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.