Fig. 8.

Post-translational modifications of Orc1 suppressed its ability to induce exposure of phosphatidylserine on cell surfaces and caspase-3 activity. (A) The five CDK consensus phosphorylation sites in hamster Orc1 were mutated cumulatively, either (S/T→D) or (S/T→A). Thus, D1 contains a single amino acid change (S277D), whereas D5 contains five amino acid changes, one at each site. FCgOrc1 and FHsOrc1 also were modified by addition of either a single human ubiquitin (Ub) fused to their C-terminus, or a single ubiquitin in which all seven lysines had been ‘knocked-out’ by changing them to arginines [Ub(KO)]. CHO cells were transfected with 1 µg of the indicated phosphorylation site mutant, and HeLa cells were transfected with 1 µg of the indicated Ub-fusion protein. Cells were incubated for 12 hours before staining the expressed protein with anti-FLAG antibody to determine their relative expression levels; wt, wild type. (B) The fraction of annexin-V-positive cells was determined at 12 hours (light bars) and 24 hours (dark bars) post-transfection for the indicated plasmid. (C) Caspase-3 activity was determined at 12 hours post-transfection for the indicated plasmid, and corrected for activity produced by pCI transfection.