Cellular damage signals promote sequential changes at the N-terminus and BH-1 domain of the pro-apoptotic protein Bak.
The pro-apoptotic protein Bak is converted from a latent to an active form by damage-induced signals. This process involves an early exposure of an occluded N-terminal epitope of Bak in intact cells. Here we report a subsequent damage-induced change in Bak, detected using an antibody to the central BH-1 domain. Bak co-immunoprecipitated with Bc1-x(L) both in undamaged cells and early after damage, when the N-terminal epitope was exposed but the BH-1 epitope remained occluded. A subsequent decrease in binding of Bak to Bc1-x(L) correlated with exposure of an epitope in the Bak BH-1 domain. Overexpression of Bc1-x(L) did not affect the kinetics of exposure of the Bak N-terminal epitope but delayed exposure of the BH-1 domain. Cytochrome c release from mitochondria facilitates the activation of apoptotic caspases. The majority of cells with exposed Bak BH-1 domains contained cytosolic cytochrome c. However, a small proportion of cells exhibited exposed Bak BH-1 domains that co-localized with mitochondrial cytochrome c. The data are consistent with a two-step model for the activation of Bak by drug-induced damage signals where dissociation of Bc1-x(L) from the BH-1 domain of Bak occurs immediately prior to or concomitantly with cytochrome c release.
Griffiths
G J
GJ
Cancer Research Campaign Molecular Pharmacology Group, School of Biological Sciences, University of Manchester, Manchester M13 9PT, UK.
Corfe
B M
BM
Savory
P
P
Leech
S
S
Esposti
M D
MD
Hickman
J A
JA
Dive
C
C
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D017209:Apoptosis; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003574:Cytochrome c Group / Q000378:metabolism; D018985:Epitopes, B-Lymphocyte / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D017434:Protein Structure, Tertiary; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D015398:Signal Transduction; D014407:Tumor Cells, Cultured; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051020:bcl-X Protein
D000590:Amino Acid Chloromethyl Ketones; C494772:BAK1 protein, human; C494811:BCL2L1 protein, human; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D003574:Cytochrome c Group; D018985:Epitopes, B-Lymphocyte; D008565:Membrane Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051020:bcl-X Protein; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone