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Molecules 2015, 20(1), 1643-1660; doi:10.3390/molecules20011643

Stability of the Human Hsp90-p50Cdc37 Chaperone Complex against Nucleotides and Hsp90 Inhibitors, and the Influence of Phosphorylation by Casein Kinase 2

1
Drug Discovery Department, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612, USA
2
Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota, Minneapolis, MN 55414, USA
3
Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, KS 66160, USA
*
Author to whom correspondence should be addressed.
Academic Editor: Philippe Belmont
Received: 1 December 2014 / Accepted: 12 January 2015 / Published: 19 January 2015
(This article belongs to the Special Issue Design and Study of Kinase Inhibitors)
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Abstract

The molecular chaperone Hsp90 is regulated by co-chaperones such as p50Cdc37, which recruits a wide selection of client protein kinases. Targeted disruption of the Hsp90-p50Cdc37 complex by protein–protein interaction (PPI) inhibitors has emerged as an alternative strategy to treat diseases characterized by aberrant Hsp90 activity. Using isothermal microcalorimetry, ELISA and GST-pull down assays we evaluated reported Hsp90 inhibitors and nucleotides for their ability to inhibit formation of the human Hsp90β-p50Cdc37 complex, reconstituted in vitro from full-length proteins. Hsp90 inhibitors, including the proposed PPI inhibitors gedunin and H2-gamendazole, did not affect the interaction of Hsp90 with p50Cdc37 in vitro. Phosphorylation of Hsp90 and p50Cdc37 by casein kinase 2 (CK2) did not alter the thermodynamic signature of complex formation. However, the phosphorylated complex was vulnerable to disruption by ADP (IC50 = 32 µM), while ATP, AMPPNP and Hsp90 inhibitors remained largely ineffective. The differential inhibitory activity of ADP suggests that phosphorylation by CK2 primes the complex for dissociation in response to a drop in ATP/ADP levels. The approach applied herein provides robust assays for a comprehensive biochemical evaluation of potential effectors of the Hsp90-p50Cdc37 complex, such as phosphorylation by a kinase or the interaction with small molecule ligands. View Full-Text
Keywords: heat shock proteins; protein–protein interactions; protein phosphorylation; protein kinase; small molecule inhibitors heat shock proteins; protein–protein interactions; protein phosphorylation; protein kinase; small molecule inhibitors
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Olesen, S.H.; Ingles, D.J.; Zhu, J.-Y.; Martin, M.P.; Betzi, S.; Georg, G.I.; Tash, J.S.; Schönbrunn, E. Stability of the Human Hsp90-p50Cdc37 Chaperone Complex against Nucleotides and Hsp90 Inhibitors, and the Influence of Phosphorylation by Casein Kinase 2. Molecules 2015, 20, 1643-1660.

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