A homogeneous, nonradioactive high-throughput fluorogenic protein phosphatase assay.

Abstract

Protein phosphatases are critical components in cellular regulation; they do not only act as antioncogenes by antagonizing protein kinases, but they also play a positive regulatory role in a variety of cellular processes that require dephosphorylation. Thus, assessing the function of these enzymes necessitates the need for a robust, sensitive assay that accurately measures their activities. The authors present a novel, homogeneous, and nonradioactive assay to measure the enzyme activity of low concentrations of several protein phosphatases (phosphoserine/phosphothreonine phosphatases and phosphotyrosine phosphatases). The assay is based on the use of fluorogenic peptide substrates (rhodamine 110, bis-phosphopeptide amide) that do not fluoresce in their conjugated form, which is resistant to cleavage by aminopeptidases. However, upon dephosphorylation by the phosphatase of interest, the peptides become cleavable by the protease and release the highly fluorescent-free rhodamine 110. The assay is rapid, can be completed in less than 2 h, and can be carried out in multiwell plate formats such as 96-, 384-, and 1536-well plates. The assay has an excellent dynamic range, high signal-to-noise ratio, and a Z' of more than 0.8, and it is easily adapted to a robotic system for drug discovery programs targeting protein phosphatases.