A Combined Approach Reveals a Regulatory Mechanism Coupling Src's Kinase Activity, Localization, and Phosphotransferase-Independent Functions.

Multiple layers of regulation modulate the activity and localization of protein kinases. However, many details of kinase regulation remain incompletely understood. Here, we apply saturation mutagenesis and a chemical genetic method for allosterically modulating kinase global conformation to Src kinase, providing insight into known regulatory mechanisms and revealing a previously undiscovered interaction between Src's SH4 and catalytic domains. Abrogation of this interaction increased phosphotransferase activity, promoted membrane association, and provoked phosphotransferase-independent alterations in cell morphology. Thus, Src's SH4 domain serves as an intramolecular regulator coupling catalytic activity, global conformation, and localization, as well as mediating a phosphotransferase-independent function. Sequence conservation suggests that the SH4 domain regulatory interaction exists in other Src-family kinases. Our combined approach's ability to reveal a regulatory mechanism in one of the best-studied kinases suggests that it could be applied broadly to provide insight into kinase structure, regulation, and function.

A Chemically Disrupted Proximity System for Controlling Dynamic Cellular Processes.

Chemical methods that allow the spatial proximity of proteins to be temporally modulated are powerful tools for studying biology and engineering synthetic cellular behaviors. Here, we describe a new chemically controlled method for rapidly disrupting the interaction between two basally colocalized protein binding partners. Our chemically disrupted proximity (CDP) system is based on the interaction between the hepatitis C virus protease (HCVp) NS3a and a genetically encoded peptide inhibitor. Using clinically approved antiviral inhibitors as chemical disrupters of the NS3a/peptide interaction, we demonstrate that our CDP system can be used to confer temporal control over diverse intracellular processes. This NS3a-based CDP system represents a new modality for engineering chemical control over intracellular protein function that is complementary to currently available techniques.

Chemoproteomic Method for Profiling Inhibitor-Bound Kinase Complexes.

Small molecule inhibitors often only block a subset of the cellular functions of their protein targets. In many cases, how inhibiting only a portion of a multifunctional protein's functions affects the state of the cell is not well-understood. Therefore, tools that allow the systematic characterization of the cellular interactions that inhibitor-bound proteins make would be of great utility, especially for multifunctional proteins. Here, we describe a chemoproteomic strategy for interrogating the cellular localization and interactomes of inhibitor-bound kinases. By developing a set of orthogonal inhibitors that contain a trans-cyclooctene (TCO) click handle, we are able to enrich and characterize the proteins complexed to a drug-sensitized variant of the multidomain kinase Src. We show that Src's cellular interactions are highly influenced by the intermolecular accessibility of its regulatory domains, which can be allosterically modulated through its ATP-binding site. Furthermore, we find that the signaling status of the cell also has a large effect on Src's interactome. Finally, we demonstrate that our TCO-conjugated probes can be used as a part of a proximity ligation assay to study Src's localization and interactions in situ. Together, our chemoproteomic strategy represents a comprehensive method for studying the localization and interactomes of inhibitor-bound kinases and, potentially, other druggable protein targets.

A chemically-controlled system for activating RAS GTPases.

RAS GTPases are involved in a number of dynamic signaling processes and have been a major focus of research due to the prevalence of activating RAS mutations in cancer. However, despite decades of research, some fundamental aspects of RAS biology are still not well understood. Difficulty in fully defining RAS-driven signaling stems from the overall complexity of downstream pathways and a lack of tools for specifically perturbing RAS function. To better characterize RAS-driven signaling, we recently developed a chemical genetic system for activating endogenous RAS with a small molecule. In this chapter, we describe the use of chemically inducible activator of RAS (CIAR), a single-protein, chemical genetic system that allows the rapid and dose-dependent activation of endogenous RAS. Methods in this chapter also describe the validation of RAS activation with CIAR through the analysis of downstream signaling.

"Examining RAS pathway rewiring with a chemically inducible activator of RAS".

RAS signaling pathways govern diverse cellular processes, are dynamic, and exhibit marked plasticity. Yet, these features also present a considerable obstacle to their study. Here, we report the use of a recently described RAS rheostat, Chemically Inducible Activator of RAS (CIAR), to study two poorly understood phenomena in RAS biology. First, we show that short-term activation of wild type endogenous RAS can desensitize cells to EGF stimulation. Second, we examine the phenomena of paradoxical activation of RAS/ERK signaling by RAF inhibitors. Specifically, we characterize the effects on RAS/ERK signaling kinetics of four RAF inhibitors, which stabilize distinct ATP-binding site conformations. These results demonstrate the utility of CIAR in conducting quantitative studies of complex features of RAS biology.

Relative quantification of biomarkers using mixed-isotope labeling coupled with MS.

The identification and quantification of important biomarkers is a critical first step in the elucidation of biological systems. Biomarkers take many forms as cellular responses to stimuli and can be manifested during transcription, translation, and/or metabolic processing. Increasingly, researchers have relied upon mixed-isotope labeling (MIL) coupled with MS to perform relative quantification of biomarkers between two or more biological samples. MIL effectively tags biomarkers of interest for ease of identification and quantification within the mass spectrometer by using isotopic labels that introduce a heavy and light form of the tag. In addition to MIL coupled with MS, a number of other approaches have been used to quantify biomarkers including protein gel staining, enzymatic labeling, metabolic labeling, and several label-free approaches that generate quantitative data from the MS signal response. This review focuses on MIL techniques coupled with MS for the quantification of protein and small-molecule biomarkers.