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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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Associations of p28 with Bcl-2,  Bcl-XL, and procaspase-8 (pro-FLICE).  (A) GST (lane 1) or GST fused to p28  amino acids 165–246 (lane 2), 122–164  (lane 3), 1–164 (lane 4), and 1–246 (lane 5)  were expressed in bacteria, purified, and  transferred to nitrocellulose in duplicate  after SDS-PAGE. One blot was stained  with Ponceau S and the other probed by  ligand blotting (Far Western) with 32P-Bcl-2Δc22/his6/HMK, as indicated. Constructs and results are summarized below  the blots. (B) Standard recombinant DNA  manipulations were used to create cDNAs  encoding Bcl-XL tagged at the COOH terminus with the Myc epitope EQKLISEEDL (Chinnayan et al., 1997); pro-FLICE tagged at the COOH terminus  with the hemagglutinin (HA) epitope,  YPYDVPDYA (Chinnayan et al., 1997);  Bcl-2 tagged at the NH2 terminus with the  HA epitope (Nguyen et al., 1994); and p28  tagged with the Flag epitope, in which the  Flag sequence MDYKDDDDKA was inserted between Pro240 and Met241 of p28.  The recombinant cDNAs, or Flag DNA  alone (Control-Flag), were inserted into  pcDNA 3 (Invitrogen) or RcRSV (Pharmacia Fine Chemicals) (HA-Bcl-2) and  transfected into 293T cells, as indicated  (pluses and minuses). After incubation of  cell lysates with anti-Flag antibody, immunoprecipitates (ip) and lysates were resolved by SDS-PAGE, transferred to nitrocellulose, and the blot developed with  the indicated antibody and visualized by  enhanced chemiluminescence. Ig HC, immunoglobulin heavy chain. (C) Same as in  B, except that Bax was included in cotransfections, as indicated.
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Figure 4: Associations of p28 with Bcl-2, Bcl-XL, and procaspase-8 (pro-FLICE). (A) GST (lane 1) or GST fused to p28 amino acids 165–246 (lane 2), 122–164 (lane 3), 1–164 (lane 4), and 1–246 (lane 5) were expressed in bacteria, purified, and transferred to nitrocellulose in duplicate after SDS-PAGE. One blot was stained with Ponceau S and the other probed by ligand blotting (Far Western) with 32P-Bcl-2Δc22/his6/HMK, as indicated. Constructs and results are summarized below the blots. (B) Standard recombinant DNA manipulations were used to create cDNAs encoding Bcl-XL tagged at the COOH terminus with the Myc epitope EQKLISEEDL (Chinnayan et al., 1997); pro-FLICE tagged at the COOH terminus with the hemagglutinin (HA) epitope, YPYDVPDYA (Chinnayan et al., 1997); Bcl-2 tagged at the NH2 terminus with the HA epitope (Nguyen et al., 1994); and p28 tagged with the Flag epitope, in which the Flag sequence MDYKDDDDKA was inserted between Pro240 and Met241 of p28. The recombinant cDNAs, or Flag DNA alone (Control-Flag), were inserted into pcDNA 3 (Invitrogen) or RcRSV (Pharmacia Fine Chemicals) (HA-Bcl-2) and transfected into 293T cells, as indicated (pluses and minuses). After incubation of cell lysates with anti-Flag antibody, immunoprecipitates (ip) and lysates were resolved by SDS-PAGE, transferred to nitrocellulose, and the blot developed with the indicated antibody and visualized by enhanced chemiluminescence. Ig HC, immunoglobulin heavy chain. (C) Same as in B, except that Bax was included in cotransfections, as indicated.

Mentions: Various p28 fusion proteins were constructed in which GST was linked to p28 amino acids 1–246 (full length p28), 1–164 (p20), 122–164, and 165–246. These constructs, together with GST itself, were purified, and equal amounts were examined for their ability to bind to the cytosolic domain of Bcl-2 in a ligand blot assay. As shown in Fig. 4 A, reactivity was observed for both GST-p28 (lane 5) and GST-p20 (lane 4), with weak activity possibly registering with the COOH-terminal 165–246 amino acid domain (lane 2), and none detected for the middle 122–164 amino acid domain (lane 3) or for GST alone (lane 1).


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)

Associations of p28 with Bcl-2,  Bcl-XL, and procaspase-8 (pro-FLICE).  (A) GST (lane 1) or GST fused to p28  amino acids 165–246 (lane 2), 122–164  (lane 3), 1–164 (lane 4), and 1–246 (lane 5)  were expressed in bacteria, purified, and  transferred to nitrocellulose in duplicate  after SDS-PAGE. One blot was stained  with Ponceau S and the other probed by  ligand blotting (Far Western) with 32P-Bcl-2Δc22/his6/HMK, as indicated. Constructs and results are summarized below  the blots. (B) Standard recombinant DNA  manipulations were used to create cDNAs  encoding Bcl-XL tagged at the COOH terminus with the Myc epitope EQKLISEEDL (Chinnayan et al., 1997); pro-FLICE tagged at the COOH terminus  with the hemagglutinin (HA) epitope,  YPYDVPDYA (Chinnayan et al., 1997);  Bcl-2 tagged at the NH2 terminus with the  HA epitope (Nguyen et al., 1994); and p28  tagged with the Flag epitope, in which the  Flag sequence MDYKDDDDKA was inserted between Pro240 and Met241 of p28.  The recombinant cDNAs, or Flag DNA  alone (Control-Flag), were inserted into  pcDNA 3 (Invitrogen) or RcRSV (Pharmacia Fine Chemicals) (HA-Bcl-2) and  transfected into 293T cells, as indicated  (pluses and minuses). After incubation of  cell lysates with anti-Flag antibody, immunoprecipitates (ip) and lysates were resolved by SDS-PAGE, transferred to nitrocellulose, and the blot developed with  the indicated antibody and visualized by  enhanced chemiluminescence. Ig HC, immunoglobulin heavy chain. (C) Same as in  B, except that Bax was included in cotransfections, as indicated.
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Related In: Results  -  Collection

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Figure 4: Associations of p28 with Bcl-2, Bcl-XL, and procaspase-8 (pro-FLICE). (A) GST (lane 1) or GST fused to p28 amino acids 165–246 (lane 2), 122–164 (lane 3), 1–164 (lane 4), and 1–246 (lane 5) were expressed in bacteria, purified, and transferred to nitrocellulose in duplicate after SDS-PAGE. One blot was stained with Ponceau S and the other probed by ligand blotting (Far Western) with 32P-Bcl-2Δc22/his6/HMK, as indicated. Constructs and results are summarized below the blots. (B) Standard recombinant DNA manipulations were used to create cDNAs encoding Bcl-XL tagged at the COOH terminus with the Myc epitope EQKLISEEDL (Chinnayan et al., 1997); pro-FLICE tagged at the COOH terminus with the hemagglutinin (HA) epitope, YPYDVPDYA (Chinnayan et al., 1997); Bcl-2 tagged at the NH2 terminus with the HA epitope (Nguyen et al., 1994); and p28 tagged with the Flag epitope, in which the Flag sequence MDYKDDDDKA was inserted between Pro240 and Met241 of p28. The recombinant cDNAs, or Flag DNA alone (Control-Flag), were inserted into pcDNA 3 (Invitrogen) or RcRSV (Pharmacia Fine Chemicals) (HA-Bcl-2) and transfected into 293T cells, as indicated (pluses and minuses). After incubation of cell lysates with anti-Flag antibody, immunoprecipitates (ip) and lysates were resolved by SDS-PAGE, transferred to nitrocellulose, and the blot developed with the indicated antibody and visualized by enhanced chemiluminescence. Ig HC, immunoglobulin heavy chain. (C) Same as in B, except that Bax was included in cotransfections, as indicated.
Mentions: Various p28 fusion proteins were constructed in which GST was linked to p28 amino acids 1–246 (full length p28), 1–164 (p20), 122–164, and 165–246. These constructs, together with GST itself, were purified, and equal amounts were examined for their ability to bind to the cytosolic domain of Bcl-2 in a ligand blot assay. As shown in Fig. 4 A, reactivity was observed for both GST-p28 (lane 5) and GST-p20 (lane 4), with weak activity possibly registering with the COOH-terminal 165–246 amino acid domain (lane 2), and none detected for the middle 122–164 amino acid domain (lane 3) or for GST alone (lane 1).

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