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

Area of Science:

• Molecular Biology
• Proteomics
• Cell Signaling

Background:

• Heat shock protein 90 (HSP90) is a molecular chaperone crucial for cell signaling by stabilizing client proteins, particularly kinases.
• Characterizing the full spectrum of HSP90-kinase interactions in humans has been limited by the lack of high-throughput proteomic methods.

Purpose of the Study:

• To systematically characterize HSP90-kinase interactions across the human kinome using quantitative proteomic approaches.
• To investigate the impact of HSP90 inhibition on kinase expression and identify direct HSP90 clients.

Main Methods:

• Employed parallel-reaction monitoring (PRM)-based targeted proteomics to quantify kinase expression changes upon HSP90 inhibition.
• Utilized CRISPR-engineered cells with a C-terminal tandem affinity tag on endogenous HSP90β for affinity pull-down and subsequent PRM analysis.

Main Results:

• Treatment with the HSP90 inhibitor ganetespib led to diminished expression of 99 out of 249 detected kinases.
• Affinity pull-down experiments identified 86 kinases enriched in cells expressing tagged HSP90β, indicating direct interactions.

Conclusions:

• The study provides the first proteome-wide characterization of HSP90-kinase interactions in human cells.
• Results demonstrate extensive and previously unappreciated interactions between HSP90 and the human kinome, impacting kinase stability and cellular signaling pathways.