Enzyme Activity Assays for Protein Kinases: Strategies to Identify Active Substrates

Abstract

Protein kinases are an important class of enzymes and drug targets. New opportunities to discover medicines for neglected diseases can be leveraged by the extensive kinase tools and knowledge created in targeting human kinases. A valuable tool for kinase drug discovery is an enzyme assay that measures catalytic function. The functional assay can be used to identify inhibitors, estimate affinity, characterize molecular mechanisms of action (MMOAs) and evaluate selectivity. However, establishing an enzyme assay for a new kinases requires identification of a suitable substrate. Identification of a new kinase’s endogenous physiologic substrate and function can be extremely costly and time consuming. Fortunately, most kinases are promiscuous and will catalyze the phosphotransfer from ATP to alternative substrates with differing degrees of catalytic efficiency. In this manuscript we review strategies and successes in the identification of alternative substrates for kinases from organisms responsible for many of the neglected tropical diseases (NTDs) towards the goal of informing strategies to identify substrates for new kinases. Approaches for establishing a functional kinase assay include measuring auto-activation and use of generic substrates and peptides. The most commonly used generic substrates are casein, myelin basic protein, and histone. Sequence homology modeling can provide insights into the potential substrates and the requirement for activation. Empirical approaches that can identify substrates include screening of lysates (which may also help identify native substrates) and use of peptide arrays. All of these approaches have been used with a varying degree of success to identify alternative substrates.

Insights into the non-mitotic functions of Aurora kinase A: more than just cell division

“…This can be achieved in different ways: among the most common approaches, the activity of a kinase can be tested in vitro. The kinase is purified from bacteria, cells or tissues, and it is used to phosphorylate a putative substrate in presence of ATP [20,21]. In this experimental context, the use of radioactive ATP can help discriminating the phosphorylated form of the substrate by the non-phosphorylated one in western-blotting analyses.…”

“…The purified kinase can also be added directly to cell or tissue lysates, and then processed by biochemical approaches. This allows to explore the capacity of the kinase to phosphorylate a given substrate in its endogenous environment [20].…”

New Approach Methods to Evaluate Health Risks of Air Pollutants: Critical Design Considerations for In Vitro Exposure Testing

“…In addition to expression, the testing of protein function may also be relevant to inhalation exposure, for instance as indicators of signal transduction or oxidative stress. As examples, enzymatic assays can be utilized in vitro to measure kinases (MAPK, JNK), proteases (MMPs), oxidases (NOXs), and metabolic activities (mitochondrial, CYP450s), among others, that may be altered after certain exposure conditions [137][138][139][140].…”

From the research laboratory to the database: the Caenorhabditis elegans kinome in UniProtKB

“…One possible explanation for the discrepancy is that immunoprecipitation of drl-1 from COS cells pulled down another kinase that is responsible for the activity detected. Moreover, they used a promiscuous substrate, myelin basic protein (MBP), which is known to be phosphorylated by multiple kinases [ 29 ]. As there is no other evidence to suggest that dlr-1 is an active kinase, we annotated the protein as an inactive kinase by adding a ‘Domain’ comment to describe that the protein kinase domain is predicted to be catalytically inactive and a ‘Caution’ comment mentioning that some activity has been detected although residues involved in the catalytic activity are absent.…”