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Mitotic phosphorylation regulates Hsp72 spindle localization by uncoupling ATP binding from substrate release

DOI: 10.1126/scisignal.aao2464 DOI Help

Authors: Manjeet Mukherjee (University of Leeds) , Sarah Sabir (University of Leeds) , Laura O’regan (University of Leicester) , Josephina Sampson (University of Leeds) , Mark W. Richards (University of Leeds) , Nicolas Huguenin-dezot (Medical Research Council Laboratory of Molecular Biology) , James R. Ault (University of Leeds) , Jason W. Chin (Medical Research Council Laboratory of Molecular Biology) , Anastasia Zhuravleva (University of Leeds) , Andrew M. Fry (University of Leicester) , Richard Bayliss (University of Leeds)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Science Signaling , VOL 11

State: Published (Approved)
Published: August 2018

Abstract: Hsp72 is a member of the 70-kDa heat shock family of molecular chaperones (Hsp70s) that comprise a nucleotide-binding domain (NBD) and a substrate-binding domain (SBD) connected by a linker that couples the exchange of adenosine diphosphate (ADP) for adenosine triphosphate (ATP) with the release of the protein substrate. Mitotic phosphorylation of Hsp72 by the kinase NEK6 at Thr66 located in the NBD promotes the localization of Hsp72 to the mitotic spindle and is required for efficient spindle assembly and chromosome congression and segregation. We determined the crystal structure of the Hsp72 NBD containing a genetically encoded phosphoserine at position 66. This revealed structural changes that stabilized interactions between subdomains within the NBD. ATP binding to the NBD of unmodified Hsp72 resulted in the release of substrate from the SBD, but phosphorylated Hsp72 retained substrate in the presence of ATP. Mutations that prevented phosphorylation-dependent subdomain interactions restored the connection between ATP binding and substrate release. Thus, phosphorylation of Thr66 is a reversible mechanism that decouples the allosteric connection between nucleotide binding and substrate release, providing further insight into the regulation of the Hsp70 family. We propose that phosphorylation of Hsp72 on Thr66 by NEK6 during mitosis promotes its localization to the spindle by stabilizing its interactions with components of the mitotic spindle.

Subject Areas: Biology and Bio-materials, Chemistry

Instruments: I04-Macromolecular Crystallography