Quantification of Protein Kinase Enzymatic Activity in Unfractionated Cell Lysates Using CSox‐Based Sensors

Abstract

Defining perturbations in protein kinase activity within biological samples can provide insight into disease mechanisms as well as potential targets for drug development. In this article, we present a method that utilizes a phosphorylation‐sensitive amino acid, termed CSox, to afford kinase‐selective biosensors capable of reporting on enzymatic activity directly in biological samples. These sensors produce an increase in fluorescence in response to phosphorylation of an amino acid residue adjacent to CSox. Probes can be designed for either serine/threonine or tyrosine kinases, and analysis can be performed using standard fluorescence equipment. The procedures provided herein represent our optimized protocols for the design, validation, and application of CSox‐based protein kinase activity sensors. Curr. Protoc. Chem. Biol. 6:135‐156 © 2014 by John Wiley & Sons, Inc.

Design and evaluation of a real-time activity probe for focal adhesion kinase

“…S2). These data highlight the need to evaluate the Mg 2þ affinity of newly synthesized CSoxbased probes due to the influence of sequence context on Mg 2þ affinity [37]. In this case, the relatively tight affinity of these FAK sensors for Mg 2þ compared to previously described CSox-based chemosensors is likely attributable to the frequency of acidic residues within the FAK autophosphorylation site.…”

“…However, it is important to recognize that FAKtide-S2 could be utilized as a substrate by tyrosine kinases not present in the current panel. Thus, validation of this assay in heterogeneous biological samples will require a more detailed analysis of probe specificity in cell lysates [37].…”

“…Peptides were synthesized as previously described [33,37] using Fmoc-CSox[TBDPS]-OH and were purified using preparative reverse-phase HPLC (Fig. S1).…”

“…1) [33e36]. CSox-based sensors can be readily accessed through standard Fmoc solid-phase peptide synthesis, allowing for the facile synthesis of direct reporters of kinase enzymatic activity [37], making these probes ideally suited for HTS. Indeed, recent work from the Dalby laboratory has detailed the use of Sox-based sensors to identify eEF-2 inhibitor scaffolds from a 32,960 member small molecule library [38].…”

The 8-Silyloxyquinoline Scaffold as a Versatile Platform for the Sensitive Detection of Aqueous Fluoride

“…This scaffold has also been used to provide a sensitive fluorescent readout in aqueous samples. [60][61][62][63] Therefore, we chose to investigate the ability of 8-hydroxyquinoline derivatives to function as fluoride sensors.…”