ATM kinase phosphorylates HSP90 on T297 changing its conformation dynamics and promoting its interaction with HER2 receptor tyrosine kinase

Heat Shock Protein 90 (HSP90) is an essential molecular chaperone whose activity is regulated not only by co-chaperones but also by distinct post-translational modifications. Interestingly, its chaperone activity is essential for the stability of several oncogenes, among which the receptor tyrosine kinase HER2. HER2 is overexpressed in 20–30% of breast and ovarian cancers. Its overexpression triggers proliferative and transforming pathways aberrant activation and therefore frequently correlates with invasive and poor prognostic features, and associates with shorter patient survival. Of note, HSP90 inhibitors have been studied in HER2-positive breast cancer and have shown promising results. Unexpectedly, we previously reported that ATM promotes the interaction of HER2 with HSP90 therefore sustaining HER2 protein stability and tumorigenicity. To further investigate the interplay between HER2-HSP90 and ATM, we tested the hypothesis that ATM could phosphorylate HSP90. We confirmed that ATM activation can induce the phosphorylation of HSP90 in HER2 positive breast cancer models. Point mutagenesis showed that T297 is the major site targeted by ATM kinase and importantly the unphosphorylatable mutant HSP90-T297A displays a reduced ability to interact with HER2, and to prevent its ubiquitination and degradation. Consistently, the overexpression of HSP90-T297A impinges on the viability of HER2-overexpressing cells, further supporting a role of this phosphorylation in the modulation of HER2 tumorigenicity. T297 is located in the middle domain of HSP90, a region that is involved in the interaction of HSP90 with clients. Consistently, structural studies indicate that T297 phosphorylation can indeed favor the chaperone's interaction with HER2, further supporting our hypothesis. • Targeting HSP90 activity is a promising therapeutic strategy for HER2 positive Breast cancer treatment • HSP90 activity is regulated by post-translational modification like phosphorylation • ATM kinase regulates HSP90β chaperone activity through phosphorylation on Threonine 297 • HSP90β structural analysis and comparative Molecular Dynamics (MD) simulations indicate that T297 phosphorylation impacts on the internal dynamics of the chaperone, preorganizing the client binding site in a favorable dynamic state to lock HER2 once recruited • ATM dependent phosphorylation of HSP90 on T297 is a novel mechanism to promote HSP90 chaperone functionality, sustaining HER2 protein levels, stability and in vitro tumorigenicity.