Conformational change in the active center region of GST P1-1, due to binding of a synthetic conjugate of DXR with GSH, enhanced JNK-mediated apoptosis.
Treatment of cells with a synthetic conjugate of DXR with GSH via glutaraldehyde (GSH-DXR) caused cytochrome c to be released from the mitochondria to the cytosol following potent activation of caspase-3 and -9 by typical DNA fragmentation. This apoptosis was regulated by the JNK-signaling pathway. In the present experiment, binding of GSH-DXR to GST P1-1 allosterically led to the disappearance of its enzyme activity and activated the kinase activity of JNK without dissociation of the JNK-GST P1-1 complex. The recombinant GST P1-1 molecule with a mutation in the active center region (W38H and C47S) lost its GST activity when bound to JNK to the same degree as the wild-type, with the mutated GST P1-1 molecule failing to inhibit the activity of JNK. It has been reported that JNK-signaling is regulated by GST P1-1 via interaction with the C-terminus. We confirmed that GST P1-1 deletion mutant (Delta194-209) and a site-directed mutant (R201A) in the C-terminal region failed to bind and inhibit JNK. These results indicated that not only binding of the C-terminal region of GST P1-1 to the JNK molecule, but also the active center region of GST P1-1 play important roles in the regulation of JNK enzyme activity. The findings suggested that allosteric inhibition of GST P1-1 activity by the binding of GSH-DXR following conformational change may activate JNK and induce apoptosis via the mitochondrial pathway in the cells.
Asakura
Tadashi
T
Department of Biochemistry (I), Jikei University School of Medicine, 3-25-8 Nishi-shinbashi, Minato-ku, Tokyo, 105-8461, Japan. tad_asakura@jikei.ac.jp
Sasagawa
Atsuko
A
Takeuchi
Hitoshi
H
Shibata
Shun-Ichi
S
Marushima
Hideki
H
Mamori
Satoshi
S
Ohkawa
Kiyoshi
K
D000818:Animals; D017209:Apoptosis; D002460:Cell Line; D004317:Doxorubicin / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D004789:Enzyme Activation; D005978:Glutathione / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D051549:Glutathione S-Transferase pi / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D048031:JNK Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009154:Mutation; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection
D011993:Recombinant Fusion Proteins; C436625:glutathione-doxorubicin conjugate; D004317:Doxorubicin; D051549:Glutathione S-Transferase pi; D048031:JNK Mitogen-Activated Protein Kinases; D005978:Glutathione