Mutational analysis of N-Bak reveals different structural requirements for antiapoptotic activity in neurons and proapoptotic activity in nonneuronal cells.
N-Bak, a neuron-specific BH3-only splice variant of Bak, is proapoptotic when overexpressed in nonneuronal cells, but antiapoptotic in NGF-deprived sympathetic neurons. We generated mutants of N-Bak and compared their activities in COS-7 or Neuro2A cells to those in NGF-deprived sympathetic neurons. A C-terminal deletion shortly after the BH3 domain of N-Bak compromised its neuroprotective activity but had little effect on its cytotoxic activity in nonneuronal cells. Amino acid changes in the BH3 domain of N-Bak differently affected its function in nonneuronal cells and in neurons. The same changes in the BH3 domain of longer Bak isoform affected its function similarly in nonneuronal cells and neurons. C-terminally truncated Bax, a structural analogue of N-Bak, was also neuroprotective, whereas Blk, a different BH3-only protein was apoptotic in neurons. Thus, neuron-specific antiapoptotic interactions require a "N-Bak-type" conformation, not just a BH3 domain, whereas the presence of a BH3 domain in the Bak protein is sufficient to kill nonneuronal cells.
Sun
Yun-Fu
YF
Program in Molecular Neurobiology, Institute of Biotechnology, University of Helsinki, P.O. Box 56, Viikki Biocenter, Finland.
Yu
Li-Ying
LY
Saarma
Mart
M
Arumäe
Urmas
U
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D019556:COS Cells; D004252:DNA Mutational Analysis; D015870:Gene Expression; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics*; D051379:Mice; D016296:Mutagenesis; D009447:Neuroblastoma; D009474:Neurons / Q000166:cytology* / Q000502:physiology; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000235:genetics; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics; D014407:Tumor Cells, Cultured; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein
C494773:Bak1 protein, mouse; C494827:Bax protein, mouse; D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein