A multiplexed homogeneous fluorescence-based assay for protein kinase activity in cell lysates

Abstract

New methods to quantify protein kinase activities directly from complex cellular mixtures are critical for understanding biological regulatory pathways. Herein, a fluorescence-based chemosensor strategy for the direct measurement of kinase activities in crude mammalian cell lysates is described. We first designed a new fluorescent peptide reporter substrate for each target kinase. These kinase chemosensors were readily phosphorylated by recombinant target enzyme and underwent a several-fold fluorescence increase upon phosphorylation. Then, using unfractionated cell lysates, a homogeneous kinase assay was developed that was reproducible, linear and highly preferential for monitoring changes in cellular activity of the target kinase. The general protocol was developed for the kinase Akt and then easily extended to measure protein kinase A (PKA) and mitogen-activated protein kinase-associated protein kinase 2 (MK2) activities. This assay platform is immediately useful for studying protein kinase signaling in crude cellular extracts.

Fluorescent Amino Acids: Modular Building Blocks for the Assembly of New Tools for Chemical Biology

“…Peptide probes containing the Sox/C-Sox amino acid for a variety of kinases have been synthesized and implemented for kinase measurements with recombinant enzymes and in unfractionated cell lysates, yielding fluorescence enhancements of ~2-12-fold. [62][63][64] It was also found that substitution of the sulfonamide moiety on C-Sox by various substituted triazole groups (installed via click chemistry) gave rise to a multitude of novel Mg 2+ -chelating hydroxyquinoline FlAAs with fluorescence emission maxima that are shifted up to 40 nm. 65 These new click derivatives were incorporated into peptide substrates for study of the MK2 kinase and shown to be as efficiently turned over as C-Sox-containing peptides.…”

Stable Isotope Labeling of Phosphoproteins for Large-scale Phosphorylation Rate Determination

“…Both of these techniques provide useful information on whether the total amount of phosphorylated protein is increasing or decreasing over time; however, they do not directly measure new phosphorylation events or phosphorylation rates (12). MS techniques and fluorescence techniques have been developed to measure phosphorylation rates on synthetic peptides with known kinase consensus motifs in cell lysates (13,14). These techniques provide a read-out of kinase activity in cell lysates under different biological conditions.…”

Profile of Barbara Imperiali

“…They often malfunction in disease states, such as cancer. Through interactions with colleagues at both MIT and other universities, Imperiali and her team fine-tuned the approach into a method that can screen kinase inhibitors and profile kinase activities in cell and tissue lysates (7,8). "The approach that we developed has been commercialized, and we are now looking towards ways to further development of the methods for diagnostic applications," she says.…”