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			PubMed Journals: Oncogene

  Source:		PMID: 11420704


    		Oncogene. 2001 May 24;20(23):2918-26.
     
			Sequential two-step cleavage of the retinoblastoma
			protein by caspase-3/-7 during etoposide-induced
			apoptosis.

			Fattman CL(1), Delach SM, Dou QP, Johnson
			DE.

			Author Information
			(1) Department of Pharmacology,
			University of Pittsburgh School of Medicine
			and the University of Pittsburgh Cancer
			Institute, Pittsburgh, Pennsylvania, PA
			15213, USA.

			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).

			DOI: 10.1038/sj.onc.1204414 PMID: 11420704
			[Indexed for MEDLINE]

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