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 |