Evaluation of Kinase Activity Profiling Using Chemical
Proteomics
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Abstract
Protein
kinases are important mediators of intracellular signaling
and are reversibly activated by phosphorylation. Immobilized kinase
inhibitors can be used to enrich these often low-abundance proteins,
to identify targets of kinase inhibitors, or to probe their selectivity.
It has been suggested that the binding of kinases to affinity beads
reflects a kinase’s activation status, a concept that is under
considerable debate. To assess the merits of the idea, we performed
a series of experiments including quantitative phosphoproteomics and
purification of kinases by single or mixed affinity matrices from
signaling activated or resting cancer cells. The data show that mixed
affinity beads largely bind kinases independent of their activation
status, and experiments using individual immobilized kinase inhibitors
show mixed results in terms of preference for binding the active or
inactive conformation. Taken together, activity- or conformation-dependent
binding to such affinity resins depends (i) on the kinase, (ii) on
the affinity probe, and (iii) on the activation status of the lysate
or cell. As a result, great caution should be exercised when inferring
kinase activity from such binding data. The results also suggest that
assaying kinase activity using binding data is restricted to a limited
number of well-chosen cases