Related Experiment Video
Updated: May 30, 2026
Real-Time Monitoring of Aurora kinase A Activation using Conformational FRET Biosensors in Live Cells
Published on: July 30, 2020
A novel cell-based, high-content assay for phosphorylation of Lats2 by Aurora A
Amy Emery1, David A Sorrell, Stacy Lawrence
1Cylacel Ltd, James Lindsay Place, Dundee, UK.
Abstract:
Aurora A kinase is a key regulator of mitosis, which is upregulated in several human cancers, making it a potential target for anticancer therapeutics. Consequently, robust medium- to high-throughput cell-based assays to measure Aurora A kinase activity are critical for the development of small-molecule inhibitors. Here the authors compare measurement of the phosphorylation of two Aurora A substrates previously used in high-content screening Aurora A assays, Aurora A itself and TACC3, with a novel substrate Lats2. Using antibodies directed against phosphorylated forms of Aurora A (pThr288), P-TACC3 (pSer558), and P-Lats2 (pSer83), the authors investigate their suitability in parallel for development of a cell-based assay using several reference Aurora inhibitors: MLN8054, VX680, and AZD1152-HQPA. They validate a combined assay of target-specific phosphorylation of Lats2 at the centrosome and an increase in mitotic index as a measure of Aurora A activity. The assay is both sensitive and robust and has acceptable assay performance for high-throughput screening or potency estimation from concentration-response assays. It has the advantage that it can be carried out using a commercially available monoclonal antibody against phospho-Lats2 and the widely available Cellomics ArrayScan HCS reader and thus represents a significant addition to the tools available for the identification of Aurora A specific inhibitors.
Insights
A new cell-based assay effectively measures Aurora A kinase activity by monitoring Lats2 phosphorylation. This sensitive and robust assay aids in developing targeted anticancer therapeutics by identifying Aurora A inhibitors.
Area of Science:
- Biochemistry
- Cell Biology
- Cancer Therapeutics
Background:
- Aurora A kinase is crucial for mitosis and often overexpressed in cancers.
- Developing targeted anticancer drugs requires reliable assays for Aurora A kinase activity.
Purpose of the Study:
- To compare the efficacy of different Aurora A kinase substrates (Aurora A, TACC3, Lats2) in cell-based assays.
- To validate a novel, sensitive, and robust assay for measuring Aurora A kinase activity.
Main Methods:
- Compared phosphorylation of Aurora A (pThr288), TACC3 (pSer558), and Lats2 (pSer83) using specific antibodies.
- Utilized reference Aurora inhibitors (MLN8054, VX680, AZD1152-HQPA) to validate assay performance.
- Developed a combined assay measuring Lats2 phosphorylation at the centrosome and mitotic index.
Main Results:
- Validated a novel assay combining Lats2 phosphorylation and mitotic index as a measure of Aurora A activity.
- The developed assay is sensitive, robust, and suitable for high-throughput screening.
- Demonstrated the assay's utility with commercially available reagents and equipment.
Conclusions:
- A new cell-based assay using Lats2 phosphorylation is effective for measuring Aurora A kinase activity.
- This assay is a valuable tool for identifying and developing Aurora A specific inhibitors for cancer therapy.
- The assay's sensitivity and robustness make it suitable for drug discovery and potency estimation.
