TY - JOUR
T1 - Mitochondria are required for ATM activation by extranuclear oxidative stress in cultured human hepatoblastoma cell line Hep G2 cells
AU - Morita, Akinori
AU - Tanimoto, Keiji
AU - Murakami, Tomoki
AU - Morinaga, Takeshi
AU - Hosoi, Yoshio
N1 - Funding Information:
This study was supported in part by KAKENHI (23659588) from the Japan Society for the Promotion of Science (to Y. Hosoi). We are grateful to Dr. Nobuo Watanabe for helpful discussions with him. The expert technical assistance and administrative support of Ms. Chiyo Oda, Ms. Ikuko Fukuba, and Ms. Hikaru Hirono are gratefully acknowledged. Electron microscopic analyses were carried out at the Analysis Center of Life Science (Acols), Hiroshima University.
PY - 2014/1/24
Y1 - 2014/1/24
N2 - Ataxia-telangiectasia mutated (ATM) is a serine/threonine protein kinase that plays a central role in DNA damage response (DDR). A recent study reported that oxidized ATM can be active in the absence of DDR. However, the issue of where ATM is activated by oxidative stress remains unclear. Regarding the localization of ATM, two possible locations, namely, mitochondria and peroxisomes are possible. We report herein that ATM can be activated when exposed to hydrogen peroxide without inducing nuclear DDR in Hep G2 cells, and the oxidized cells could be subjected to subcellular fractionation. The first detergent-based fractionation experiment revealed that active, phosphorylated ATM was located in the second fraction, which also contained both mitochondria and peroxisomes. An alternative fractionation method involving homogenization and differential centrifugation, which permits the light membrane fraction containing peroxisomes to be produced, but not mitochondria, revealed that the light membrane fraction contained only traces of ATM. In contrast, the heavy membrane fraction, which mainly contained mitochondrial components, was enriched in ATM and active ATM, suggesting that the oxidative activation of ATM occurs in mitochondria and not in peroxisomes. In Rho 0-Hep G2 cells, which lack mitochondrial DNA and functional mitochondria, ATM failed to respond to hydrogen peroxide, indicating that mitochondria are required for the oxidative activation of ATM. These findings strongly suggest that ATM can be activated in response to oxidative stress in mitochondria and that this occurs in a DDR-independent manner.
AB - Ataxia-telangiectasia mutated (ATM) is a serine/threonine protein kinase that plays a central role in DNA damage response (DDR). A recent study reported that oxidized ATM can be active in the absence of DDR. However, the issue of where ATM is activated by oxidative stress remains unclear. Regarding the localization of ATM, two possible locations, namely, mitochondria and peroxisomes are possible. We report herein that ATM can be activated when exposed to hydrogen peroxide without inducing nuclear DDR in Hep G2 cells, and the oxidized cells could be subjected to subcellular fractionation. The first detergent-based fractionation experiment revealed that active, phosphorylated ATM was located in the second fraction, which also contained both mitochondria and peroxisomes. An alternative fractionation method involving homogenization and differential centrifugation, which permits the light membrane fraction containing peroxisomes to be produced, but not mitochondria, revealed that the light membrane fraction contained only traces of ATM. In contrast, the heavy membrane fraction, which mainly contained mitochondrial components, was enriched in ATM and active ATM, suggesting that the oxidative activation of ATM occurs in mitochondria and not in peroxisomes. In Rho 0-Hep G2 cells, which lack mitochondrial DNA and functional mitochondria, ATM failed to respond to hydrogen peroxide, indicating that mitochondria are required for the oxidative activation of ATM. These findings strongly suggest that ATM can be activated in response to oxidative stress in mitochondria and that this occurs in a DDR-independent manner.
KW - ATM
KW - Hydrogen peroxide
KW - Mitochondria
KW - Oxidative stress
KW - Peroxisome
UR - http://www.scopus.com/inward/record.url?scp=84893725571&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84893725571&partnerID=8YFLogxK
U2 - 10.1016/j.bbrc.2013.12.139
DO - 10.1016/j.bbrc.2013.12.139
M3 - Article
C2 - 24406161
AN - SCOPUS:84893725571
VL - 443
SP - 1286
EP - 1290
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
SN - 0006-291X
IS - 4
ER -