High-Throughput Targeted Quantitative Analysis of the Interaction between HSP90 and Kinases

Kinases, which function
in numerous cell signaling processes, are
among the best characterized groups of client proteins for the 90-kDa
heat shock protein (HSP90), a molecular chaperone that suppresses
the aggregation and maintains the proper folding of its substrate
proteins (i.e., clients). No high-throughput proteomic method, however,
has been developed for the characterizations of the interactions between
HSP90 and the human kinome. Herein, by employing a parallel-reaction
monitoring (PRM)-based targeted proteomic method, we found that 99
out of the 249 detected kinase proteins display diminished expression
in cultured human cells upon treatment with ganetespib, a small-molecule
HSP90 inhibitor. PRM analysis of kinase proteins in the affinity pull-down
samples showed that 86 out of the 120 detected kinases are enriched
from the CRISPR-engineered cells where a tandem affinity tag was conjugated
with the C-terminus of endogenous HSP90β protein over the parental
cells. Together, our results from the two complementary quantitative
proteomic experiments offer systematic characterizations about the
HSP90–kinase interactions at the entire proteome scale and
reveal extensive interactions between HSP90 and kinase proteins in
human cells.