title: Hsp90 inhibition differentially destabilises MAP kinase and TGF-beta signalling components in cancer cells revealed by kinase-targeted chemoproteomics.
abstract: BACKGROUND
  The heat shock protein 90 (Hsp90) is required for the stability of many signalling kinases. As a target for cancer therapy it allows the simultaneous inhibition of several signalling pathways. However, its inhibition in healthy cells could also lead to severe side effects. This is the first comprehensive analysis of the response to Hsp90 inhibition at the kinome level.
  
  
  METHODS
  We quantitatively profiled the effects of Hsp90 inhibition by geldanamycin on the kinome of one primary (Hs68) and three tumour cell lines (SW480, U2OS, A549) by affinity proteomics based on immobilized broad spectrum kinase inhibitors ("kinobeads"). To identify affected pathways we used the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway classification. We combined Hsp90 and proteasome inhibition to identify Hsp90 substrates in Hs68 and SW480 cells. The mutational status of kinases from the used cell lines was determined using next-generation sequencing. A mutation of Hsp90 candidate client RIPK2 was mapped onto its structure.
  
  
  RESULTS
  We measured relative abundances of > 140 protein kinases from the four cell lines in response to geldanamycin treatment and identified many new potential Hsp90 substrates. These kinases represent diverse families and cellular functions, with a strong representation of pathways involved in tumour progression like the BMP, MAPK and TGF-beta signalling cascades. Co-treatment with the proteasome inhibitor MG132 enabled us to classify 64 kinases as true Hsp90 clients. Finally, mutations in 7 kinases correlate with an altered response to Hsp90 inhibition. Structural modelling of the candidate client RIPK2 suggests an impact of the mutation on a proposed Hsp90 binding domain.
  
  
  CONCLUSIONS
  We propose a high confidence list of Hsp90 kinase clients, which provides new opportunities for targeted and combinatorial cancer treatment and diagnostic applications.
authors.affiliation: Department of Vertebrate Genomics, Max-Planck Institute for Molecular Genetics, Berlin, Germany.
authors.forename: Armin
authors.initials: A
authors.lastname: Haupt
authors.forename: Gerard
authors.initials: G
authors.lastname: Joberty
authors.forename: Marcus
authors.initials: M
authors.lastname: Bantscheff
authors.forename: Holger
authors.initials: H
authors.lastname: Fröhlich
authors.forename: Henning
authors.initials: H
authors.lastname: Stehr
authors.forename: Michal R
authors.initials: MR
authors.lastname: Schweiger
authors.forename: Axel
authors.initials: A
authors.lastname: Fischer
authors.forename: Martin
authors.initials: M
authors.lastname: Kerick
authors.forename: Stefan T
authors.initials: ST
authors.lastname: Boerno
authors.forename: Andreas
authors.initials: A
authors.lastname: Dahl
authors.forename: Michael
authors.initials: M
authors.lastname: Lappe
authors.forename: Hans
authors.initials: H
authors.lastname: Lehrach
authors.forename: Cayetano
authors.initials: C
authors.lastname: Gonzalez
authors.forename: Gerard
authors.initials: G
authors.lastname: Drewes
authors.forename: Bodo Mh
authors.initials: BM
authors.lastname: Lange
mesh_terms: D016227:Benzoquinones / Q000494:pharmacology; D045744:Cell Line, Tumor; D004791:Enzyme Inhibitors / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000235:genetics / Q000378:metabolism*; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry; D006801:Humans; D047029:Lactams, Macrocyclic / Q000494:pharmacology; D009154:Mutation; D009369:Neoplasms / Q000188:drug therapy / Q000378:metabolism; D040901:Proteomics; D053475:Receptor-Interacting Protein Serine-Threonine Kinase 2 / Q000235:genetics*; D015398:Signal Transduction / Q000187:drug effects; D016212:Transforming Growth Factor beta / Q000378:metabolism*
chemical_list: D016227:Benzoquinones; D004791:Enzyme Inhibitors; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D016212:Transforming Growth Factor beta; C506253:RIPK2 protein, human; D053475:Receptor-Interacting Protein Serine-Threonine Kinase 2; D048049:Extracellular Signal-Regulated MAP Kinases; C001277:geldanamycin