Research ArticleBiochemistry

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

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Science Signaling  14 Aug 2018:
Vol. 11, Issue 543, eaao2464
DOI: 10.1126/scisignal.aao2464

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Modified Hsp72 structure-function in mitosis

Chaperone proteins support protein folding, mediate the formation of multiprotein complexes, and enable protein degradation. The functions of the chaperone Hsp72 are coupled; that is, nucleotide exchange triggers the release of bound substrate proteins. Upon phosphorylation, Hsp72 is localized to the mitotic spindle in dividing cells. Changes in Hsp72 abundance or activity are implicated in aging, inflammation, and cancer. Mukherjee et al. used a modified Hsp72 construct to examine structure-function relationships affecting nucleotide exchange and substrate binding during mitosis. Crystal structure analysis showed that phosphorylation induced conformational changes in Hsp72 that enabled nucleotide exchange but maintained substrate binding, which were critical to its function at the mitotic spindle. Exploring ways to mimic or disrupt phosphorylation-dependent changes in Hsp72 may lead to new research and therapeutic tools.


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.

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