Sequential two-step cleavage of the retinoblastoma protein by caspase-3/-7 during etoposide-induced apoptosis.
During cellular apoptosis, retinoblastoma protein (RB) is subjected to cleavage near the carboxyl terminus by a caspase-3-like protease. In addition, an heretofore unidentified protease cleaves RB internally, generating fragments of 68 and 48 kDa. Internal cleavage abrogates the ability of RB to associate with E2F. To investigate the mechanism of RB internal cleavage, we developed and employed an in vitro cleavage assay. Incubation of in vitro translated (35)S-RB with apoptotic cell extracts led to RB cleavage at the C-terminus, followed by internal cleavage. The caspase peptide inhibitors z-VAD-FMK or z-DEVD-FMK blocked both cleavage events. Rapid C-terminal and internal cleavage were also observed when recombinant caspase-3 was added to (35)S-RB. Moreover, when caspase-3 was added to nonapoptotic cell extract, efficient internal cleavage of cellular RB was observed. Caspase-mediated internal cleavage occurred following RB residue aspartate(349) in the sequence DSID(349). This sequence is consistent with a DXXD recognition motif for caspase-3-like enzymes. Interestingly, we also observed RB internal cleavage in caspase-3-deficient MCF-7 cells, indicating that other caspases are capable of cleaving RB internally. Indeed, caspase-7, a member of the caspase-3 subfamily, was found to cleave (35)S-RB at both the carboxyl terminus, and following aspartate(349). By contrast, caspases that are not members of the caspase-3 subfamily failed to cleave RB. Taken together, our findings demonstrate that during apoptosis, a caspase-3-like protease is responsible for degradation and functional inactivation of RB by cleaving the protein internally following aspartate(349).
Fattman
C L
CL
Department of Pharmacology, University of Pittsburgh School of Medicine and the University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania, PA 15213, USA.
Delach
S M
SM
Dou
Q P
QP
Johnson
D E
DE
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3; D053179:Caspase 7; D061945:Caspase Inhibitors; D020169:Caspases / Q000235:genetics / Q000378:metabolism*; D002453:Cell Cycle / Q000187:drug effects; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D005047:Etoposide / Q000494:pharmacology*; D006801:Humans; D008969:Molecular Sequence Data; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology*; D009842:Oligopeptides / Q000494:pharmacology; D010446:Peptide Fragments / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000187:drug effects / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D000590:Amino Acid Chloromethyl Ketones; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D019384:Nucleic Acid Synthesis Inhibitors; D009842:Oligopeptides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D016160:Retinoblastoma Protein; C110772:benzoylcarbonyl-aspartyl-glutamyl-valyl-aspartyl-fluoromethyl ketone; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D005047:Etoposide; C505407:CASP3 protein, human; C505520:CASP7 protein, human; D053148:Caspase 3; D053179:Caspase 7; D020169:Caspases