Interrogating Signaling Nodes Involved in Cellular Transformations Using Kinase Activity Probes

Abstract

Summary Protein kinases catalyze protein phosphorylation and thereby control the flow of information through signaling cascades. Currently available methods for concomitant assessment of the enzymatic activities of multiple kinases in complex biological samples rely upon indirect proxies for enzymatic activity, such as posttranslational modifications to protein kinases. Our laboratories have recently described a method for directly quantifying the enzymatic activity of kinases in unfractionated cell lysates using substrates containing a phosphorylation-sensitive unnatural amino acid termed CSox, which can be monitored using fluorescence. Herein, we demonstrate the utility of this methodology using a probe set encompassing p38α, MK2, ERK1/2, Akt, and PKA. This panel of chemosensors provides activity measurements of individual kinases in a model of skeletal muscle differentiation and can be readily used to generate individualized kinase activity profiles for tissue samples from clinical cancer patients.

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

“…66 Based on this scaffold approach and the benefits of the C-Sox amino acid, further sensors for specific kinases (p38, JNK, MAPK family) have been developed, providing insight into the modulation of kinase activities in diseased tissue samples. 67 Chelating FlAAs have found use in other applications as well. For example, Wang and coworkers have reported the synthesis and incorporation of the metal-chelating 2-amino-3-hydroxyquinoline-3-yl)-propanoic acid (HQ-Ala) into proteins not only as a CHEF fluorescent reporter, but also a handle for introduction of a heavy metal ion for Xray crystallography phasing.…”

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

“…Consensus phosphorylation sites often do not provide adequate selectivity amongst endogenous kinases; well-characterized substrates should be employed when possible. As a general guideline, we have found that peptide substrates with k cat values of >20 min −1 provide a sufficient turnover in order to visualize endogenous kinase activity in cell lysates within one hour (Stains et al, 2012). In addition, K M values in the low μM range (<5 μM) typically provide selectivity for the KOI while minimizing off-target activity if appropriate sensor concentrations are used (i.e.…”

“…There are two situations that can arise in which obtained data may not produce a hyperbolic plot. First, the data may exhibit an initial lag phase prior to the linear region of the data that should be used for work-up (Lukovic et al, 2009; Shults et al, 2005; Stains et al, 2011; Stains et al, 2012). In this case, the initial lag phase should not be included in the analysis as this will bias the fit.…”

“…In addition, ATP is not a selective substrate for protein kinases, rendering this approach incompatible with complex cell lysates. In contrast, CSox-based sensors can be used on standard fluorescence equipment without specialized training and can be tuned to selectively report on the activity of a KOI in unfractionated cell lysates (Lukovic et al, 2009; Stains et al, 2011; Stains et al, 2012). Moreover, phosphoryl transfer can be monitored in real-time, allowing for straightforward kinetic analysis.…”

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.…”