PubMed Journals: EMBO J
Source: PMID: 9351817
⇦ ⇨ EMBO J. 1997 Nov 3;16(21):6346-54.
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.
Rhéaume E(1), Cohen LY, Uhlmann F, Lazure
C, Alam A, Hurwitz J, Sékaly RP, Denis F.
(1) Laboratoire d'Immunologie, Institut
de recherches cliniques de Montréal, Québec,
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.
DOI: 10.1093/emboj/16.21.6346 PMCID: PMC1170241
PMID: 9351817 [Indexed for MEDLINE]