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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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Ectopic expression of p20 induces apoptosis. (A) CHO  LR73 cells expressing neomycin resistance, either alone (− Bcl-2)  or together with Bcl-2 (+ Bcl-2), were cotransfected with a luciferase reporter plasmid and Rc/RSV expressing either full  length p28 or p28 amino acids 1–164 (i.e., p20). After 2 d, cells  were recovered, analyzed for luciferase activity, and the enzyme  activity expressed relative to the values obtained in the presence  of p28 (arbitrarily set at 100). The results shown are the average  of two separate experiments. (B) CHO cells were transfected  with the p28 and p20 expression plasmids together with pHook  (Invitrogen). 24 h later, transfected cells were recovered with  Capture-Tec beads, cultured on coverslips, stained with 4′,6′-diamidino-2-phenyl indole (DAPI), and visualized under a microscope.
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Figure 7: Ectopic expression of p20 induces apoptosis. (A) CHO LR73 cells expressing neomycin resistance, either alone (− Bcl-2) or together with Bcl-2 (+ Bcl-2), were cotransfected with a luciferase reporter plasmid and Rc/RSV expressing either full length p28 or p28 amino acids 1–164 (i.e., p20). After 2 d, cells were recovered, analyzed for luciferase activity, and the enzyme activity expressed relative to the values obtained in the presence of p28 (arbitrarily set at 100). The results shown are the average of two separate experiments. (B) CHO cells were transfected with the p28 and p20 expression plasmids together with pHook (Invitrogen). 24 h later, transfected cells were recovered with Capture-Tec beads, cultured on coverslips, stained with 4′,6′-diamidino-2-phenyl indole (DAPI), and visualized under a microscope.

Mentions: CHO (neo) cells were transiently cotransfected with a luciferase reporter gene together with RcRSV expressing either p28 or p20. Subsequent measurements revealed that coexpression of p20 with the reporter severely depressed the amount of luciferase activity obtained relative to coexpression with p28 (Fig. 7 A). p28, on the other hand, had no deleterious effect on the recovery of luciferase activity compared to a control RcRSV plasmid that did not encode protein (not shown). If these same transfections were conducted in cells stably expressing Bcl-2, however, Bcl-2 largely overcame the dominant negative influence of p20 on luciferase activity (Fig. 7 A), presumably because p20 could no longer interfere with normal p28 function. Because of this protective effect by Bcl-2, we conclude that the negative influence of p20 on luciferase activity was the result of induction of apoptosis. This was confirmed by microscopic analysis, which revealed apoptotic nuclei in p20- but not p28-transfected cells (Fig. 7 B). The findings described in Fig. 7 have been consistently observed many times and in different cell types.


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)

Ectopic expression of p20 induces apoptosis. (A) CHO  LR73 cells expressing neomycin resistance, either alone (− Bcl-2)  or together with Bcl-2 (+ Bcl-2), were cotransfected with a luciferase reporter plasmid and Rc/RSV expressing either full  length p28 or p28 amino acids 1–164 (i.e., p20). After 2 d, cells  were recovered, analyzed for luciferase activity, and the enzyme  activity expressed relative to the values obtained in the presence  of p28 (arbitrarily set at 100). The results shown are the average  of two separate experiments. (B) CHO cells were transfected  with the p28 and p20 expression plasmids together with pHook  (Invitrogen). 24 h later, transfected cells were recovered with  Capture-Tec beads, cultured on coverslips, stained with 4′,6′-diamidino-2-phenyl indole (DAPI), and visualized under a microscope.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139787&req=5

Figure 7: Ectopic expression of p20 induces apoptosis. (A) CHO LR73 cells expressing neomycin resistance, either alone (− Bcl-2) or together with Bcl-2 (+ Bcl-2), were cotransfected with a luciferase reporter plasmid and Rc/RSV expressing either full length p28 or p28 amino acids 1–164 (i.e., p20). After 2 d, cells were recovered, analyzed for luciferase activity, and the enzyme activity expressed relative to the values obtained in the presence of p28 (arbitrarily set at 100). The results shown are the average of two separate experiments. (B) CHO cells were transfected with the p28 and p20 expression plasmids together with pHook (Invitrogen). 24 h later, transfected cells were recovered with Capture-Tec beads, cultured on coverslips, stained with 4′,6′-diamidino-2-phenyl indole (DAPI), and visualized under a microscope.
Mentions: CHO (neo) cells were transiently cotransfected with a luciferase reporter gene together with RcRSV expressing either p28 or p20. Subsequent measurements revealed that coexpression of p20 with the reporter severely depressed the amount of luciferase activity obtained relative to coexpression with p28 (Fig. 7 A). p28, on the other hand, had no deleterious effect on the recovery of luciferase activity compared to a control RcRSV plasmid that did not encode protein (not shown). If these same transfections were conducted in cells stably expressing Bcl-2, however, Bcl-2 largely overcame the dominant negative influence of p20 on luciferase activity (Fig. 7 A), presumably because p20 could no longer interfere with normal p28 function. Because of this protective effect by Bcl-2, we conclude that the negative influence of p20 on luciferase activity was the result of induction of apoptosis. This was confirmed by microscopic analysis, which revealed apoptotic nuclei in p20- but not p28-transfected cells (Fig. 7 B). The findings described in Fig. 7 have been consistently observed many times and in different cell types.

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