title: Stability of the human Hsp90-p50Cdc37 chaperone complex against nucleotides and Hsp90 inhibitors, and the influence of phosphorylation by casein kinase 2.
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.
authors.affiliation: Drug Discovery Department, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612, USA. sanne.olesen@agilent.com.
authors.forename: Sanne H
authors.initials: SH
authors.lastname: Olesen
authors.affiliation: Drug Discovery Department, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612, USA. Donna.Ingles@moffitt.org.
authors.forename: Donna J
authors.initials: DJ
authors.lastname: Ingles
authors.affiliation: Drug Discovery Department, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612, USA. Jinyi.Zhu@moffitt.org.
authors.forename: Jin-Yi
authors.initials: JY
authors.lastname: Zhu
authors.affiliation: Drug Discovery Department, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612, USA. Mathew.Martin@newcastle.ac.uk.
authors.forename: Mathew P
authors.initials: MP
authors.lastname: Martin
authors.affiliation: Drug Discovery Department, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612, USA. stephane.betzi@inserm.fr.
authors.forename: Stephane
authors.initials: S
authors.lastname: Betzi
authors.affiliation: Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota, Minneapolis, MN 55414, USA. georg@umn.edu.
authors.forename: Gunda I
authors.initials: GI
authors.lastname: Georg
authors.affiliation: Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, KS 66160, USA. JTASH@kumc.edu.
authors.forename: Joseph S
authors.initials: JS
authors.lastname: Tash
authors.affiliation: Drug Discovery Department, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612, USA. ernst.schonbrunn@moffitt.org.
authors.forename: Ernst
authors.initials: E
authors.lastname: Schönbrunn
mesh_terms: D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D002151:Calorimetry; D047390:Casein Kinase II / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000378:metabolism*; D045744:Cell Line, Tumor; D018833:Chaperonins / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D009711:Nucleotides / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D060066:Protein Interaction Maps / Q000187:drug effects; D055550:Protein Stability / Q000187:drug effects; D017434:Protein Structure, Tertiary; D054852:Small Molecule Libraries / Q000494:pharmacology; D013816:Thermodynamics
chemical_list: C498362:CDC37 protein, human; D018797:Cell Cycle Proteins; D018841:HSP90 Heat-Shock Proteins; D009711:Nucleotides; D054852:Small Molecule Libraries; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D047390:Casein Kinase II; D018833:Chaperonins