Correlation between structure of Bcl-2 and its inhibitory function of JNK and caspase activity in dopaminergic neuronal apoptosis.
To examine the correlation between the structure of Bcl-2 and its inhibitory function of c-Jun N-terminal kinase (JNK) and caspase activity, we established a dopaminergic neuronal cell line, MN9D overexpressing Bcl-2 (MN9D/Bcl-2) or its structural mutants. The mutants comprised a point mutation in the BH1 (G145A; MN9D/BH1) or BH2 (W188A; MN9D/BH2) domain and a deletion mutation in the C-terminal (MN9D/C22), BH3 (MN9D/BH3), or BH4 (MN9D/BH4) domain. As determined by the TUNEL (terminal deoxynucleotidyltransferase nick end-labeling) and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reduction assay, apoptotic death of MN9D/Neo cells reached 80-90% within 24 h in response to 1 microM staurosporine. Upon staurosporine treatment, JNK activity increased six- to sevenfold over the basal level within 2-4 h. Treatment of MN9D/Neo with both staurosporine and a caspase inhibitor, Z-VAD, attenuated cell death without suppressing JNK activation. Both staurosporine-induced cell death and JNK activation were attenuated in MN9D/Bcl-2. As determined by cleavage of poly(ADP-ribose) polymerase into 85 kDa, Bcl-2 blocked caspase activity as well. When cells overexpressing one of the Bcl-2 mutants were treated with staurosporine, death was attenuated in MN9D/BH1, MN9D/BH2, and MN9D/C22 but not in MN9D/BH3 and MN9D/BH4. Similarly, both JNK and caspase activation were blocked in MN9D/BH1, MN9D/BH2, and MN9D/C22, whereas they were not suppressed in MN9D/BH3 and MN9D/BH4. Taken together, our data indicate that there exists a close structural and functional correlation of Bcl-2 to JNK and caspase activity in staurosporine-induced dopaminergic neuronal cell death.
Choi
W S
WS
Department of Biology, Yonsei University College of Science and Institute of Life Science, Seoul, Korea.
Yoon
S Y
SY
Chang
I I
II
Choi
E J
EJ
Rhim
H
H
Jin
B K
BK
Oh
T H
TH
Krajewski
S
S
Reed
J C
JC
Oh
Y J
YJ
D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D020169:Caspases / Q000378:metabolism*; D002478:Cells, Cultured; D004298:Dopamine / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D020287:In Situ Nick-End Labeling; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D009154:Mutation / Q000502:physiology; D009474:Neurons / Q000166:cytology* / Q000201:enzymology*; D010300:Parkinson Disease / Q000378:metabolism; D017434:Protein Structure, Tertiary; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019311:Staurosporine / Q000494:pharmacology; D013329:Structure-Activity Relationship; D014162:Transfection
D004791:Enzyme Inhibitors; D019253:Proto-Oncogene Proteins c-bcl-2; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D020169:Caspases; D019311:Staurosporine; D004298:Dopamine