A peptide biosensor for detecting intracellular Abl kinase activity using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Abstract

Many cancers are characterized by changes in protein phosphorylation as a result of kinase dysregulation. Disruption of Abl kinase signaling through the Philadelphia chromosome (causing the Bcr-Abl mutation) in chronic myeloid leukemia (CML) has provided a paradigm for development of kinase inhibitor drugs such as the specific inhibitor imatinib (also known as STI571 or Gleevec). However, since patients are treated indefinitely with this drug to maintain remission, resistance is increasingly becoming an issue. While there are many ways to detect kinase activity, most lack the ability to ‘multiplex’ the analysis (to detect more than one substrate simultaneously). Here we report a novel biosensor for detecting Abl kinase activity and sensitivity to inhibitor in live, intact cells overexpressing a CML model Abl kinase construct. This straightforward methodology could eventually provide a new tool for detecting kinase activity and inhibitor drug response in cancer cells that overexpress oncogenic kinases.

Detection of Early Abl Kinase Activation after Ionizing Radiation by Using a Peptide Biosensor

“…The primary advantage of such FRET-based sensors is that subcellular kinase activities can be imaged in real time—however, these techniques require sophisticated quantitative imaging and can suffer from low dynamic range in the fold-response that can be detected, since changes in FRET signal are sometimes small even upon dramatic changes in the level of phosphorylation of the sensor. Because the phosphorylation of the peptide biosensor described here can be monitored in a standard, simple manner using either the generic phosphotyrosine antibody 4G10 or label-free mass spectrometry (as we have previously demonstrated [25] ), this strategy could be advantageous for molecular biology laboratories that want a simple, straightforward assay for exploring the initial timecourse of Abl’s role in the DNA damage response.…”

“…The Abl SH3 ligand sequence acts to further promote this specificity by providing interaction with the Abl SH3 domain, making the biosensor peptide a better mimic of native substrates, which include such protein-protein interaction modules. K biotin = lysine biotinylated on the γ-amino group (side chain) is included for detecting the total amount of biosensor via streptavidin blotting; The photolinker (a 3-(2-nitrobenzyl)-3-aminopropionyl residue) is incorporated in case detection with mass spectrometry is used (as we have previously reported [25] ) however this is not important for the work described in this manuscript. B) Shows a cartoon representation of the interactions between Abl, ATM and DNAPK after DNA damage.…”

“…To follow the intracellular activation of Abl in response to IR-induced DNA damage, we used our previously reported peptide-based biosensor substrate. [25] This biosensor (Figure 1) has the following functional modules: a known peptide substrate for Abl, [26] a photocleavable linker, an Abl SH3 domain-binding ligand peptide, [27] a biotin tag and a cell-penetrating peptide, TAT, [28] to aid delivery of the biosensor across the plasma membrane. To distinguish between the contributions of ATM and DNAPK to Abl activation in the DNA damage pathway, we generated HEK293 cell lines with stable, Tet-ON inducible expression of Abl-WT-EGFP and Abl(S465A)-EGFP, which lacks the known ATM phosphorylation site.…”

Cdc14 Phosphatases Preferentially Dephosphorylate a Subset of Cyclin-dependent kinase (Cdk) Sites Containing Phosphoserine

“…Peptide Synthesis and Purification-Peptide synthesis and purification was performed using solid-phase Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry on a Prelude parallel peptide synthesizer (Protein Technologies, Tucson, AZ) essentially as described (14) with the following changes. Coupling times for phosphorylated amino acids were increased to 3 h. Completed peptides were cleaved with 5 ml of 95% TFA, 2.5% water, and 2.5% triisopropylsilane, precipitated, and washed three times with 35 ml of diethyl ether prior to purification by reverse phase HPLC.…”

A general strategy for studying multisite protein phosphorylation using label-free selected reaction monitoring mass spectrometry

“…Peptides were synthesized using CLEAR-Amide resin (Peptides International) at 50 μmol scale by solid-phase FMOC chemistry on a Prelude peptide synthesizer (Protein Technologies, Inc.) essentially as described previously [29]. The only modification was that coupling times for FMOC-phosphoserine and -phosphothreonine (Anaspec, Inc.) were increased to 3 hours.…”