Bcl-2 acts upstream of the PARP protease and prevents its activation
Bcl-2 acts upstream of the PARP protease and prevents its activation
Apoptosis has recently been extensively studied and multiple factors have been implicated in its regulation. It remains unclear how these factors are ordered in the cell death pathway. Here we investigate the relationship between the inhibitor of apoptosis, bcl-2, and the PARP protease, prlCE/CPP32, recently implicated in apoptosis. Using PARP proteolysis as an indicator of the activation of the PARP protease, we find that the chemotherapeutic agent, etoposide, induces apoptosis and PARP proteolysis in Molt4 cells as early as 4 h with cell death lagging behind this event. In contrast, Molt4 cells that over-express bcl-2 show no PARP proteolysis or cell death. In order to determine if bcl-2 inhibits the PARP protease or its activation, we developed a cell-free system. Using this system with extracts from etoposide-treated cells and purified bovine PARP, we demonstrate that extracts from bcl-2 over-expressing cells cause little or no PARP proteolysis. Whereas, extracts from control vector cells contain an active PARP protease. This protease is inhibited by the tetrapeptide ICE-like protease inhibitor, YVAD-chloromethylketone. Interestingly, this protease is not inhibited by the addition of purified bcl-2 protein. These results rule out that bcl-2 directly inhibits the active protease or that it has an effect downstream of prlCE/CPP32 such as preventing access to the PARP substrate. These results also demonstrate a role of bcl-2 in interfering with an upstream signal required to activate the PARP protease and allow us to begin to order the components in the apoptotic pathway.
apoptosis, bcl-2, parp, proteases
29-33
Perry, David K.
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Smyth, Miriam J.
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Wang, Hong-Gang
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Reed, John C.
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Duriez, Patrick
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Poirier, Guy G.
5151b4a5-5657-412d-88c4-e3b9127ac42c
Obeid, LinaM.
e3315025-b5df-4ef2-a129-5d30010c3577
Hannun, Yusuf A.
b89a0b1f-006e-4e76-947e-de213df9d2f9
January 1997
Perry, David K.
4391bf02-c3b7-4364-87f2-34e15657b07d
Smyth, Miriam J.
517c2a9a-666b-48c7-a668-79a6684e0350
Wang, Hong-Gang
f15184c5-b067-490f-80c9-7926e41305af
Reed, John C.
e8d7330b-77fa-454e-949b-90f52a898e94
Duriez, Patrick
4cf499bc-007a-43b3-b180-d6e5dc3d151b
Poirier, Guy G.
5151b4a5-5657-412d-88c4-e3b9127ac42c
Obeid, LinaM.
e3315025-b5df-4ef2-a129-5d30010c3577
Hannun, Yusuf A.
b89a0b1f-006e-4e76-947e-de213df9d2f9
Perry, David K., Smyth, Miriam J., Wang, Hong-Gang, Reed, John C., Duriez, Patrick, Poirier, Guy G., Obeid, LinaM. and Hannun, Yusuf A.
(1997)
Bcl-2 acts upstream of the PARP protease and prevents its activation.
Cell Death and Differentiation, 4 (1), .
(PMID:16465207)
Abstract
Apoptosis has recently been extensively studied and multiple factors have been implicated in its regulation. It remains unclear how these factors are ordered in the cell death pathway. Here we investigate the relationship between the inhibitor of apoptosis, bcl-2, and the PARP protease, prlCE/CPP32, recently implicated in apoptosis. Using PARP proteolysis as an indicator of the activation of the PARP protease, we find that the chemotherapeutic agent, etoposide, induces apoptosis and PARP proteolysis in Molt4 cells as early as 4 h with cell death lagging behind this event. In contrast, Molt4 cells that over-express bcl-2 show no PARP proteolysis or cell death. In order to determine if bcl-2 inhibits the PARP protease or its activation, we developed a cell-free system. Using this system with extracts from etoposide-treated cells and purified bovine PARP, we demonstrate that extracts from bcl-2 over-expressing cells cause little or no PARP proteolysis. Whereas, extracts from control vector cells contain an active PARP protease. This protease is inhibited by the tetrapeptide ICE-like protease inhibitor, YVAD-chloromethylketone. Interestingly, this protease is not inhibited by the addition of purified bcl-2 protein. These results rule out that bcl-2 directly inhibits the active protease or that it has an effect downstream of prlCE/CPP32 such as preventing access to the PARP substrate. These results also demonstrate a role of bcl-2 in interfering with an upstream signal required to activate the PARP protease and allow us to begin to order the components in the apoptotic pathway.
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Published date: January 1997
Keywords:
apoptosis, bcl-2, parp, proteases
Identifiers
Local EPrints ID: 164825
URI: http://eprints.soton.ac.uk/id/eprint/164825
ISSN: 1350-9047
PURE UUID: 9a3b10a9-a6ca-415c-a6ca-b88828ec1ae5
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Date deposited: 05 Oct 2010 09:10
Last modified: 12 Jul 2022 01:41
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Contributors
Author:
David K. Perry
Author:
Miriam J. Smyth
Author:
Hong-Gang Wang
Author:
John C. Reed
Author:
Patrick Duriez
Author:
Guy G. Poirier
Author:
LinaM. Obeid
Author:
Yusuf A. Hannun
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