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Prepublished online as a Blood First Edition Paper on May 8, 2003; DOI 10.1182/blood-2002-11-3499.
Submitted November 19, 2002
Accepted May 1, 2003
Heat-shock protein 70 binds caspase-activated DNase and enhances its activity in TCR-stimulated T cells
Qing-Li Liu, Hiroyuki Kishi, Kenzo Ohtsuka, and Atsushi Muraguchi*
Department of Immunology, Faculty of Medicine, Toyama Medical and Pharmaceutical University, Toyama, Japan
Laboratory of Cell & Stress Biology, Department of Environmental Biology, College of Bioscience & Biotechnology, Chubu University, Kasugai, Japan
* Corresponding author; email: gucci{at}ms.toyama-mpu.ac.jp.
DNA-fragmentation is a hallmark of cells undergoing apoptosis and mainly mediated by the caspase-activated DNase (CAD or DFF40), which is activated when released from its inhibitor protein (ICAD or DFF45) upon apoptosis signals. Here we analyzed the effect of heat shock protein 70 (Hsp70) on CAD activity in TCR-induced apoptosis using a T cell line (TAg-Jurkat). Overexpression of Hsp70 significantly augmented the apoptotic cell death as well as DNA-fragmentation in CD3/CD28- or staurosporine-stimulated cells. Following stimulation of cells with CD3/CD28- or staurosporine, Hsp70 was co-precipitated with free CAD, but not with CAD associated with ICAD. Furthermore, the purified Hsp70 dose-dependently augmented DNA-fragmentation activity of caspase-3-activated CAD in a cell-free system. Peptide-binding domain-deleted Hsp70 could neither bind nor augment its activity, while ATP binding domain-deleted Hsp70 or the peptide-binding domain itself bound CAD and augmented its activity. These results indicate that the binding of Hsp70 to the activated CAD via peptide-binding domain augments its activity. Although CAD lost its activity in an hour after being released from ICAD in vitro, its activity was retained after an hour-incubation in the presence of Hsp70, suggesting that Hsp70 may be involved in stabilization of CAD activity. Finally, CAD that had been coprecipitated with Hsp70 from the cell lysate of staurosporine-activated 293T cells induced chromatin DNA-fragmentation and its activity was not inhibited by ICAD. These results suggest that Hsp70 binds free CAD in TCR-stimulated T cells to stabilize and augment its activity.
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