BRCC2, a novel BH3-like domain-containing protein, induces apoptosis in a caspase-dependent manner.
We report here the structure-functional characterization of a novel intronless gene, BRCC2, located on human chromosome 11q24.1. BRCC2 open reading frame (327 bp) codes for an approximately 12-kDa protein (108 amino acids (aa)) localized predominantly in the cytosol and to a lesser extent in the mitochondria. Ectopic expression of BRCC2 cDNA also was found in both the cytosol and mitochondria. Exogenous expression of BRCC2 caused apoptotic cell death in three different cell lines as evidenced by enhanced chromatin condensation, DNA fragmentation, or an enhanced number of cells in the sub-G(1) phase. In human prostate cancer cells (PC-3), BRCC2-induced DNA fragmentation was blocked efficiently by coexpression of the anti-apoptotic molecule, Bcl-X(L). Transient transfection of BRCC2 cDNA into PC-3 cells in the presence of a broad-range caspase inhibitor, Z-VAD-fmk (100 microM, 24 h), abrogated DNA fragmentation. Consistently, BRCC2 expression correlated with the activation of caspase-3 and caspase-9. An N-terminal deletion mutant of BRCC2 (10.2 kDa, Delta1-16 aa) lacking a BH3-like domain (5-12 aa, LPIEGQEI) or BRCC2 containing a mutant BH3-like domain (leucine 5-->glutamate) failed to induce apoptosis, whereas a C-terminal deletion mutant (6.8 kDa, Delta62-108 aa) retained the apoptotic activity comparable to the full-length BRCC2. Finally, the treatment of HeLa cells with doxorubicin or hydrogen peroxide (H(2)O(2)) led to an increase in the mitochondrial (heavy membrane) level of endogenous BRCC2 (doxorubicin (100 ng/ml), 5 h, approximately 2-fold; H(2)O(2) (200 microM), 2 h, approximately 2-fold). These findings demonstrate that BRCC2 functions as a proapoptotic molecule and suggest that BRCC2 induces a caspase-dependent mitochondrial pathway of cell death.
Broustas
Constantinos G
CG
Department of Radiation Medicine, Lombardi Cancer Center, Georgetown University, Washington, D. C. 20057, USA.
Gokhale
Prafulla C
PC
Rahman
Aquilur
A
Dritschilo
Anatoly
A
Ahmad
Imran
I
Kasid
Usha
U
D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D024682:BRCA2 Protein; D015153:Blotting, Western; D019556:COS Cells; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism*; D002460:Cell Line; D045744:Cell Line, Tumor; D002843:Chromatin / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D053938:DNA Fragmentation; D018076:DNA, Complementary / Q000378:metabolism; D004317:Doxorubicin / Q000494:pharmacology; D005260:Female; D017353:Gene Deletion; D006367:HeLa Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008297:Male; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000502:physiology*; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism; D016366:Open Reading Frames; D010957:Plasmids / Q000378:metabolism; D011471:Prostatic Neoplasms / Q000378:metabolism; D017434:Protein Structure, Tertiary; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D014162:Transfection; D051020:bcl-X Protein
D000903:Antibiotics, Antineoplastic; D051017:Apoptosis Regulatory Proteins; C494811:BCL2L1 protein, human; C118024:BLID protein, human; D024682:BRCA2 Protein; C551750:BRCA2 protein, human; D002843:Chromatin; D018076:DNA, Complementary; D008565:Membrane Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051020:bcl-X Protein; D004317:Doxorubicin; D006861:Hydrogen Peroxide; C505407:CASP3 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases