In the most basic of terms a kinase is an enzyme that can transfer a phosphate group from ATP to a biomolecule (fig. 1).
Although this biomolecule could be a protein, lipid, or carbohydrate most research is performed on
protein kinases so that will be our focus.
The transfer of the phosphate group is called phosphorylation. In protein phosphorylation, the transfer can only occur onto 3 amino acid residues, serine, threonine, and tyrosine. Kinases, for the most part, are divided into those that can phosphorylate serine/threonine and those that can phosphorylate tyrosine.
In addition, kinases are not able to phosphorylate every possible residue but are restrained by a variety of sequence motifs around the phosphorylatable residue.
These sequences determine if a kinase, specifically the kinases active site, can interact with the protein, coordinate ATP-hydrolysis and phosphate transfer that characterize the phosphorylation event.
Fig. 1: Reaction catalyzed by kinases: Kinases transfer phosphate groups from ATP to a biomolecule
Addition of a phosphate causes changes to the local environment in the protein due to charge on the phosphate group as well as simply addition of a ‘bulky’ extension.
These local changes can result in conformational changes in the protein that alter its function. This altered function includes changes in the ability of the protein to
interact with binding partners.
The classic example is transcription factors whose ability to bind specific regulatory elements in DNA are changed by phosphorylation events. Changes in protein function
can also be seen in changes to enzyme activity, including changes to kinase activity. From the changes in protein function, extensive webs of interaction and interplay between various kinase signaling pathways have been drawn and are enough to make your head spin
Fig 2. The mitogen activated protein kinase (MAPK) pathway.
The final cellular effect of these pathways is the phosphorylation of proteins that are important in the regulation of vital processes such as differentiation, proliferation and apoptosis.
As evidenced by the complex signaling webs associated with kinases, their activity is a tightly regulated process. This regulation is important due to the essential nature of the cellular processes in which they are involved.
The involvement of kinases in essential processes also explains the interest in them as targets of therapeutics. Especially to remedy diseases where dysregulation of kinases has been identified as a key component.
In particular, cancers are frequently associated with kinase malfunction so discovery of drugs that target kinases is desirable, and to achieve this assays that can accurately represent kinase activity are required.
Using these assays, the ability of test compounds to inhibit the kinase activity can be determined.
In general, the search for kinase inhibitors requires
a very targeted approach to decrease the possibility of confounding effects.
The targeted approach usually requires the isolation of the kinase from the complex signaling milieu found within cells.
Therefore, the typical kinase assay is referred to as an in vitro kinase assay which has been optimized for enzyme / substrate concentrations and buffer conditions,
including optimized ATP and co-factor concentrations.
There are several steps involved in the process of a
protein being phosphorylated.
Protein kinase assays have taken advantage of this fact to develop assays that, monitor interactions with the
kinase active site, evaluate conversion of ATP to ADP and attachment of a phosphate group onto the substrate.
Choice of which approach you should use depends on the question you wish to answer, the type of sample to which you have access and the detection capability you prefer. Fortunately, Eutheria Bioscience has the options that employ all the detection capabilities available on
There is a huge selection of binding assays available. To give you an idea, here two popular and commonly used kinase binding assays are being highlighted.
The LanthaScreenTM Eu Kinase Binding assay platform (ThermoFisher Scientific) was designed as an HTS platform that can identify any compound that alters binding to the
active site (aka ATP-site) on a kinase.
The platform employs a fluorescently labeled tracer of which there are now six. The tracers are based on known inhibitors that bind to kinases. Specificity for a kinase is achieved
using a tagged version of the kinase.
This tag can bind a Europium-labeled antibody such that when antibody and tracer are both bound to the kinase a
high TR-FRET signal is observed. Potential inhibitors are discovered based on their ability to displace the tracer resulting in a decreased TR-FRET
Fig. 3: Schematic of LanthaScreen Eu Kinase Binding Assay.
Currently over 300 validated assays are listed. These include several important mutant versions of some of the kinases. Furthermore, it has been shown that this assay platform can be used to study both active and inactive kinases and to assess the role of phosphorylation on the interaction with the tracer.
And as we saw in figure 2 the regulation of kinases by phosphorylation is essential. The TR-FRET based method to measure kinases is further used to analyze the kinetic binding parameters of kinase interactions.
To this end, the binding parameters association rate and dissociation rate are initially determined for a combination of Europium-labeled kinase and a fluorescently labeled tracer.
Then an unknown potential kinase inhibitor is added to the measurement to investigate whether it competes with the known tracer. If this is the case, the association curve of the tracer changes and association and dissociation rate of the unknown inhibitor can be calculated.
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