Limits...
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.

Show MeSH

Related in: MedlinePlus

Identification of p20. (A) Preparative SDS-PAGE of differentially solubilized protein from KB cells 60 h after infection with  adenovirus pm1760/2072. Aliquots of fractions eluted from the gel were assayed by 32P-Bcl-2Δc21/his6/HMK Far Western, and the radioactive bands corresponding to p20 and p21 Bax were detected and quantified by phosphorimaging. The levels relative to the maximal  signal detected (set at 100) were plotted as a bar graph (upper panel). Equal aliquots from the same fractions were also subjected to analytical 12% SDS-PAGE, and the gels were stained with Coomassie brilliant blue (lower panel). The positions of molecular mass marker  proteins are indicated. black, p20; gray, Bax. (B) Proteins eluting from preparative SDS-PAGE between 190 and 220 ml were concentrated and resolved by reverse-phase HPLC. The upper panel shows the A280 profile. Equal aliquots from all fractions were assayed for  32P-Bcl-2Δc21/his6/HMK–interacting protein by Far Western, for which only p20 was detected. Amounts relative to the maximal signal  detected (set at 100) were plotted as a bar graph (lower panel). Fractions 52, 53, 54 (peak activity), and 55 were individually subjected to  NH2-terminal peptide sequence analysis. (C) Polypeptide sequence of p28 Bap31/CDM (single-letter code). Peptide sequencing of p20  revealed a perfect match with amino acids 2–11 of human Bap31(underlined; these sequence data available from GenBank/EMBL/ DDBJ under accession number X81817). This was the only detectable sequence in fraction 54, was detectable together with other sequences in fraction 53, and was not detected in fractions 52 and 55. Predicted TM segments are boxed and contain charged amino acids  in TM1 and TM3 (asterisks). The predicted caspase recognition sites, AAVD·G, are highlighted, and cleavage is denoted by arrows following Asp at positions 164 and 238. A potential leucine zipper located between the caspase recognition sites is denoted by bold letters,  as is the KKXX ER retention signal at the COOH terminus. (D) Comparison of putative death effector domain sequences for the indicated proteins. The sequences, given in the single-letter amino acid code, were obtained from GenBank/EMBL/DDBJ, and their relative positions in the molecule are shown in parentheses. Sequences were aligned using the PILEUP program of the GCG software package and were optimized by spacing (shown as dashes). Identical residues and conserved substitutions that were recorded for at least half  of the sequences analyzed are shaded in gray.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139787&req=5

Figure 2: Identification of p20. (A) Preparative SDS-PAGE of differentially solubilized protein from KB cells 60 h after infection with adenovirus pm1760/2072. Aliquots of fractions eluted from the gel were assayed by 32P-Bcl-2Δc21/his6/HMK Far Western, and the radioactive bands corresponding to p20 and p21 Bax were detected and quantified by phosphorimaging. The levels relative to the maximal signal detected (set at 100) were plotted as a bar graph (upper panel). Equal aliquots from the same fractions were also subjected to analytical 12% SDS-PAGE, and the gels were stained with Coomassie brilliant blue (lower panel). The positions of molecular mass marker proteins are indicated. black, p20; gray, Bax. (B) Proteins eluting from preparative SDS-PAGE between 190 and 220 ml were concentrated and resolved by reverse-phase HPLC. The upper panel shows the A280 profile. Equal aliquots from all fractions were assayed for 32P-Bcl-2Δc21/his6/HMK–interacting protein by Far Western, for which only p20 was detected. Amounts relative to the maximal signal detected (set at 100) were plotted as a bar graph (lower panel). Fractions 52, 53, 54 (peak activity), and 55 were individually subjected to NH2-terminal peptide sequence analysis. (C) Polypeptide sequence of p28 Bap31/CDM (single-letter code). Peptide sequencing of p20 revealed a perfect match with amino acids 2–11 of human Bap31(underlined; these sequence data available from GenBank/EMBL/ DDBJ under accession number X81817). This was the only detectable sequence in fraction 54, was detectable together with other sequences in fraction 53, and was not detected in fractions 52 and 55. Predicted TM segments are boxed and contain charged amino acids in TM1 and TM3 (asterisks). The predicted caspase recognition sites, AAVD·G, are highlighted, and cleavage is denoted by arrows following Asp at positions 164 and 238. A potential leucine zipper located between the caspase recognition sites is denoted by bold letters, as is the KKXX ER retention signal at the COOH terminus. (D) Comparison of putative death effector domain sequences for the indicated proteins. The sequences, given in the single-letter amino acid code, were obtained from GenBank/EMBL/DDBJ, and their relative positions in the molecule are shown in parentheses. Sequences were aligned using the PILEUP program of the GCG software package and were optimized by spacing (shown as dashes). Identical residues and conserved substitutions that were recorded for at least half of the sequences analyzed are shaded in gray.

Mentions: Identification of the p20 Bcl-2–binding polypeptide was obtained by NH2-terminal peptide sequence analysis of p20 after its purification by a combination of differential solubilization in detergent, preparative SDS-PAGE, and reverse-phase HPLC (Fig. 2, A and B). Several individual HPLC fractions were subjected to peptide sequence analysis to detect a polypeptide sequence whose appearance correlated with the appearance of p20 Bcl-2–binding activity (Fig. 2). One candidate sequence emerged, and it was the only sequence that was detected in the peak fraction of Bcl-2–binding activity (Fig. 2 B, fraction 54). It showed a perfect match with amino acids 2–11 of human Bap31 (these sequence data available from GenBank/EMBL/DDBJ under accession number X81817)/CDM (these sequence data available from GenBank/EMBL/DDBJ under accession number Z31696), suggesting that p20 derives from the NH2 terminus of this 27,991-kD (p28) protein. Reverse transcription-PCR analysis of the p28 coding region, using total RNA obtained from KB cells after induction of apoptosis, showed no evidence that p20 arose by differential splicing of p28 mRNA (data not shown). As demonstrated below, Bcl-2 also associates with full length p28 in vitro and in vivo; failure to observe this interaction in the original ligand blot analyses (Fig. 1) was likely the result of relatively inefficient transfer of p28 to nitrocellulose blots.


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)

Identification of p20. (A) Preparative SDS-PAGE of differentially solubilized protein from KB cells 60 h after infection with  adenovirus pm1760/2072. Aliquots of fractions eluted from the gel were assayed by 32P-Bcl-2Δc21/his6/HMK Far Western, and the radioactive bands corresponding to p20 and p21 Bax were detected and quantified by phosphorimaging. The levels relative to the maximal  signal detected (set at 100) were plotted as a bar graph (upper panel). Equal aliquots from the same fractions were also subjected to analytical 12% SDS-PAGE, and the gels were stained with Coomassie brilliant blue (lower panel). The positions of molecular mass marker  proteins are indicated. black, p20; gray, Bax. (B) Proteins eluting from preparative SDS-PAGE between 190 and 220 ml were concentrated and resolved by reverse-phase HPLC. The upper panel shows the A280 profile. Equal aliquots from all fractions were assayed for  32P-Bcl-2Δc21/his6/HMK–interacting protein by Far Western, for which only p20 was detected. Amounts relative to the maximal signal  detected (set at 100) were plotted as a bar graph (lower panel). Fractions 52, 53, 54 (peak activity), and 55 were individually subjected to  NH2-terminal peptide sequence analysis. (C) Polypeptide sequence of p28 Bap31/CDM (single-letter code). Peptide sequencing of p20  revealed a perfect match with amino acids 2–11 of human Bap31(underlined; these sequence data available from GenBank/EMBL/ DDBJ under accession number X81817). This was the only detectable sequence in fraction 54, was detectable together with other sequences in fraction 53, and was not detected in fractions 52 and 55. Predicted TM segments are boxed and contain charged amino acids  in TM1 and TM3 (asterisks). The predicted caspase recognition sites, AAVD·G, are highlighted, and cleavage is denoted by arrows following Asp at positions 164 and 238. A potential leucine zipper located between the caspase recognition sites is denoted by bold letters,  as is the KKXX ER retention signal at the COOH terminus. (D) Comparison of putative death effector domain sequences for the indicated proteins. The sequences, given in the single-letter amino acid code, were obtained from GenBank/EMBL/DDBJ, and their relative positions in the molecule are shown in parentheses. Sequences were aligned using the PILEUP program of the GCG software package and were optimized by spacing (shown as dashes). Identical residues and conserved substitutions that were recorded for at least half  of the sequences analyzed are shaded in gray.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139787&req=5

Figure 2: Identification of p20. (A) Preparative SDS-PAGE of differentially solubilized protein from KB cells 60 h after infection with adenovirus pm1760/2072. Aliquots of fractions eluted from the gel were assayed by 32P-Bcl-2Δc21/his6/HMK Far Western, and the radioactive bands corresponding to p20 and p21 Bax were detected and quantified by phosphorimaging. The levels relative to the maximal signal detected (set at 100) were plotted as a bar graph (upper panel). Equal aliquots from the same fractions were also subjected to analytical 12% SDS-PAGE, and the gels were stained with Coomassie brilliant blue (lower panel). The positions of molecular mass marker proteins are indicated. black, p20; gray, Bax. (B) Proteins eluting from preparative SDS-PAGE between 190 and 220 ml were concentrated and resolved by reverse-phase HPLC. The upper panel shows the A280 profile. Equal aliquots from all fractions were assayed for 32P-Bcl-2Δc21/his6/HMK–interacting protein by Far Western, for which only p20 was detected. Amounts relative to the maximal signal detected (set at 100) were plotted as a bar graph (lower panel). Fractions 52, 53, 54 (peak activity), and 55 were individually subjected to NH2-terminal peptide sequence analysis. (C) Polypeptide sequence of p28 Bap31/CDM (single-letter code). Peptide sequencing of p20 revealed a perfect match with amino acids 2–11 of human Bap31(underlined; these sequence data available from GenBank/EMBL/ DDBJ under accession number X81817). This was the only detectable sequence in fraction 54, was detectable together with other sequences in fraction 53, and was not detected in fractions 52 and 55. Predicted TM segments are boxed and contain charged amino acids in TM1 and TM3 (asterisks). The predicted caspase recognition sites, AAVD·G, are highlighted, and cleavage is denoted by arrows following Asp at positions 164 and 238. A potential leucine zipper located between the caspase recognition sites is denoted by bold letters, as is the KKXX ER retention signal at the COOH terminus. (D) Comparison of putative death effector domain sequences for the indicated proteins. The sequences, given in the single-letter amino acid code, were obtained from GenBank/EMBL/DDBJ, and their relative positions in the molecule are shown in parentheses. Sequences were aligned using the PILEUP program of the GCG software package and were optimized by spacing (shown as dashes). Identical residues and conserved substitutions that were recorded for at least half of the sequences analyzed are shaded in gray.
Mentions: Identification of the p20 Bcl-2–binding polypeptide was obtained by NH2-terminal peptide sequence analysis of p20 after its purification by a combination of differential solubilization in detergent, preparative SDS-PAGE, and reverse-phase HPLC (Fig. 2, A and B). Several individual HPLC fractions were subjected to peptide sequence analysis to detect a polypeptide sequence whose appearance correlated with the appearance of p20 Bcl-2–binding activity (Fig. 2). One candidate sequence emerged, and it was the only sequence that was detected in the peak fraction of Bcl-2–binding activity (Fig. 2 B, fraction 54). It showed a perfect match with amino acids 2–11 of human Bap31 (these sequence data available from GenBank/EMBL/DDBJ under accession number X81817)/CDM (these sequence data available from GenBank/EMBL/DDBJ under accession number Z31696), suggesting that p20 derives from the NH2 terminus of this 27,991-kD (p28) protein. Reverse transcription-PCR analysis of the p28 coding region, using total RNA obtained from KB cells after induction of apoptosis, showed no evidence that p20 arose by differential splicing of p28 mRNA (data not shown). As demonstrated below, Bcl-2 also associates with full length p28 in vitro and in vivo; failure to observe this interaction in the original ligand blot analyses (Fig. 1) was likely the result of relatively inefficient transfer of p28 to nitrocellulose blots.

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