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

Jing Jin*, Ruijun Tian, Adrian Pasculescu, Anna Yue Dai, Kelly Williton, Lorne Taylor, Mikhail M. Savitski, Marcus Bantscheff, James R. Woodgett, Tony Pawson, Karen Colwill

*Corresponding author for this work

Research output: Contribution to journalArticle

6 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)1007-1018
Number of pages12
JournalMolecular and cellular biology
Volume36
Issue number6
DOIs
StatePublished - Jan 1 2016

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ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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