title: Mutational Analysis of Glycogen Synthase Kinase 3β Protein Kinase Together with Kinome-Wide Binding and Stability Studies Suggests Context-Dependent Recognition of Kinases by the Chaperone Heat Shock Protein 90.
abstract: The heat shock protein 90 (HSP90) and cell division cycle 37 (CDC37) chaperones are key regulators of protein kinase folding and maturation. Recent evidence suggests that thermodynamic properties of kinases, rather than primary sequences, are recognized by the chaperones. In concordance, we observed a striking difference in HSP90 binding between wild-type (WT) and kinase-dead (KD) glycogen synthase kinase 3β (GSK3β) forms. Using model cell lines stably expressing these two GSK3β forms, we observed no interaction between WT GSK3β and HSP90, in stark contrast to KD GSK3β forming a stable complex with HSP90 at a 1:1 ratio. In a survey of 91 ectopically expressed kinases in DLD-1 cells, we compared two parameters to measure HSP90 dependency: static binding and kinase stability following HSP90 inhibition. We observed no correlation between HSP90 binding and reduced stability of a kinase after pharmacological inhibition of HSP90. We expanded our stability study to >50 endogenous kinases across four cell lines and demonstrated that HSP90 dependency is context dependent. These observations suggest that HSP90 binds to its kinase client in a particular conformation that we hypothesize to be associated with the nucleotide-processing cycle. Lastly, we performed proteomics profiling of kinases and phosphopeptides in DLD-1 cells to globally define the impact of HSP90 inhibition on the kinome.
authors.affiliation: The Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada jing.jin@northwestern.edu tian.rj@sustc.edu.cn.
authors.forename: Jing
authors.initials: J
authors.lastname: Jin
authors.affiliation: The Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada jing.jin@northwestern.edu tian.rj@sustc.edu.cn.
authors.forename: Ruijun
authors.initials: R
authors.lastname: Tian
authors.affiliation: The Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.
authors.forename: Adrian
authors.initials: A
authors.lastname: Pasculescu
authors.affiliation: The Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.
authors.forename: Anna Yue
authors.initials: AY
authors.lastname: Dai
authors.affiliation: The Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.
authors.forename: Kelly
authors.initials: K
authors.lastname: Williton
authors.affiliation: The Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.
authors.forename: Lorne
authors.initials: L
authors.lastname: Taylor
authors.affiliation: Cellzome, Heidelberg, Germany.
authors.forename: Mikhail M
authors.initials: MM
authors.lastname: Savitski
authors.affiliation: Cellzome, Heidelberg, Germany.
authors.forename: Marcus
authors.initials: M
authors.lastname: Bantscheff
authors.affiliation: The Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada.
authors.forename: James R
authors.initials: JR
authors.lastname: Woodgett
authors.affiliation: The Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Tony
authors.initials: T
authors.lastname: Pawson
authors.affiliation: The Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.
authors.forename: Karen
authors.initials: K
authors.lastname: Colwill
mesh_terms: D000818:Animals; D002460:Cell Line; D038362:Glycogen Synthase Kinase 3 / Q000235:genetics* / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D051379:Mice; D009154:Mutation; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism
chemical_list: D018841:HSP90 Heat-Shock Proteins; D011494:Protein Kinases; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; C000605507:Gsk3b protein, mouse; D038362:Glycogen Synthase Kinase 3