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Prepublished online as a Blood First Edition Paper on May 8, 2003; DOI 10.1182/blood-2002-11-3499.
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Blood, 1 September 2003, Vol. 102, No. 5, pp. 1788-1796
IMMUNOBIOLOGY
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
From the Department of Immunology, Faculty of Medicine, Toyama Medical
and Pharmaceutical University, Sugitani, Toyama, Japan; and the Laboratory of
Cell & Stress Biology, Department of Environmental Biology, College of
Bioscience & Biotechnology, Chubu University, Matsumoto-cho, Kasugai,
Aichi, Japan.
DNA fragmentation is a hallmark of cells undergoing apoptosis and is
mediated mainly by the caspase-activated DNase (CAD or DNA-fragmentation
factor 40 [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 T-cell receptor
(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 coprecipitated
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
adenosine triphosphate (ATP)–binding domain–deleted Hsp70 or the
peptide-binding domain itself bound CAD and augmented its activity. These
results indicate that the the binding of Hsp70 to the activated CAD via the
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 of 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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