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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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Induction of p28 cleavage and procaspase-3 (pro-CPP32) processing during apoptosis in vivo. (A) Cell extracts  were obtained from KB cells that had either been infected for 60 h  with adenovirus pm1716/2072 lacking expression of E1B 19K or  had been mock infected (+ or − Apoptosis, respectively). After  12% SDS-PAGE and transfer to nitrocellulose, blots were incubated with affinity-purified chicken antibody against p28 amino  acids 165–246 (α p28-C) or p28 amino acids 122–164 (α p28-M)  and were developed with secondary antibody conjugated either  to HRP and visualized by electrochemiluminescence (Amersham  Intl., Arlington Heights, IL) (α p28-M) or to alkaline phosphatase and visualized with NBT/BCIP (Boehringer Mannheim  Biochemicals, Indianapolis, IN) (α p28-C), according to the manufacturer's instructions. Bands corresponding to p28 are indicated. Arrows labeled a and b denote products whose sizes are  consistent with cleavage of p28 at the sites designated a and b in  the schematic. (B) KB cells expressing neomycin resistance either  alone (minus Bcl-2, lanes 6–10) or together with Bcl-2 (plus Bcl-2,  lanes 1–5) were infected with adenovirus pm1716/2072 lacking  expression of E1B 19K and cell extracts prepared at 0, 24, 36, 48,  and 60 h postinfection (p.i.; lanes 1 and 6, 2 and 7, 3 and 8, 4 and  9, and 5 and 10, respectively). Aliquots (15 μg protein) were subjected to 12% SDS-PAGE, transferred to nitrocellulose, and  blots were probed with antibody against p28-M or against the 17-kD subunit of CPP32 (Boulakia et al., 1996), and the products  were developed as described in A. The positions of p28 and the  cleavage products a and b are indicated in the upper panels. The  arrow denotes a cross-reacting product whose appearance is variable (e.g., it did not appear in A). The positions of full length pro-CPP32 and the processed 17-kD subunit (p17) and putative  29-kD processing intermediate (asterisk) are indicated in the lower  panels.
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Figure 6: Induction of p28 cleavage and procaspase-3 (pro-CPP32) processing during apoptosis in vivo. (A) Cell extracts were obtained from KB cells that had either been infected for 60 h with adenovirus pm1716/2072 lacking expression of E1B 19K or had been mock infected (+ or − Apoptosis, respectively). After 12% SDS-PAGE and transfer to nitrocellulose, blots were incubated with affinity-purified chicken antibody against p28 amino acids 165–246 (α p28-C) or p28 amino acids 122–164 (α p28-M) and were developed with secondary antibody conjugated either to HRP and visualized by electrochemiluminescence (Amersham Intl., Arlington Heights, IL) (α p28-M) or to alkaline phosphatase and visualized with NBT/BCIP (Boehringer Mannheim Biochemicals, Indianapolis, IN) (α p28-C), according to the manufacturer's instructions. Bands corresponding to p28 are indicated. Arrows labeled a and b denote products whose sizes are consistent with cleavage of p28 at the sites designated a and b in the schematic. (B) KB cells expressing neomycin resistance either alone (minus Bcl-2, lanes 6–10) or together with Bcl-2 (plus Bcl-2, lanes 1–5) were infected with adenovirus pm1716/2072 lacking expression of E1B 19K and cell extracts prepared at 0, 24, 36, 48, and 60 h postinfection (p.i.; lanes 1 and 6, 2 and 7, 3 and 8, 4 and 9, and 5 and 10, respectively). Aliquots (15 μg protein) were subjected to 12% SDS-PAGE, transferred to nitrocellulose, and blots were probed with antibody against p28-M or against the 17-kD subunit of CPP32 (Boulakia et al., 1996), and the products were developed as described in A. The positions of p28 and the cleavage products a and b are indicated in the upper panels. The arrow denotes a cross-reacting product whose appearance is variable (e.g., it did not appear in A). The positions of full length pro-CPP32 and the processed 17-kD subunit (p17) and putative 29-kD processing intermediate (asterisk) are indicated in the lower panels.

Mentions: Two cleavage products of p28, similar in size to those seen in vitro, were also observed in cells that had been induced to undergo apoptotic cell death in response to infection by 19K-defective adenovirus (Fig. 6). In Fig. 6 A, p28 cleavage during apoptosis in vivo was analyzed using antibodies raised in chicken to either of two regions of the protein: p28 amino acids 122–164 (α p28-M) and 165–246 (α p28-C). Cleavage products were detected with α p28-M but not with α p28-C, a finding consistent with the suggestion from peptide sequence analysis that the p20 cleavage product derives from the NH2 terminus of p28 (Fig. 2). α p28-C also failed to detect the larger of the two cleavage products (designated a in Fig. 6 A) despite the predicted overlap of this product with the sequence injected into chickens. Presumably, this means that the extreme eight amino acids of p28 are critically important for epitope recognition by this antibody. Finally, protein electrophoretic blots were developed from apoptotic cell extracts, cut in half along the vertical midline of a protein lane, and one half probed with α p28-M and the other with 32P-Bcl-2Δc21/his6/HMK. p20 detected by the Bcl-2 probe migrated exactly with p20 detected by α p28-M immunoblotting (not shown).


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)

Induction of p28 cleavage and procaspase-3 (pro-CPP32) processing during apoptosis in vivo. (A) Cell extracts  were obtained from KB cells that had either been infected for 60 h  with adenovirus pm1716/2072 lacking expression of E1B 19K or  had been mock infected (+ or − Apoptosis, respectively). After  12% SDS-PAGE and transfer to nitrocellulose, blots were incubated with affinity-purified chicken antibody against p28 amino  acids 165–246 (α p28-C) or p28 amino acids 122–164 (α p28-M)  and were developed with secondary antibody conjugated either  to HRP and visualized by electrochemiluminescence (Amersham  Intl., Arlington Heights, IL) (α p28-M) or to alkaline phosphatase and visualized with NBT/BCIP (Boehringer Mannheim  Biochemicals, Indianapolis, IN) (α p28-C), according to the manufacturer's instructions. Bands corresponding to p28 are indicated. Arrows labeled a and b denote products whose sizes are  consistent with cleavage of p28 at the sites designated a and b in  the schematic. (B) KB cells expressing neomycin resistance either  alone (minus Bcl-2, lanes 6–10) or together with Bcl-2 (plus Bcl-2,  lanes 1–5) were infected with adenovirus pm1716/2072 lacking  expression of E1B 19K and cell extracts prepared at 0, 24, 36, 48,  and 60 h postinfection (p.i.; lanes 1 and 6, 2 and 7, 3 and 8, 4 and  9, and 5 and 10, respectively). Aliquots (15 μg protein) were subjected to 12% SDS-PAGE, transferred to nitrocellulose, and  blots were probed with antibody against p28-M or against the 17-kD subunit of CPP32 (Boulakia et al., 1996), and the products  were developed as described in A. The positions of p28 and the  cleavage products a and b are indicated in the upper panels. The  arrow denotes a cross-reacting product whose appearance is variable (e.g., it did not appear in A). The positions of full length pro-CPP32 and the processed 17-kD subunit (p17) and putative  29-kD processing intermediate (asterisk) are indicated in the lower  panels.
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Figure 6: Induction of p28 cleavage and procaspase-3 (pro-CPP32) processing during apoptosis in vivo. (A) Cell extracts were obtained from KB cells that had either been infected for 60 h with adenovirus pm1716/2072 lacking expression of E1B 19K or had been mock infected (+ or − Apoptosis, respectively). After 12% SDS-PAGE and transfer to nitrocellulose, blots were incubated with affinity-purified chicken antibody against p28 amino acids 165–246 (α p28-C) or p28 amino acids 122–164 (α p28-M) and were developed with secondary antibody conjugated either to HRP and visualized by electrochemiluminescence (Amersham Intl., Arlington Heights, IL) (α p28-M) or to alkaline phosphatase and visualized with NBT/BCIP (Boehringer Mannheim Biochemicals, Indianapolis, IN) (α p28-C), according to the manufacturer's instructions. Bands corresponding to p28 are indicated. Arrows labeled a and b denote products whose sizes are consistent with cleavage of p28 at the sites designated a and b in the schematic. (B) KB cells expressing neomycin resistance either alone (minus Bcl-2, lanes 6–10) or together with Bcl-2 (plus Bcl-2, lanes 1–5) were infected with adenovirus pm1716/2072 lacking expression of E1B 19K and cell extracts prepared at 0, 24, 36, 48, and 60 h postinfection (p.i.; lanes 1 and 6, 2 and 7, 3 and 8, 4 and 9, and 5 and 10, respectively). Aliquots (15 μg protein) were subjected to 12% SDS-PAGE, transferred to nitrocellulose, and blots were probed with antibody against p28-M or against the 17-kD subunit of CPP32 (Boulakia et al., 1996), and the products were developed as described in A. The positions of p28 and the cleavage products a and b are indicated in the upper panels. The arrow denotes a cross-reacting product whose appearance is variable (e.g., it did not appear in A). The positions of full length pro-CPP32 and the processed 17-kD subunit (p17) and putative 29-kD processing intermediate (asterisk) are indicated in the lower panels.
Mentions: Two cleavage products of p28, similar in size to those seen in vitro, were also observed in cells that had been induced to undergo apoptotic cell death in response to infection by 19K-defective adenovirus (Fig. 6). In Fig. 6 A, p28 cleavage during apoptosis in vivo was analyzed using antibodies raised in chicken to either of two regions of the protein: p28 amino acids 122–164 (α p28-M) and 165–246 (α p28-C). Cleavage products were detected with α p28-M but not with α p28-C, a finding consistent with the suggestion from peptide sequence analysis that the p20 cleavage product derives from the NH2 terminus of p28 (Fig. 2). α p28-C also failed to detect the larger of the two cleavage products (designated a in Fig. 6 A) despite the predicted overlap of this product with the sequence injected into chickens. Presumably, this means that the extreme eight amino acids of p28 are critically important for epitope recognition by this antibody. Finally, protein electrophoretic blots were developed from apoptotic cell extracts, cut in half along the vertical midline of a protein lane, and one half probed with α p28-M and the other with 32P-Bcl-2Δc21/his6/HMK. p20 detected by the Bcl-2 probe migrated exactly with p20 detected by α p28-M immunoblotting (not shown).

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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Related in: MedlinePlus