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p28 Bap31, a Bcl-2/Bcl-XL- and procaspase-8-associated protein in the endoplasmic reticulum.

Ng FW, Nguyen M, Kwan T, Branton PE, Nicholson DW, Cromlish JA, Shore GC - J. Cell Biol. (1997)

Bottom Line: Bax, a pro-apoptotic member of the Bcl-2 family, does not associate with the complex; however, it prevents Bcl-2 from doing so.The resulting NH2-terminal p20 fragment induces apoptosis when expressed ectopically in otherwise normal cells.Taken together, the results suggest that p28 Bap31 is part of a complex in the endoplasmic reticulum that mechanically bridges an apoptosis-initiating caspase, like procaspase-8, with the anti-apoptotic regulator Bcl-2 or Bcl-XL.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, McIntyre Medical Sciences Building, McGill University, Montreal, Quebec, Canada H3G 1Y6.

ABSTRACT
We have identified a human Bcl-2-interacting protein, p28 Bap31. It is a 28-kD (p28) polytopic integral protein of the endoplasmic reticulum whose COOH-terminal cytosolic region contains overlapping predicted leucine zipper and weak death effector homology domains, flanked on either side by identical caspase recognition sites. In cotransfected 293T cells, p28 is part of a complex that includes Bcl-2/Bcl-XL and procaspase-8 (pro-FLICE). Bax, a pro-apoptotic member of the Bcl-2 family, does not associate with the complex; however, it prevents Bcl-2 from doing so. In the absence (but not presence) of elevated Bcl-2 levels, apoptotic signaling by adenovirus E1A oncoproteins promote cleavage of p28 at the two caspase recognition sites. Purified caspase-8 (FLICE/MACH/Mch5) and caspase-1(ICE), but not caspase-3 (CPP32/apopain/ Yama), efficiently catalyze this reaction in vitro. The resulting NH2-terminal p20 fragment induces apoptosis when expressed ectopically in otherwise normal cells. Taken together, the results suggest that p28 Bap31 is part of a complex in the endoplasmic reticulum that mechanically bridges an apoptosis-initiating caspase, like procaspase-8, with the anti-apoptotic regulator Bcl-2 or Bcl-XL. This raises the possibility that the p28 complex contributes to the regulation of procaspase-8 or a related caspase in response to E1A, dependent on the status of the Bcl-2 setpoint within the complex.

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Appearance of a  Bcl-2–interacting polypeptide during E1A-induced apoptosis. KB cells expressing  neomycin resistance, either  alone (Neo) or together with  Bcl-2, were infected with either adenovirus dl520E1B−  (expressing 12S E1A and no  E1B products) or pm1760/ 2072 (expressing 12S and 13S  E1A but not E1B 19K). At  the indicated times after infection, samples of cells were  either assessed for viability  by exclusion of trypan blue  (graph) or prepared for  ligand blot (Far Western)  analysis, as described in Materials and Methods, using  32P-Bcl-2Δc21/his6/HMK as a  probe (upper panel, Bcl- 2–expressing cells; lower  panel, Neo control cells).  Ligand blots were visualized  by phosphorimaging. The radioactive band associated  with a polypeptide of Mr 20  kD is labeled p20, whereas  that which comigrates with  Bax is designated p21 Bax.  The latter was determined  using a blot cut along the vertical midline of a protein lane  and developing one half by  immunoblot analysis with  anti–human Bax (Chen et al.,  1996) and the other by ligand  blotting with 32P-Bcl-2Δc21/ his6/HMK (results not shown).
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Figure 1: Appearance of a Bcl-2–interacting polypeptide during E1A-induced apoptosis. KB cells expressing neomycin resistance, either alone (Neo) or together with Bcl-2, were infected with either adenovirus dl520E1B− (expressing 12S E1A and no E1B products) or pm1760/ 2072 (expressing 12S and 13S E1A but not E1B 19K). At the indicated times after infection, samples of cells were either assessed for viability by exclusion of trypan blue (graph) or prepared for ligand blot (Far Western) analysis, as described in Materials and Methods, using 32P-Bcl-2Δc21/his6/HMK as a probe (upper panel, Bcl- 2–expressing cells; lower panel, Neo control cells). Ligand blots were visualized by phosphorimaging. The radioactive band associated with a polypeptide of Mr 20 kD is labeled p20, whereas that which comigrates with Bax is designated p21 Bax. The latter was determined using a blot cut along the vertical midline of a protein lane and developing one half by immunoblot analysis with anti–human Bax (Chen et al., 1996) and the other by ligand blotting with 32P-Bcl-2Δc21/ his6/HMK (results not shown).

Mentions: Fig. 1 shows one such example after infection of neo- and BCL-2–expressing human KB cells with adenovirus type 5 producing either 12S E1A mRNA (which encodes 243R E1A protein) or both 12S and 13S E1A mRNAs (which encode 243R and 289R E1A proteins, respectively), but lacking expression of the dominant suppressor of apoptotic cell death, E1B 19-kD protein (19K). Induction of apoptotic cell death by either virus (Nguyen et al., 1994; Teodoro et al., 1995) was accompanied by the appearance of p20 Bcl-2–binding activity, whereas apparent binding to Bax did not change significantly during the time course of infection. Of note, however, is the observation that stable expression of Bcl-2 in these cells countered cell death and prevented the appearance of p20 Bcl-2–binding activity after viral infection.


p28 Bap31, a Bcl-2/Bcl-XL- and procaspase-8-associated protein in the endoplasmic reticulum.

Ng FW, Nguyen M, Kwan T, Branton PE, Nicholson DW, Cromlish JA, Shore GC - J. Cell Biol. (1997)

Appearance of a  Bcl-2–interacting polypeptide during E1A-induced apoptosis. KB cells expressing  neomycin resistance, either  alone (Neo) or together with  Bcl-2, were infected with either adenovirus dl520E1B−  (expressing 12S E1A and no  E1B products) or pm1760/ 2072 (expressing 12S and 13S  E1A but not E1B 19K). At  the indicated times after infection, samples of cells were  either assessed for viability  by exclusion of trypan blue  (graph) or prepared for  ligand blot (Far Western)  analysis, as described in Materials and Methods, using  32P-Bcl-2Δc21/his6/HMK as a  probe (upper panel, Bcl- 2–expressing cells; lower  panel, Neo control cells).  Ligand blots were visualized  by phosphorimaging. The radioactive band associated  with a polypeptide of Mr 20  kD is labeled p20, whereas  that which comigrates with  Bax is designated p21 Bax.  The latter was determined  using a blot cut along the vertical midline of a protein lane  and developing one half by  immunoblot analysis with  anti–human Bax (Chen et al.,  1996) and the other by ligand  blotting with 32P-Bcl-2Δc21/ his6/HMK (results not shown).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139787&req=5

Figure 1: Appearance of a Bcl-2–interacting polypeptide during E1A-induced apoptosis. KB cells expressing neomycin resistance, either alone (Neo) or together with Bcl-2, were infected with either adenovirus dl520E1B− (expressing 12S E1A and no E1B products) or pm1760/ 2072 (expressing 12S and 13S E1A but not E1B 19K). At the indicated times after infection, samples of cells were either assessed for viability by exclusion of trypan blue (graph) or prepared for ligand blot (Far Western) analysis, as described in Materials and Methods, using 32P-Bcl-2Δc21/his6/HMK as a probe (upper panel, Bcl- 2–expressing cells; lower panel, Neo control cells). Ligand blots were visualized by phosphorimaging. The radioactive band associated with a polypeptide of Mr 20 kD is labeled p20, whereas that which comigrates with Bax is designated p21 Bax. The latter was determined using a blot cut along the vertical midline of a protein lane and developing one half by immunoblot analysis with anti–human Bax (Chen et al., 1996) and the other by ligand blotting with 32P-Bcl-2Δc21/ his6/HMK (results not shown).
Mentions: Fig. 1 shows one such example after infection of neo- and BCL-2–expressing human KB cells with adenovirus type 5 producing either 12S E1A mRNA (which encodes 243R E1A protein) or both 12S and 13S E1A mRNAs (which encode 243R and 289R E1A proteins, respectively), but lacking expression of the dominant suppressor of apoptotic cell death, E1B 19-kD protein (19K). Induction of apoptotic cell death by either virus (Nguyen et al., 1994; Teodoro et al., 1995) was accompanied by the appearance of p20 Bcl-2–binding activity, whereas apparent binding to Bax did not change significantly during the time course of infection. Of note, however, is the observation that stable expression of Bcl-2 in these cells countered cell death and prevented the appearance of p20 Bcl-2–binding activity after viral infection.

Bottom Line: Bax, a pro-apoptotic member of the Bcl-2 family, does not associate with the complex; however, it prevents Bcl-2 from doing so.The resulting NH2-terminal p20 fragment induces apoptosis when expressed ectopically in otherwise normal cells.Taken together, the results suggest that p28 Bap31 is part of a complex in the endoplasmic reticulum that mechanically bridges an apoptosis-initiating caspase, like procaspase-8, with the anti-apoptotic regulator Bcl-2 or Bcl-XL.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, McIntyre Medical Sciences Building, McGill University, Montreal, Quebec, Canada H3G 1Y6.

ABSTRACT
We have identified a human Bcl-2-interacting protein, p28 Bap31. It is a 28-kD (p28) polytopic integral protein of the endoplasmic reticulum whose COOH-terminal cytosolic region contains overlapping predicted leucine zipper and weak death effector homology domains, flanked on either side by identical caspase recognition sites. In cotransfected 293T cells, p28 is part of a complex that includes Bcl-2/Bcl-XL and procaspase-8 (pro-FLICE). Bax, a pro-apoptotic member of the Bcl-2 family, does not associate with the complex; however, it prevents Bcl-2 from doing so. In the absence (but not presence) of elevated Bcl-2 levels, apoptotic signaling by adenovirus E1A oncoproteins promote cleavage of p28 at the two caspase recognition sites. Purified caspase-8 (FLICE/MACH/Mch5) and caspase-1(ICE), but not caspase-3 (CPP32/apopain/ Yama), efficiently catalyze this reaction in vitro. The resulting NH2-terminal p20 fragment induces apoptosis when expressed ectopically in otherwise normal cells. Taken together, the results suggest that p28 Bap31 is part of a complex in the endoplasmic reticulum that mechanically bridges an apoptosis-initiating caspase, like procaspase-8, with the anti-apoptotic regulator Bcl-2 or Bcl-XL. This raises the possibility that the p28 complex contributes to the regulation of procaspase-8 or a related caspase in response to E1A, dependent on the status of the Bcl-2 setpoint within the complex.

Show MeSH
Related in: MedlinePlus