Said A. Goueli, Kevin Hsiao, and Bob Bulleit
Promega Corp., Madison, WI USA 53711
Protein phosphorylation and dephosphorylation are well recognized to play an important role in many cellular processes. The enzymes catalyzing the phosphorylation and dephosphorylation of cellular proteins are pivotal to cell growth, development, differentiation, and apoptosis. Many of the protein kinases are receptors for growth factors, oncogenes, or substrates for other kinases. Similarly, protein phosphatases are not only anti-oncogenes, but they also can act to stimulate oncogenesis. Thus, the identification of such enzymes will help in understanding their function. Hence, there is a need for an assay that measures the activity of these enzymes accurately and with high sensitivity in order to screen compounds for new inhibitors of these enzymes and the development of future drugs. Here we present a novel, robust assay to measure the enzyme activity of low concentrations of several protein kinases and phosphatases with high specificity. The principle of the assay is based on the use of enzyme substrates (Rhodamine 110, bis-peptide amide) that are fluorogenic (no fluorescence) and the generation of reaction products that are fluorescent; and that the fluorescence output in the reactions are directly related to the amount of enzymes. This is because the peptide/R110 conjugate can be cleaved by a protease if it is not phosphorylated and generate a fluorescent product but cannot be cleaved if it is in the phosphorylated form. Thus upon phosphorylation, the phosphopeptide is not cleaved, resulting in a very low fluorescence, and the decrease in fluorescence output can be used as a measure of the activity of the kinase under study. The same principle is used to assay for protein phosphatases using phosphopeptide/R110 conjugate, but here the increase in fluorescence output is directly related to the activity of the phosphatase in the reaction. We were able to detect the activity of kinases and phosphatases at low nanogram level and accurately assess the IC50 values for known inhibitors. In addition, on screening of a chemical library of 640 compounds for their effect as inhibitors of kinases or phosphatases, we successfully identified the selective inhibitors for these enzymes. We will show representative data we generated with multiple kinases and phosphatases. Finally, the assay is rapid, can be completed in less than 2 hours, and can be carried out in multiwell plate formats such as 96-, 384-, and 1536-well plates. The signal-to-noise ratio is very high (40) with a Z′ of over 0.8. Finally, the assays are easily adaptable to robotic systems for drug discovery programs targeting protein kinases and protein phosphatases.