C-terminal region of Bfl-1 induces cell death that accompanies caspase activation when fused with GFP.
Previously, we reported that anti-apoptotic Bfl-1 is converted to a pro-apoptotic protein following fusion at its N-terminus with green fluorescent protein (GFP) (GFP-Bfl-1). In this study, we performed a Bfl-1 deletion study in order to elucidate the underlying mechanism of GFP-Bfl-1-induced cell death. We found that the Bcl-2 homology (BH) domains in Bfl-1 are dispensable with respect to cell death and that GFP fusion with the 29 amino acids of the C-terminal region of Bfl-1 (GFP-BC) is sufficient to induce cell death. Moreover, when BC was fused with other tagging partners like GST or MBP, little cell death was observed, implying that the GFP region is as important as the BC region for GFP-BC-induced cell death. Further deletion analysis defined a region of GFP as a determinant of GFP-BC-induced cell death. Confocal microscopic analysis showed that GFP-chimeras containing the BC region of Bfl-1 are located mainly in mitochondria. The GFP-BC-induced cell death accompanied cellular caspase activation, and treatment with the pan-caspase inhibitor, Boc-D-FMK, partially inhibited GFP-BC-induced cell death. However, the over-expression of anti-apoptotic molecules, such as Bcl-x(L) and CrmA, did not block GFP-BC-induced cell death. In summary, GFP-BC induces cell death with caspase activation through mitochondria dependent process.
Yang
Wan Seok
WS
Department of Pathology, Tumor Immunity Medical Research Center and Cancer Research Institute, Seoul National University College of Medicine, Seoul, Korea.
Ko
Jae-Kyun
JK
Park
Sue-O
SO
Choi
Hye-Young
HY
Kim
Yong-Nyun
YN
Kim
Chul-Woo
CW
D017209:Apoptosis / Q000502:physiology*; D001483:Base Sequence; D020169:Caspases / Q000378:metabolism*; D002460:Cell Line; D017931:DNA Primers; D004789:Enzyme Activation; D049452:Green Fluorescent Proteins / Q000235:genetics*; D006801:Humans; D008856:Microscopy, Fluorescence; D015778:Minor Histocompatibility Antigens; D008928:Mitochondria / Q000201:enzymology / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000737:chemistry / Q000235:genetics / Q000502:physiology*
C096816:BCL2-related protein A1; D017931:DNA Primers; D015778:Minor Histocompatibility Antigens; D019253:Proto-Oncogene Proteins c-bcl-2; D049452:Green Fluorescent Proteins; D020169:Caspases