Peptide reporters of kinase activity in whole cell lysates

Abstract

Kinase assays are used to screen for small-molecule inhibitors that may show promise as targeted pharmaceutical therapies. Using cell lysates instead of purified kinases provides a more accurate estimate of inhibitor sensitivity and selectivity in a biological setting. This review summarizes the range of homogeneous (solution-phase) and heterogeneous (solid-supported) formats available for using peptide substrates to monitor kinase activities in cell lysates. With a focus on heterogeneous kinase assays, the peptide substrate Abltide is used as a model to optimize presentation geometries and the modular arrangement of short sequences for kinase recognition. We present results from peptides immobilized on two- and three-dimensional surfaces such as hydrogels on 96-well plates and glass slides, and fluorescent Luminex beads. We discuss methods to increase assay sensitivity using chemifluorescent ELISAs, antibody-based recognition, and label-free mass spectrometry. Monitoring the activity of specific kinases in cell lysates presents challenges that can be overcome by manipulating peptide substrates to optimize assay conditions. In particular, signal-to-background ratios were improved by 1) adding long branched hydrophilic linkers between the substrate and the surface, 2) changing the orientation of peptides relative to the surface, and 3) including peptide ligands in cis or in trans to recruit kinases to the surface. By improving the accessibility of immobilized peptide substrates to kinases in solution, the apparent rate of phosphorylation increased and assays were more sensitive to changes in endogenous kinase activities. These strategies can be generalized to improve the reactivity of most peptide substrates used in heterogeneous kinase assays with cell lysates.

A magnetic bead-based protein kinase assay with dual detection techniques

“…In particular, streptavidin on the bead surface may occlude the bound peptide substrate from easy access to kinases in solution. In general, solid-phase kinase assays have been characterized as less efficient that solution-phase kinase reactions [46]. Post-reaction capture of phosphorylated substrates, after solution-phase kinase reactions, has been used as an alternative to solid-phase kinase assays.…”

Fluorescence Lifetime Imaging of Biosensor Peptide Phosphorylation in Single Live Cells

“…[11] The substrate portion (bold) is relatively selective for the c-Abl kinase (Abl1) over other tyrosine kinases, however it is phosphorylated by the Abl family member named Abl-related gene (Arg, also known as Abl2). [12] Abl1 and Abl2 are highly homologous and share many functions in normal cells. [13] In this work, “Abl kinase” denotes both Abl1 and Abl2.…”

A Bead-Based Activity Screen for Small-Molecule Inhibitors of Signal Transduction in Chronic Myelogenous Leukemia Cells

“…Previous work using immobilized Abltide to measure Bcr-Abl activity in cell lysates demonstrated that altered orientations resulted in increased signal intensities (35). It was also shown that inclusion of p40, the high-affinity peptide ligand for the Abl kinase SH3 domain, led to more efficient phosphorylation of Abltide (17, 36).…”