Abstract
Cdc6 is the bifunctional AAA+ ATPase that assembles prereplicative complexes on origins of replication and activates p21(CIP1)- or p27(KIP1)-bound Cdk2. During the G(1)-S transition, the Cdc6 gene essential for chromosomal replication is activated by the E2F transcriptional factor. Paradoxically, Apaf-1 encoding the central component of the apoptosome is also activated at the same time and by E2F. Consequently, genes for antipodal life and death are regulated in the same manner by the same transcriptional factor. Here we report a striking solution to this paradox. Besides performing prereplicative complex assembly and Cdk2 activation, Cdc6 obstructed apoptosome assembly by forming stable complexes very likely with a monomer of cytochrome c-activated Apaf-1 molecules. This function depended on its own ATPase domain but not on the cyclin-binding motif. In proliferating rodent fibroblasts, Cdc6 continued to block apoptosome assembly induced by a non-cytochrome c or some other mechanism, suppressing seemingly unintended apoptosis when promoting cell proliferation. Thus, Cdc6 is an AAA+ ATPase with three functions, all working for life.
Highlights
The apoptosome is a major apparatus for intrinsic stimulus-induced apoptosis
Antibodies and Reagents—Anti-caspase-3, anti-caspase-9, anti-cleaved caspase-9 (Asp353), anti-cytochrome c, anti-Bad, anti-S6K1, anti-phospho-S6K1(Thr389), and anti-Rheb antibodies were purchased from Cell Signaling Technology; anti-actin antibody was from Santa Cruz Biotechnology; anti-Cdc6 antibody was from NeoMarkers; anti-Apaf-1 antibody was from Alexis Biochemicals; anti-phospho-Rb (Ser780) and anticaspase-9 mouse monoclonal antibodies were from MBL International; anti-caspase-8 antibody was from BioVision; anti-XIAP antibody was from R&D Systems; anti-Rb, anti-Bax, anti-Bcl-2, and anti-Bcl-X antibodies were from BD Biosciences; anti-Bak antibody was from Millipore; and antip53 antibody was from Oncogene
Enforced Expression of Cdc6 Suppresses Anoikis of mTORC1activated Fibroblasts—To confirm the initial finding that overexpression of Cdc6 suppresses anoikis of mTORC1-activated rat embryonic fibroblasts irrespective of how mTORC1 is activated, we first compared the anoikis susceptibility of the REFs activated for mTORC1 by a Tsc2 mutation or active Rheb overexpression in the presence or absence of overexpression of Cdc6
Summary
Results: Cdc, known to assemble prereplicative complexes on replication origins and activate p21CIP1-/p27KIP1-bound Cdk, obstructs cytochrome c- or some other mechanism-induced apoptosome assembly by forming stable complexes with activated Apaf-1. Besides performing prereplicative complex assembly and Cdk activation, Cdc obstructed apoptosome assembly by forming stable complexes very likely with a monomer of cytochrome c-activated Apaf-1 molecules This function depended on its own ATPase domain but not on the cyclin-binding motif. Cdc is an AAA؉ ATPase with three functions, all working for life Cells execute their own death in response to stress-induced intrinsic and death receptor-mediated extrinsic apoptotic stimuli [1, 2]. The resulting open shaped Apaf-1 molecules with cytc attached are allowed to form heptameric complexes, which bind via their caspase-recruiting domain and activate caspase-9 This ternary complex is the mature apoptosome fully capable of converting procaspase-3 to the active two-chain form. Subsequent investigation led us to resolve this discrepancy and discover an entirely new function for Cdc
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