Hamster Bcl-2 protein is cleaved in vitro and in cells by caspase-9 and caspase-3.
Full-length cDNA of hamster bcl-2 (771 nt) was cloned by RT-PCR and inserted into pGEX-4T-1 to produce the recombinant hamster Bcl-2 protein. The purified recombinant Bcl-2 protein (26.4 kDa) was used as a substrate for the active human caspase-3 and caspase-9 in vitro. It is shown here that Bcl-2 is efficiently cleaved by caspase-3 to a 23 kDa fragment. Although not possessing a putative caspase-9 cleavage site in its sequence, hamster Bcl-2 was also cleaved by caspase-9 into exactly the same 23 kDa cleavage product, indicating that cleavage occurred at the same site. Caspase-3- and caspase-9-mediated cleavage of Bcl-2 was efficiently blocked by caspase-3 (zDEVD) and caspase-9 (zLEHD) inhibitor, respectively. We also show that caspase-9/-3-mediated cleavage of Bcl-2 occurs in vivo during apoptosis in CHO-HSV-TK cells after exposure to the antiviral drug ganciclovir.
Tomicic
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Division of Applied Toxicology, Institute of Toxicology, Medical Faculty, University of Mainz, D-55131 Mainz, Germany.
Kaina
B
B
D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D053148:Caspase 3; D053453:Caspase 9; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D006224:Cricetinae; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009842:Oligopeptides / Q000494:pharmacology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism
D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D009842:Oligopeptides; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins; C110772:benzoylcarbonyl-aspartyl-glutamyl-valyl-aspartyl-fluoromethyl ketone; C403754:benzyloxycarbonyl-leucyl-glutamyl-histidyl-aspartic acid fluoromethyl ketone; D005982:Glutathione Transferase; D013917:Thrombin; C505407:CASP3 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases