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.

激酶（kinase）是参与众多细胞信号转导过程的蛋白质类群，同时也是90kDa热休克蛋白（90-kDa heat shock protein, HSP90）研究最为深入的客户蛋白类群之一；HSP90作为一类分子伴侣，可抑制其底物蛋白（即客户蛋白）聚集并维持其正确折叠。然而目前尚未开发出可用于表征HSP90与人类激酶组之间相互作用的高通量蛋白质组学方法。本研究采用基于平行反应监测（parallel-reaction monitoring, PRM）的靶向蛋白质组学方法，发现在经小分子HSP90抑制剂格奈特西布（ganetespib）处理的培养人细胞中，249个被检测到的激酶蛋白中有99个的表达水平显著下调。对亲和沉淀样本中的激酶蛋白进行PRM分析后发现，在通过CRISPR基因编辑技术将串联亲和标签连接至内源性HSP90β蛋白C端的工程细胞中，120个被检测到的激酶中有86个相较于亲本细胞得到了显著富集。综上，本研究通过两项互补的定量蛋白质组学实验，实现了全蛋白质组规模下HSP90与激酶相互作用的系统表征，并揭示了人类细胞中HSP90与激酶蛋白之间广泛的相互作用。