependent signaling pathways in normal and cancer cells and forms the basis for the generation of compounds amenable for preclinical and patient trials.
Insights
The focal adhesion kinase (FAK) inhibitor PF-573,228 effectively blocks cell migration by disrupting adhesion signaling. This FAK inhibitor shows promise for cancer therapy by targeting cell movement without affecting growth or survival.
Area of Science:
- Biochemistry
- Cell Biology
- Cancer Research
Background:
- Focal adhesion kinase (FAK) is a key regulator of integrin and growth factor signaling pathways.
- Elevated FAK expression is observed in various cancers, including breast and prostate cancer.
- FAK plays a crucial role in cell migration, proliferation, and survival.
Purpose of the Study:
- To investigate the effects of the FAK inhibitor PF-573,228 on adhesion-mediated signaling.
- To evaluate the potential of PF-573,228 as a therapeutic agent for cancer.
Main Methods:
- In vitro inhibition assays using purified FAK.
- Cell-based assays to measure FAK phosphorylation, cell growth, apoptosis, and migration.
- Assessment of focal adhesion turnover.
Main Results:
- PF-573,228 demonstrated potent inhibition of purified FAK (IC50 = 4 nM).
- In cultured cells, PF-573,228 inhibited FAK phosphorylation (Tyr397) with an IC50 of 30-100 nM.
- PF-573,228 inhibited chemotactic and haptotactic migration and focal adhesion turnover, but did not affect cell growth or induce apoptosis.
Conclusions:
- PF-573,228 is a potent FAK inhibitor that disrupts cell migration.
- This compound serves as a valuable tool for studying FAK functions in integrin-dependent signaling.
- PF-573,228 provides a basis for developing novel cancer therapeutics targeting FAK.
