December 1, 2012 (Vol. 32, No. 21)
Timothy J. Manning
Zhengliang L. Wu
Kinases catalyze the transfer of the γ-phosphate from adenosine 5´-triphosphate (ATP) to proteins, lipids, sugars, or other acceptors. This process, termed phosphorylation, forms the cornerstone of most signal transduction cascades. Kinases regulate virtually all cellular activities and have important roles in cell survival, proliferation, differentiation, and migration. Dysregulated kinase activity contributes to a wide range of human disorders including cancer. As such, kinases are currently prime pharmacological targets, and simple, accurate assays used to measure kinase activity are crucial for rapid drug discovery.
Currently available kits typically fall into two general categories. Most are kinase-specific and are based on antibodies that recognize phosphorylated products. In contrast, universal kinase assays are designed to measure the common donor substrate ATP, or the product ADP, and can be used for virtually all kinases. Because of their flexibility, universal assays are desirable for high-throughput screening (HTS) and are the focus of this article.
A new Universal Kinase Activity Kit (Catalog # EA004) has been developed by R&D Systems. This assay utilizes a specific phosphatase to release the β-phosphate from adenosine 5´-diphosphate (ADP) produced during the kinase reaction. The amount of free phosphate is then measured and is used as an indicator of kinase activity. This new assay offer several advantages over currently available universal kinase assays.
Drawbacks of Commonly Used Universal Kinase Assays
Most commercially available kits have shortcomings that restrict their use in studying enzyme kinetics or for drug screening. Traditional kinase activity assays have used the incorporation of a radioactive 32P or 33P into ATP, and followed the transfer of the radioisotope to the substrate. The key to this assay is separating the phosphorylated product and ATP. This can be challenging when the acceptor substrate is similar to ATP in size or in its biochemical properties. Some example substrates that fall into this category include simple sugars or nucleotides. Substrate-product separation is usually a time-consuming process and can make the assay impractical for HTS. In addition, these assays suffer from the same drawbacks inherent in all radioisotope experiments, including strict government regulation, the potential for radioactive contamination, and the need for proper waste disposal.
There are three major types of nonradioactive universal kinase assays. The first type relies on an ADP-binding reagent or sensor, such as anti-ADP antibodies. Because binding of the antibody to ADP does not follow a linear relationship with ADP concentration, this type of assay is not considered to be quantitative. This is especially the case when ADP concentrations are high.
The second type relies on the measurement of ATP. If the donor substrate ATP is measured directly, ATP levels decrease during the reaction and the signal will be inversely related to product formation. This assay can exhibit reduced sensitivity, especially when the ATP concentration is high and consumption is low. ADP produced during the kinase reaction can also be converted back to ATP for measurement. The assay will require the complete elimination of the donor substrate ATP. The ADP-ATP conversion and the elimination of donor ATP add an additional layer of complexity to the experiment, potentially making the assay less quantitative and prone to false positives during inhibitor screening. A third type of kinase assay relies on coupling the ADP-ATP conversion to the phosphoenolpyruvate-pyruvate reaction. The reduction or oxidation of pyruvate can be used as a measure of kinase activity. The multiple coupling reactions required in this type of assay can similarly make it less quantitative and more prone to false positives.
A New Solution for Measuring Kinase Activity
The new Universal Kinase Assay Kit from R&D Systems is suitable for use in any kinase reaction that produces ADP. The assay couples the kinase reaction with the nucleotidase CD39L2, releasing phosphate from the ADP that is produced. The free phosphate is then detected using colorimetric malachite green-based reagents. In its most simple format, CD39L2 can be directly added to the kinase reaction. In this case the coupling reaction takes place simultaneously with the primary reaction, and the phosphate generated can be measured directly without additional substrate-product separation steps. In many cases this assay can be complete within 30 minutes. CD39L2 can also be added after the kinase reaction if a longer reaction time (>20 minutes) is needed to produce sufficient product for detection. This simplicity, combined with microplate-based colorimetric detection, makes the assay amenable to HTS.
This assay format can also be used to accurately quantify enzyme kinetics. This is possible because only one first-order coupling reaction is involved. The signal produced by a first-order coupling reaction will faithfully reflect the progress of the primary reaction. Coupling reactions used in existing kinase assays are typically second- or even third-order reactions. Furthermore, because only one coupling reaction is necessary, the assay is also much less susceptible to false positives that might occur during pharmacological screening. The more coupling reactions required in an assay, the greater the possibility for a false positive caused by non-specific effects at any coupling step.
The Universal Kinase Activity Kit (Catalog # EA004) provides a convenient, nonradioactive, HTS-compatible format for assaying kinase activity. The assay can be used to characterize enzyme kinetics for any kinase reaction that produces ADP. The simple format avoids many of the issues typically associated with current assays and offers an ideal solution for drug or inhibitor screening. For more details and sample data, please see our recent publication1 or visit our website at www.RnDSystems.com/KinaseAssay.
Timothy J. Manning
1. Wu, Z.L. (2011) Phosphatase-Coupled Universal Kinase Assay and Kinetics for First-Order-Rate Coupling Reaction. PLoS One 6(8): e23172. doi:10.1371/journal.pone.0023172.
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