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

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 Thr 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 Thr 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 Thr by NEK6 during mitosis promotes its localization to the spindle by stabilizing its interactions with components of the mitotic spindle.

HSP70 regulates Eg5 distribution within the mitotic spindle and modulates the cytotoxicity of Eg5 inhibitors

“…HSP70 is a highly conserved inducible molecular chaperone essential for cellular proteostasis; it has been shown to be targeted to the mitotic centrosome by multiple mitotic kinases 11 , 12 , 63 – 65 , and its pharmacological inhibition disrupts mitotic spindle assembly to induce mitotic arrest and mitotic cell death 10 , 11 . Eg5 is a kinesin MT motor that plays an essential role in spindle assembly, making it a promising target for antimitotic cancer therapeutics 4 , 14 , 25 .…”

ERK-dependent phosphorylation of the linker and substrate-binding domain of HSP70 increases folding activity and cell proliferation

“…4c). To prove this hypothesis, we conducted a limited digestion assay using proteinase K because the exposed linker region is easy to cleave with proteinase 15 . As shown in Fig.…”

“…For the limited digestion assay, purified wild type HSP70 and the S385D/S400D mutants were incubated with proteinase K according to a previously reported method 15,16 . Ten micrograms of HSP70 protein was preincubated with 200 μM ATP at room temperature, and proteinase K was added in a dose-dependent manner.…”

Hsp70 molecular chaperones: multifunctional allosteric holding and unfolding machines

“…In contrast, the endoplasmic reticulum (ER) Hsp70, bindingimmunoglobulin protein (BiP), shows less favorable domain docking than DnaK: its ATPbound state is heterogeneous with only half of the molecules adopting a domain-docked conformation [40]. In addition to evolutionary tuning, the allosteric landscapes of Hsp70s are also modulated by post-translational modifications, and this tuning of their allosteric behaviors is directly related to their physiological functions [40][41][42]. For instance, AMPylation of T518 biases BiP towards the domain-docked state, subsequently inactivating BiP and limiting its interaction with substrates [40,42].…”

“…For instance, AMPylation of T518 biases BiP towards the domain-docked state, subsequently inactivating BiP and limiting its interaction with substrates [40,42]. Phosphorylation of HspA1 at T66 promotes domain undocking and increases substrate-binding affinity in the ATP-bound state [41]. A physiological result of this modification is that the interactions of HspA1 with components of mitotic spindles are stabilized and the localization to the spindle is favored.…”