Ubiquitin-mediated degradation of the proapoptotic active form of bid. A functional consequence on apoptosis induction.
Under basal conditions, the proapoptotic protein Bid is a long-lived protein. Pro-apoptotic stimuli such as tumor necrosis factor-alpha (TNFalpha) or Fas induce its caspase-8-mediated cleavage into two fragments. The COOH-terminal cleavage fragment of Bid (tBid) becomes localized to mitochondrial membranes and triggers the release of cytochrome c. Here we show that tBid is ubiquitinated and subsequently degraded by the 26 S proteasome. Degradation of tBid is significantly inhibited by the proteasome inhibitors MG-132 and lactacystin. In contrast, caspase-specific or lysosomal inhibitors do not affect tBid stability. Furthermore, mutation of the putative ubiquitin acceptor sites within tBid results in a stabilized protein as assessed by pulse-chase analysis. To address whether tBid degradation might be regulated by interaction with other Bcl-2-like proteins, cotransfection studies were performed. However, neither the presence of proapoptotic Bax nor antiapoptotic Bcl-2 or Bcl-XL affected tBid degradation. Finally, we determined the functional role of tBid degradation. Overexpression of stabilized tBid proteins significantly enhanced cytochrome c release and subsequent apoptosis induction approximately 2-fold compared with wild type tBid. Similarly, tBid-induced apoptosis was considerably amplified by inhibition of tBid degradation using the proteasome-specific inhibitor MG-132. Thus, proteasomal degradation of tBid limits the extent of apoptosis in living cells.
Breitschopf
K
K
Division of Molecular Cardiology, Department of Internal Medicine IV, University of Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany.
Zeiher
A M
AM
Dimmeler
S
S
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D051023:BH3 Interacting Domain Death Agonist Protein; D019556:COS Cells; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D014162:Transfection; D014452:Ubiquitins / Q000378:metabolism*; D051020:bcl-X Protein
C494811:BCL2L1 protein, human; D051023:BH3 Interacting Domain Death Agonist Protein; C494814:BID protein, human; D002352:Carrier Proteins; D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011994:Recombinant Proteins; D014452:Ubiquitins; D051020:bcl-X Protein; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde