Wild-type, mitochondrial and ER-restricted Bcl-2 inhibit DNA damage-induced apoptosis but do not affect death receptor-induced apoptosis.
The proto-oncogene Bcl-2 is expressed in membranes of mitochondria and endoplasmic reticulum and mediates resistance against a broad range of apoptotic stimuli. Although several mechanisms of Bcl-2 action have been proposed, its role in different cellular organelles remains elusive. Here, we analyzed the function of Bcl-2 targeted specifically to certain subcellular compartments in Jurkat cells. Bcl-2 expression was restricted to the outer mitochondrial membrane by replacing its membrane anchor with the mitochondrial insertion sequence of ActA (Bcl-2/MT) or the ER-specific sequence of cytochrome b5 (Bcl-2/ER). Additionally, cells expressing wild-type Bcl-2 (Bcl-2/WT) or a transmembrane domain-lacking mutant (Bcl-2/DeltaTM) were employed. Apoptosis induced by ionizing radiation or by the death receptors for CD95L or TRAIL was analyzed by determination of the mitochondrial membrane potential (DeltaPsi(m)) and activation of different caspases. Bcl-2/WT and Bcl-2/MT strongly inhibited radiation-induced apoptosis and caspase activation, whereas Bcl-2/DeltaTM had completely lost its anti-apoptotic effect. Interestingly, Bcl-2/ER conferred protection against radiation-induced mitochondrial damage and apoptosis similarly to Bcl-2/MT. The finding that ER-targeted Bcl-2 interfered with mitochondrial DeltaPsi(m) breakdown and caspase-9 activation indicates the presence of a crosstalk between both organelles in radiation-induced apoptosis. By contrast, Bcl-2 in either subcellular position did not influence CD95- or TRAIL-mediated apoptosis.
Rudner
J
J
Department of Radiation Oncology, University of Tübingen, Hoppe-Seyler Str. 3, D-72076 Tübingen, Germany.
Lepple-Wienhues
A
A
Budach
W
W
Berschauer
J
J
Friedrich
B
B
Wesselborg
S
S
Schulze-Osthoff
K
K
Belka
C
C
D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D053454:Caspase 12; D020169:Caspases / Q000378:metabolism; D004249:DNA Damage; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006801:Humans; D019169:Jurkat Cells; D008562:Membrane Glycoproteins / Q000378:metabolism* / Q000494:pharmacology; D008928:Mitochondria / Q000378:metabolism*; D000090063:Proto-Oncogene Mas; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D011839:Radiation, Ionizing; D013347:Subcellular Fractions; D053221:TNF-Related Apoptosis-Inducing Ligand; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism* / Q000494:pharmacology; D019014:fas Receptor / Q000378:metabolism*
D051017:Apoptosis Regulatory Proteins; C000715470:MAS1 protein, human; D008562:Membrane Glycoproteins; D000090063:Proto-Oncogene Mas; D019253:Proto-Oncogene Proteins c-bcl-2; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; D014409:Tumor Necrosis Factor-alpha; D019014:fas Receptor; C506444:CASP12 protein, human; D053454:Caspase 12; D020169:Caspases