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

Manjeet Mukherjee1, Sarah Sabir1, Laura O'Regan2

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Summary

Heat shock protein 72 (Hsp72) phosphorylation by NEK6 stabilizes its mitotic spindle localization. This modification decouples ATP binding from substrate release, regulating Hsp70 function during cell division.

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Area of Science:

  • Molecular Biology
  • Cell Biology
  • Structural Biology

Background:

  • Hsp72, a 70-kDa heat shock protein, functions as a molecular chaperone with nucleotide-binding (NBD) and substrate-binding domains (SBD).
  • Mitotic phosphorylation of Hsp72 by NEK6 at Thr66 is crucial for its spindle localization and proper chromosome segregation.

Purpose of the Study:

  • To elucidate the structural and functional consequences of Hsp72 phosphorylation at Thr66.
  • To understand how NEK6-mediated phosphorylation regulates Hsp72's interaction with ATP and substrates during mitosis.

Main Methods:

  • Determined the crystal structure of the Hsp72 NBD with phosphoserine at position 66.
  • Investigated the effect of phosphorylation on ATP binding and substrate release using biochemical assays.
  • Utilized site-directed mutagenesis to disrupt phosphorylation-dependent structural changes.

Main Results:

  • Phosphorylation at Thr66 stabilizes NBD subdomain interactions, altering its structure.
  • Phosphorylated Hsp72 retains substrate even in the presence of ATP, unlike unmodified Hsp72.
  • Mutations preventing phosphorylation-induced structural changes restore ATP-dependent substrate release.

Conclusions:

  • Phosphorylation of Hsp72 at Thr66 by NEK6 acts as a reversible switch, decoupling nucleotide binding from substrate release.
  • This regulatory mechanism fine-tunes Hsp70 function during mitosis, promoting spindle localization and stability.
  • Provides new insights into the allosteric regulation of the Hsp70 chaperone family.