The association of deamidation of Bcl-xL and translocation of Bax to the mitochondria through activation of JNK in the induction of apoptosis by treatment with GSH-conjugated DXR.
We investigated the induction of apoptosis via deamidation of Bcl-xL and translocation of Bax to the mitochondria by treatment with GSH-DXR. GSH-DXR treatment of HepG2 cells, which did not express GST P1-1, exhibited deamidation of Bcl-xL, and the degree of deamidation was related to the activation of caspase-3. Overexpression of GST P1-1 in HepG2 cells decreased both the Bcl-xL deamidation and caspase-3 activation induced by treatment with GSH-DXR. Bcl-xL deamidation and caspase-3 activation were also suppressed by co-treatment with SP600125, a specific inhibitor of JNK activity. Overexpression of wild-type Bcl-xL in HepG2 decreased GSH-DXR-induced apoptosis although deamidation was observed. However, expression of the deamidated mutant of Bcl-xL, in which aspartic acid was substituted for both arginine 52 and 66 (N52,66D-Bcl-xL), exhibited high sensitivity for the induction of apoptosis. Expression of the Bcl-xL mutant, in which alanine was substituted for both arginine 52 and 66 (N52,66A-Bcl-xL), suppressed deamidation and showed resistance to the induction of apoptosis by treatment with GSH-DXR. On the other hand, endogenous Bax and overexpressed Flag-Bax were localized in the cytosolic fraction of HepG2 cells. Treatment of the cells with GSH-DXR caused translocation of Flag-Bax to the mitochondrial fraction following the induction of apoptosis. The induced apoptosis was enhanced by the expression of Flag-Bax. Moreover, Flag-Bax was partly located in the mitochondrial fraction in N52,66D-Bcl-xL-expressed cells without the induction of apoptosis. Therefore, the induction of apoptosis by treatment of HepG2 with GSH-DXR was enhanced, thereby facilitating the release of cytochrome c by both deamidated inactivation of Bcl-xL and functional translocation of Bax to the mitochondria via JNK activation. Deamidation of Bcl-xL might be induced in order to translocate Bax to the mitochondria.
Asakura
Tadashi
T
Department of Biochemistry, Jikei University School of Medicine, Minato-ku, Tokyo 105-8461, Japan. tad_asakura@jikei.ac.jp
Maeda
Kazuhiro
K
Omi
Hiroko
H
Matsudaira
Hiroshi
H
Ohkawa
Kiyoshi
K
D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D015153:Blotting, Western; D053148:Caspase 3 / Q000187:drug effects / Q000378:metabolism; D045744:Cell Line, Tumor; D004317:Doxorubicin / Q000031:analogs & derivatives* / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005978:Glutathione / Q000031:analogs & derivatives* / Q000494:pharmacology; D006801:Humans; D048670:MAP Kinase Kinase 4 / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D051028:bcl-2-Associated X Protein / Q000378:metabolism*; D051020:bcl-X Protein / Q000187:drug effects / Q000378:metabolism*
C494811:BCL2L1 protein, human; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein; C436625:glutathione-doxorubicin conjugate; D004317:Doxorubicin; D048670:MAP Kinase Kinase 4; D053148:Caspase 3; D005978:Glutathione