The large subunit of replication factor C is a substrate for caspase-3 in vitro and is cleaved by a caspase-3-like protease during fas-mediated apoptosis Journal Article


Authors: Rhéaume, E.; Cohen, L. Y.; Uhlmann, F.; Lazure, C.; Alam, A.; Hurwitz, J.; Sékaly, R. P.; Denis, F.
Article Title: The large subunit of replication factor C is a substrate for caspase-3 in vitro and is cleaved by a caspase-3-like protease during fas-mediated apoptosis
Abstract: Caspase-3 is an ICE-like protease activated during apoptosis induced by different stimuli. Poly(ADP-ribose) polymerase (PARP), the first characterized substrate of caspase-3, shares a region of homologly with the large subunit of Replication Factor C (RF-C), a five-subunit complex that is part of the processive eukaryotic DNA polymerase holoenzymes. Caspase-3 cleaves PARP at a DEVD-G motif present in the 140 kDa subunit of RF-C (RFC140) and evolutionarily conserved. We show that cleavage of RFC140 during Fas-mediated apoptosis in Jurkat cells and lymphocytes results in generation of multiple fragments. Cleavage is inhibited by the caspase-3-like protease inhibitor Ac-DEVD-CHO but not the caspase-1/ICE-type protease inhibitor Ac-YVAD-CHO. In addition, recombinant caspase-3 cleaves RFC140 in vitro at least at three different sites in the C-terminal half of the protein. Using amino-terminal microsequencing of radioactive fragments, we identified three sites: DEVD723G, DLVD922S and IETD1117A. We did not detect cleavage of small subunits of RF-C of 36, 37, 38 and 40 kDa by recombinant caspase-3 or by apoptotic Jurkat cell lysates. Cleavage of RFC140 during apoptosis inactivates its function in DNA replication and generates truncated forms that further inhibit DNA replication. These results identify RFC140 as a critical target for caspase-3-like proteases and suggest that caspases could mediate cell cycle arrest.
Keywords: controlled study; unclassified drug; human cell; dna-binding proteins; dna polymerase; dna replication; cell cycle; apoptosis; protein degradation; fas antigen; neoplasm proteins; homeodomain proteins; caspase 3; tumor cells, cultured; caspase; enzyme substrate; caspases; cysteine proteinase inhibitors; amino acid sequence; amino terminal sequence; recombinant fusion proteins; eukaryota; substrate specificity; saccharomyces cerevisiae proteins; replication protein c; multienzyme complex; enzyme subunit; oligopeptides; lymphocyte; lymphocytes; repressor proteins; proto-oncogene proteins c-bcl-2; proteinase inhibitor; leukemia cell line; peptide hydrolase; dna fragment; antigens, cd95; cysteine proteinase; cysteine endopeptidases; parp; leukemia, t-cell, acute; programmed cell death; humans; human; priority journal; article; cpp32; rf-c
Journal Title: EMBO Journal
Volume: 16
Issue: 21
ISSN: 0261-4189
Publisher: Wiley Blackwell  
Date Published: 1997-11-01
Start Page: 6346
End Page: 6354
Language: English
DOI: 10.1093/emboj/16.21.6346
PUBMED: 9351817
PROVIDER: scopus
PMCID: PMC1170241
DOI/URL:
Notes: Article -- Export Date: 17 March 2017 -- Source: Scopus
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  1. Jerard Hurwitz
    206 Hurwitz