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 homology 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.
authors.affiliation: Laboratoire d'Immunologie, Institut de recherches cliniques de Montréal, Québec, Canada.
authors.forename: E
authors.initials: E
authors.lastname: Rhéaume
authors.forename: L Y
authors.initials: LY
authors.lastname: Cohen
authors.forename: F
authors.initials: F
authors.lastname: Uhlmann
authors.forename: C
authors.initials: C
authors.lastname: Lazure
authors.forename: A
authors.initials: A
authors.lastname: Alam
authors.forename: J
authors.initials: J
authors.lastname: Hurwitz
authors.forename: R P
authors.initials: RP
authors.lastname: Sékaly
authors.forename: F
authors.initials: F
authors.lastname: Denis
mesh_terms: D017209:Apoptosis / Q000502:physiology*; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004261:DNA Replication / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D018398:Homeodomain Proteins; D006801:Humans; D015459:Leukemia-Lymphoma, Adult T-Cell / Q000473:pathology; D008214:Lymphocytes / Q000187:drug effects / Q000378:metabolism; D015778:Minor Histocompatibility Antigens; D009363:Neoplasm Proteins / Q000378:metabolism; D009842:Oligopeptides / Q000494:pharmacology; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D051818:Replication Protein C; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured; D019014:fas Receptor / Q000502:physiology*
chemical_list: C096816:BCL2-related protein A1; D015853:Cysteine Proteinase Inhibitors; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; C096063:MATA1 protein, S cerevisiae; D015778:Minor Histocompatibility Antigens; D009363:Neoplasm Proteins; D009842:Oligopeptides; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; C099158:acetyl-aspartyl-glutamyl-valyl-aspartal; D019014:fas Receptor; C096429:L 709049; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases; D051818:Replication Protein C