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

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:

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

Disclosure statement

The authors alone are responsible for the content and writing of this article and have no competing interests.

Additional information

Funding

This work was supported by Grants-in-Aid for Scientific Research (C) [No. 13670950, No. 23510064, No. 18K10027] (KAKENHI) from the Ministry of Education, Culture, Sports, Science and Technology of Japan and grants from Eisai Co. This work was also partly supported by the Program of the Network-type Joint Usage/Research Center for Radiation Disaster Medical Science of Hiroshima University, Nagasaki University and Fukushima Medical University.