Format

Send to

Choose Destination
Int J Hyperthermia. 2019;36(1):438-443. doi: 10.1080/02656736.2019.1587009. Epub 2019 Mar 28.

Reactivation of heat-inactivated Ku proteins by heat shock cognate protein HSC73.

Author information

1
a Department of Radioisotope Medicine, Atomic Bomb Disease and Hibakusha Medicine Unit, Atomic Bomb Disease Institute , Nagasaki University , Nagasaki , Japan.
2
b Department of Tumor and Diagnostic Pathology, Atomic Bomb Disease and Hibakusha Medicine Unit, Atomic Bomb Disease Institute , Nagasaki University , Nagasaki , Japan.
3
c Laboratory of Cell and Stress Biology, College of Bioscience and Biotechnology , Chubu University , Kasugai , Japan.

Abstract

PURPOSE:

Mouse double-stranded DNA-dependent protein kinase (DNA-PK) activity is heat sensitive. Recovery of heat-inactivated DNA repair activity is a problem after combination therapy with radiation and heat. We investigated the mechanism of recovery of heat-inactivated DNA-PK activity.

METHODS:

Hybrid cells containing a fragment of human chromosome 8 in scid cells (RD13B2) were used. DNA-PK activity was measured by an in vitro assay. Immunoprecipitation of the nuclear extract was performed with an anti-Ku80 antibody. Proteins co-precipitated with Ku80 were separated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and detected by Western blotting using anti-heat shock protein (HSP)72 and anti-heat shock cognate protein (HSC)73 antibodies. HSC73 was overexpressed with the pcDNA3.1 vector. Short hairpin (sh)RNA was used to downregulate HSC73 and HSP72.

RESULTS:

The activity of heat-inactivated DNA-PK recovered to about 50% of control during an additional incubation at 37 °C after heat treatment at 44 °C for 15 min in the presence of cycloheximide (which inhibits de novo protein synthesis). Maximal recovery was observed within 3 h of incubation at 37 °C after heat treatment. Constitutively expressed HSC73, which folds newly synthesized proteins, reached maximal levels 3 h after heat treatment using a co-immunoprecipitation assay with the Ku80 protein. Inhibiting HSC73, but not HSP72, expression with shRNA decreased the recovery of DNA-PK activity after heat treatment.

CONCLUSIONS:

These results suggest that de novo protein synthesis is unnecessary for recovery of some heat-inactivated DNA-PK. Rather, it might be reactivated by the molecular chaperone activity of HSC73, but not HSP72.

KEYWORDS:

DNA-PK activity; HSC73; Ku70/80; heat; molecular chaperone

Supplemental Content

Full text links

Icon for Taylor & Francis
Loading ...
Support Center