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First published online 14 March 2006
doi: 10.1242/jcs.02851

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Ubiquitylation, phosphorylation and Orc2 modulate the subcellular location of Orc1 and prevent it from inducing apoptosis

National Institute of Child Health and Human Development, National Institutes of Health, Building 6/3A-15, 9000 Rockville Pike, Bethesda, MD 20892-2753, USA

View larger version (56K): [in a new window]   Fig. 1. Ectopic expression of CHO (Cg) Orc1 induced loss of cell adhesion under conditions where ectopic expression of CgOrc2 did not. (A) CHO cells were transfected with the amount of either pCgOrc1 () or pCgOrc2 () indicated and then incubated for 36 hours before determining the fraction of unattached cells. (B) CHO cells were transfected with 1 µg of either pCgOrc1 () or pCgOrc2 () and then incubated for the times indicated before determining the fraction of unattached cells. Data with pCI alone was subtracted from data for pCgOrc1 and pCgOrc2. Shaded areas indicate the conditions under which pCgOrc2 and pCI gave indistinguishable results. (C) CHO cells were transfected with 1 µg of either pFCgOrc1 (FOrc1) or pFCgOrc2 (FOrc2) for the times indicated in hours post-transfection (post-T). FOrc1 and FOrc2 were quantified in the total cell population (attached plus unattached) by immunoblotting using anti-FLAG antibody. A single band was detected that migrated as 100 kDa for FOrc1 and 65 kDa for FOrc2. (D) CHO cells, transfected as in panel A, were separated into those that lifted off the dish (unattached) and those that remained attached. Half of the unattached cells and 104 attached cells were assayed for either FOrc1 or FOrc2. (E) CHO cells, transfected as in panel B, were separated into attached and unattached cells before assaying for FOrc1 and FOrc2 for the times indicated. In each experiment, attached and unattached cell proteins were fractionated in the same gel, transferred to the same immunoblot, and detected with the same anti-FLAG antibody to ensure accurate comparison.

View larger version (35K): [in a new window]   Fig. 2. Ectopic expression of human (Hs) Orc1, Orc3, Orc4, Orc5 and Orc6 induced loss of cell adhesion under conditions where ectopic expression of HsOrc2 did not. (A) HeLa cells were transfected with 1 µg of the plasmid indicated (pFHsOrc1 or pFHsOrc1) and then incubated for the times indicated before determining the fraction of unattached cells. (B) HCT116 cells (±TP53 gene, as indicated by p53-/- and p53+/+) transfected with 1 µg of pFHsOrc1 for the times indicated. (C) CHO cells were transfected with the amount of plasmid DNA indicated and then incubated for 36 hours before determining the fraction of unattached cells.

View larger version (39K): [in a new window]   Fig. 3. Ectopic expression of Orc1 induced exposure of phosphatidylserine and blebs on cell surfaces, but Orc2 did not. (A) CHO cells were transfected with 1 µg pFCgOrc1 (Orc1) or pFCgOrc2 (Orc2) DNA. At 24 hours post-transfection, they were fixed and then stained with anti-FLAG antibody (red). Inset: cells transfected with pCgOrc1 [stained with anti-CgOrc1 antibody (green)] gave results comparable with those transfected with pFCgOrc1. (B) Live transfected cells were stained with annexin-V to reveal cells undergoing apoptosis. (C) The fraction of cells that bound annexin-V was determined for the indicated plasmid or with Fugene alone. DIC, differential interference contrast.

View larger version (70K): [in a new window]   Fig. 4. Ectopic expression of Orc1 induced DNA fragmentation similar to non-transfected cells that spontaneously over-expressed Orc1. (A) Non-transfected CHO cells were stained with Hoechst to stain DNA (blue), with TUNEL assay to label double-stranded DNA breaks (green), and with anti-FLAG antibody to label FCgOrc1 (red; F-Orc). DIC, differential interference contrast. (B) CHO cells were transfected with 1 µg of either pFCgOrc1 (F-Orc1) or pFCgOrc2 (F-Orc2). At 18 hours post-transfection, the attached cells were treated as in panel A. (C) Same as in panel B, except that attached cells were analyzed at 36 hours post-transfection, and pCI-transfected cells were included. In the merged images, yellow indicates sites where DNA breaks and Orc were co-localized, and white indicates sites where DNA and Orc1 were co-localized.

View larger version (41K): [in a new window]   Fig. 5. Ectopic expression of Orc1 induced loss of cell adhesion priorto extensive DNA fragmentation, but not endoreduplication. Unattached CHO cells at the indicated times post-transfection with pCI, pFCgOrc2 (F-Orc2) or pFCgOrc1 (F-Orc1) were analyzed byfluorescence activated cell sorting (FACS). Control (C) was non-transfected cells. Cells at G1 phase (2N) and G2/M phase (4N) areindicated. Similar results were obtained with HeLa cells transfectedwith DNA expressing human Orc proteins (data not shown).

View larger version (43K): [in a new window]   Fig. 6. Low levels of ectopic Orc1 induced caspase-3 activity. (A) HeLa cells were transfected with 1 µg of either pCI, pFHsOrc1 () or pFHsOrc2 (). At the indicated times post-transfection, extracts from 106 cells were prepared by the freeze-thaw method and assayed for Z-VAD-fmk-sensitive caspase-3 activity using the colorimetric CaspACETM Assay System (Promega). Activities observed with pCI were subtracted from those observed with Orc expression vectors. The fraction of unattached transfected cells is indicated by broken line (0.5=50%). (B) CHO cells transfected with pCI, pFCgOrc1 () or pFCgOrc2 () were analyzed as in panel A. (C) Amounts of Orc1 () and Orc2 () proteins in CHO cells transfected with pFCgOrc1 were determined by densitometry of data such as those in panel E. Comparisons were made only where images were not saturating. These results were compared with the appearance of caspase-3 activity () at the beginning of transfection. Comparison with standard amounts of recombinant Orc protein fractionated in parallel was used to determine the cellular concentration of Orc. (D) Amounts of Orc1 () and Orc2 () in CHO cells transfected with pCgOrc2 were determined from data such as those in panel E, and then compared with caspase-3 activity () at the beginning of transfection. (E) Total CgOrc1 (Orc1) and CgOrc2 (Orc2) in CHO cells following transfection with pFCgOrc1 was detected by immunoblotting with both anti-CgOrc1 and anti-CgOrc2 IgG. Two independent experiments are shown. In the second, different exposures of the same gel were required to visualize both Orc1 and Orc2. (F) Total CgOrc1 and CgOrc2 in CHO cells following transfection with pFCgOrc2 was determined as in panel E. Two independent experiments are shown.

View larger version (59K): [in a new window]   Fig. 7. Co-expression of Orc2 with Orc1 suppressed induction of apoptosis by Orc1. (A) CHO cells were transfected with the indicated amounts of pFCgOrc1 and pFCgOrc2, and cell lysates were subjected to immunoblotting at 24 hours post-transfection using anti-FLAG antibody. (B) CHO cells were co-transfected with 1.5 µg pCgOrc1 and 0.5 µg pFCgOrc2. At 24 hours post-transfection, cells were stained with both anti-CgOrc1 (Orc1) and anti-FLAG (F-Orc2) antibodies. Merged images revealed common areas of localization (yellow). DIC, differential interference contrast.

View larger version (38K): [in a new window]   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/TD) or (S/TA). 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.

View larger version (88K): [in a new window]   Fig. 9. Post-translational modifications of Orc1 affected the subcellular localization of Orc1 and suppressed changes in cell morphology characteristic of apoptosis. HeLa cells were transfected with 1 µg pFHsOrc1-Ub (Orc1-Ub). CHO cells were transfected with 1 µg of either pFCgOrc1(A5) or pFCgOrc1(D5). At 12 and 24 hours post-transfection, cells were stained with anti-FLAG antibody to detect FCgOrc1 (red) and with Hoechst to detect nuclear DNA (blue). Merged images revealed FCgOrc1 localization relative to DNA, and DIC images revealed the morphology of the cell. DIC, differential interference contrast.

View larger version (33K): [in a new window]   Fig. 10. The mammalian `ORC cycle'. All six ORC subunits (shaded circles) are bound tightly to chromatin during G1 phase to provide sites for initiation of pre-replication complex (pre-RC) assembly. DNA synthesis does not begin until pre-RCs are activated by CcnE-Cdk2, a protein kinase whose activity is inhibited during G1 phase by the CDK-specific inhibitor p27/Kip1. With the onset of S phase, the affinity of Orc1 for chromatin is selectively reduced, and Orc1 is targeted for ubiquitylation by SCFSkp2. Since SCFSkp2-dependent degradation of p27 is essential for cell-cycle progression (Kossatz et al., 2004), CcnE-Cdk2 activation is accompanied by Orc1 inactivation, resulting in initiation of S phase with concomitant suppression of pre-RC assembly. Orc1 is selectively degraded during S phase in human cells, but not in hamster cells. However, even a single ubiquitin adduct can inactivate Orc1 by transporting it to the cytoplasm. In M phase, Orc1 is hyperphosphorylated and bound tightly to CcnA-Cdk1, but weakly to chromatin. Hyperphosphorylation favors cytoplasmic localization. When CcnA and CcnB are degraded during the transition from M to G1 phase, Cdk1 is inactivated, and Orc1 is dephosphorylated and rebound tightly to chromatin. If Orc1 is not associated with ORC, and if it is not ubiquitylated or hyperphosphorylated, then Orc1 can induce apoptosis.

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