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.

Figure 1. Strategy for immobilization and immuno-detection

A. Method for the immobilization of peptides on a two-dimensional hydrogel. B. Scheme for the detection of phosphorylated peptides in an ELISA-based format.

Figure 2. A branched hydrophilic linker increases substrate density and distance from the surface

A. The hydrogel surface was activated and coated with either bis-acrylamide or triacrylate (SR415). Equimolar mixtures of Albtide and Abl ligand were immobilized in duplicate wells at the indicated concentrations and phosphorylated by purified recombinant c-Abl kinase. Anti-phosphotyrosine antibody, followed by an HRP-conjugated secondary antibody, was used for chemiluminescent detection. B. The chemical structures of bis-acrylamide and SR415.

Figure 3. Molecular models predict the effect of peptide orientation and relative distance from the site of immobilization to the phosphorylated tyrosine

PyMOL was used to model the orientation of the phosphorylated tyrosine (indicated with a white arrow) relative to the site of immobilization (located at the bottom left hand corner of each figure). Carbon atoms are represented in green, nitrogen in blue, oxygen in red, and sulfur in yellow. A. Abltide with an amino-terminal cysteine (C-AT, CEAIYAAPFAKKK). B. Abltide with an amino-terminal cysteine and the inclusion of 4 amino acid spacers proximal to the site of immobilization (C-4X-AT, CSGGGKGEAIYAAPFAKKKG). C. Abltide with a carboxyl-terminal cysteine (AT-C, KGEAIYAAPFAKKKGC). D. Abltide with a carboxyl-terminal cysteine and 4 amino acids distal to the site of immobilization (4X-AT-C, SGGGKGEAIYAAPFAKKKGC).

Figure 4. Amino acid spacers increase substrate distance and accessibility at the surface

Equimolar mixtures of a peptide substrate and the p40 SH3 ligand were immobilized on a hydrogel surface at 10 μM concentrations. Substrate phosphorylation by purified c-Abl after various time points was detected by chemiluminescence using anti-phosphotyrosine antibody followed by HRP-conjugated secondary antibody.

Figure 5. Scheme for the multiplexed presentation of peptide substrates on Luminex beads

Four peptides were immobilized on distinct Luminex beads (bead regions 27, 34, 53, 65) and distributed to each well of a 96-well plate. These four peptides were treated simultaneously with cell lysates and ATP. After termination of the kinase reaction, four peptide standards (bead regions 42, 45, 61, 73) were added to each well of a 96-well plate and labeled sequentially with the biotinylated anti-phosphotyrosine clone 4G-10 and streptavidin-conjugated phycoerythrin. Peptide standards were constructed from synthetic phospho-Abltide and Abltide, mixed at molar ratios corresponding to 0%, 15%, 30% and 45% phosphorylation.

Figure 6. Modular recognition domains increase peptide reporter sensitivity

A. A Boltzmann-sigmoidal curve was used to relate increases in fluorescence intensity to the percentage of phoshorylation on standard beads. These well-specific internal standard curves were used to calculate the percentage of substrate phosphorylation by cell lysates. B. Four peptide substrates were added to each well in one half of a 96-well plate and reacted with K-562 cell lysates for up to 60 minutes. Peptides containing the p40 peptide ligand of the Abl kinase SH3 domain (C-ALAT and C-ATAL) were preferentially phosphorylated and reached the limits of quantitative detection after only 10 minutes. Abltide immobilized by the amino- (C-AT) or carboxyl-terminus (AT-C) was not phosphorylated to the same extent that it was when presented alone. Boltzmann-sigmoidal curves provided the best fit for the data, emphasizing a dose-response relationship. Points and bars represent the mean fluorescence intensity and 99% confidence interval for a minimum of 100 beads per peptide per well.

Figure 7. Label-free detection of phosphorylated peptides cleaved from a hydrogel surface by UV light

A. Representative spectra of peptides after 30-minute and 60-minute kinase reactions. Peptide substrates constructed from Abltide with a photocleavable linker and p40, the Abl SH3 ligand, were immobilized on a hydrogel surface and released after phosphorylation by K-562 cell lysates. Peak A represents un-phosphorylated Abltide, peak B represents phosphorylated Abltide, and peak C represents “heavy” Abltide used as an internal phosphorylated control (EAIpYAAGPFA). B. The scheme for using MALDI-TOF to detect multiplexed kinase activities. Unique peptide substrates can be immobilized on a hydrogel surface and specifically phosphorylated by kinases in cell lysates. Peptides can be released by the reaction of UV light with the photocleavable linker and detected by mass spectrometry.