COP9 signalosome-specific phosphorylation targets p53 to degradation by the ubiquitin system

In higher eukaryotic cells, the p53 protein is degraded by the ubiquitin-26S proteasome system mediated by Mdm2 or the human papilloma virus E6 protein. Here we show that COP9 signalosome (CSN)-specific phosphorylation targets human p53 to ubiquitin-26S proteasome-dependent degradation. As visualized by electron microscopy, p53 binds with high affinity to the native CSN complex. p53 interacts via its N-terminus with CSN subunit 5/Jab1 as shown by far-western and pull-down assays. The CSN-specific phosphorylation sites were mapped to the core domain of p53 including Thr155. A phosphorylated peptide, Deltap53(145-164), specifically inhibits CSN-mediated phosphorylation and p53 degradation. Curcumin, a CSN kinase inhibitor, blocks E6-dependent p53 degradation in reticulocyte lysates. Mutation of Thr155 to valine is sufficient to stabilize p53 against E6-dependent degradation in reticulocyte lysates and to reduce binding to Mdm2. The p53T155V mutant accumulates in both HeLa and HL 60 cells and exhibits a mutant (PAb 240+) conformation. It induces the cyclin-dependent inhibitor p21. In HeLa and MCF-7 cells, inhibition of CSN kinase by curcumin or Deltap53(145-164) results in accumulation of endogenous p53.     

Regulation of Cyclin A-Cdk2 by SCF Component Skp1 and F-Box Protein Skp2

Cyclin A-Cdk2 complexes bind to Skp1 and Skp2 during S phase, but the function of Skp1 and Skp2 is unclear. Skp1, together with F-box proteins like Skp2, are part of ubiquitin-ligase E3 complexes that target many cell cycle regulators for ubiquitination-mediated proteolysis. In this study, we investigated the potential regulation of cyclin A-Cdk2 activity by Skp1 and Skp2. We found that Skp2 can inhibit the kinase activity of cyclin A-Cdk2 in vitro, both by direct inhibition of cyclin A-Cdk2 and by inhibition of the activation of Cdk2 by cyclin-dependent kinase (CDK)-activating kinase phosphorylation. Only the kinase activity of Cdk2, not of that of Cdc2 or Cdk5, is reduced by Skp2. Skp2 is phosphorylated by cyclin A-Cdk2 on residue Ser76, but nonphosphorylatable mutants of Skp2 can still inhibit the kinase activity of cyclin A-Cdk2 toward histone H1. The F box of Skp2 is required for binding to Skp1, and both the N-terminal and C-terminal regions of Skp2 are involved in binding to cyclin A-Cdk2. Furthermore, Skp2 and the CDK inhibitor p21^Cip1/WAF1 bind to cyclin A-Cdk2 in a mutually exclusive manner. Overexpression of Skp2, but not Skp1, in mammalian cells causes a G₁/S cell cycle arrest.     

hPNAS-4 inhibits proliferation through S phase arrest and apoptosis: underlying action mechanism in ovarian cancer cells

PNAS-4, a novel pro-apoptotic gene, was activated during the early response to DNA damage. Previous studies have shown that hPNAS-4 can inhibit tumor growth when over-expressed in ovarian cancer cells. However, the underlying action mechanism remains elusive. In this work, we found that hPNAS-4 expression was significantly increased in SKOV3 cells when exposed to cisplatin, methyl methanesulfonate or mitomycin C, and that its overexpression could induce proliferation inhibition, S phase arrest and apoptosis in A2780s and SKOV3 ovarian cancer cells. The S phase arrest caused by hPNAS-4 was associated with up-regulation of p21. p21 is p53-dispensable and correlates with activation of ERK, and activation of the Cdc25A-Cdk2-Cyclin E/Cyclin A pathway, while the pro-apoptotic effects of hPNAS-4 were mediated by activation of caspase-9 and -3 other than caspase-8, and accompanied by release of AIF, Smac and cytochrome c into the cytosol. Taken together, these data suggest a new mechanism by which hPNAS-4 inhibits proliferation of ovarian cancer cells by inducing S phase arrest and apoptosis via activation of Cdc25A-Cdk2-Cyclin E/Cyclin A axis and mitochondrial dysfunction-mediated caspase-dependent and -independent apoptotic pathways. To our knowledge, we provide the first molecular evidence for the potential application of hPNAS-4 as a novel target in ovarian cancer gene therapy.     

Cdc6 determines utilization of p21(WAF1/CIP1)-dependent damage checkpoint in S phase cells

When cells traversing G(1) are irradiated with UV light, two parallel damage checkpoint pathways are activated: Chk1-Cdc25A and p53-p21(WAF1/CIP1), both targeting Cdk2, but the latter inducing a long lasting arrest. In similarly treated S phase-progressing cells, however, only the Cdc25A-dependent checkpoint is active. We have recently found that the p21-dependent checkpoint can be activated and induce a prolonged arrest if S phase cells are damaged with a base-modifying agent, such as methyl methanesulfonate (MMS) and cisplatin. But the mechanistic basis for the differential activation of the p21-dependent checkpoint by different DNA damaging agents is not understood. Here we report that treatment of S phase cells with MMS but not a comparable dose of UV light elicits proteasome-mediated degradation of Cdc6, the assembler of pre-replicative complexes, which allows induced p21 to bind Cdk2, thereby extending inactivation of Cdk2 and S phase arrest. Consistently, enforced expression of Cdc6 largely eliminates the prolonged S phase arrest and Cdk2 inactivation induced with MMS, whereas RNA interference-mediated Cdc6 knockdown not only prolongs such arrest and inactivation but also effectively activates the p21-dependent checkpoint in the UV-irradiated S phase cells.     

Chemical DNA damage activates p21 WAF1/CIP1-dependent intra-S checkpoint

When cells progressing in G₁ phase are irradiated with UV light, two damage checkpoint pathways are activated: CHK1-Cdc25A and p53-p21^WAF1/CIP1, both targeting Cdk2 but the latter inducing long lasting inactivation. In similarly irradiated S phase cells, however, p21^WAF1/CIP1-dependent checkpoint is largely inactive. We report here that p21-dependent checkpoint can effectively be activated and induce a prolonged S phase arrest with similarly extended inactivation of Cdk2 by association of p21 if mid-S phase cells are damaged with a base-modifying agent instead of UV light, indicating that the poor utilization of p21-dependent checkpoint is not an innate property of S phase cells.     

A p53-dependent checkpoint pathway prevents rereplication

Eukaryotic cells control the initiation of DNA replication so that origins that have fired once in S phase do not fire a second time within the same cell cycle. Failure to exert this control leads to genetic instability. Here we investigate how rereplication is prevented in normal mammalian cells and how these mechanisms might be overcome during tumor progression. Overexpression of the replication initiation factors Cdt1 and Cdc6 along with cyclin A-cdk2 promotes rereplication in human cancer cells with inactive p53 but not in cells with functional p53. A subset of origins distributed throughout the genome refire within 2-4 hr of the first cycle of replication. Induction of rereplication activates p53 through the ATM/ATR/Chk2 DNA damage checkpoint pathways. p53 inhibits rereplication through the induction of the cdk2 inhibitor p21. Therefore, a p53-dependent checkpoint pathway is activated to suppress rereplication and promote genetic stability.     

The role of p21(CIP1/WAF1) in growth of epithelial cells exposed to hyperoxia

Previous studies have shown that hyperoxia inhibits proliferation and increases the expression of the tumor suppressor p53 and its downstream target, the cyclin-dependent kinase inhibitor p21(CIP1/WAF1), which inhibits proliferation in the G1 phase of the cell cycle. To determine whether growth arrest was mediated through activation of the p21-dependent G1 checkpoint, the kinetics of cell cycle movement during exposure to 95% O2 were assessed in the Mv1Lu and A549 pulmonary adenocarcinoma cell lines. Cell counts, 5-bromo-2'-deoxyuridine incorporation, and cell cycle analyses revealed that growth arrest of both cell lines occurred in S phase, with A549 cells also showing evidence of a G1 arrest. Hyperoxia increased p21 in A549 but not in Mv1Lu cells, consistent with the activation of the p21-dependent G1 checkpoint. The ability of p21 to exert the G1 arrest was confirmed by showing that hyperoxia inhibited proliferation of HCT 116 colon carcinoma cells predominantly in G1, whereas an isogenic line lacking p21 arrested in S phase. The cell cycle arrest in S phase appears to be a p21-independent process caused by a gradual reduction in the rate of DNA strand elongation. Our data reveal that hyperoxia inhibits proliferation in G1 and S phase and demonstrate that p53 and p21 retain their ability to affect G1 checkpoint control during exposure to elevated O2 levels.     

The kinetics of p53 activation versus cyclin E accumulation underlies the relationship between the spindle-assembly checkpoint and the postmitotic checkpoint

Although cells can exit mitotic block aberrantly by mitotic slippage, they are prevented from becoming tetraploids by a p53-dependent postmitotic checkpoint. Intriguingly, disruption of the spindle-assembly checkpoint also compromises the postmitotic checkpoint. The precise mechanism of the interplay between these two pivotal checkpoints is not known. We found that after prolonged nocodazole exposure, the postmitotic checkpoint was facilitated by p53. We demonstrated that although disruption of the mitotic block by a MAD2-binding protein promoted slippage, it did not influence the activation of p53. Both p53 and its downstream target p21(CIP1/WAF1) were activated at the same rate irrespective of whether the spindle-assembly checkpoint was enforced or not. The accelerated S phase entry, as reflected by the premature accumulation of cyclin E relative to the activation of p21(CIP1/WAF1), is the reason for the uncoupling of the postmitotic checkpoint. In support of this hypothesis, forced premature mitotic exit with a specific CDK1 inhibitor triggered DNA replication without affecting the kinetics of p53 activation. Finally, replication after checkpoint bypass was boosted by elevating the level of cyclin E. These observations indicate that disruption of the spindle-assembly checkpoint does not directly influence p53 activation, but the shortening of the mitotic arrest allows cyclin E-CDK2 to be activated before the accumulation of p21(CIP1/WAF1). These data underscore the critical relationship between the spindle-assembly checkpoint and the postmitotic checkpoint in safeguarding chromosomal stability.     

Inhibition of cell division by the human cytomegalovirus IE86 protein: role of the p53 pathway or cyclin-dependent kinase 1/cyclin B1

The human cytomegalovirus (HCMV) IE86 protein induces the human fibroblast cell cycle from G(0)/G(1) to G(1)/S, where cell cycle progression stops. Cells with a wild-type, mutated, or null p53 or cells with null p21 protein were transduced with replication-deficient adenoviruses expressing HCMV IE86 protein or cellular p53 or p21. Even though S-phase genes were activated in a p53 wild-type cell, IE86 protein also induced phospho-Ser(15) p53 and p21 independent of p14ARF but dependent on ATM kinase. These cells did not enter the S phase. In human p53 mutant, p53 null, or p21 null cells, IE86 protein did not up-regulate p21, cellular DNA synthesis was not inhibited, but cell division was inhibited. Cells accumulated in the G(2)/M phase, and there was increased cyclin-dependent kinase 1/cyclin B1 activity. Although the HCMV IE86 protein increases cellular E2F activity, it also blocks cell division in both p53(+/+) and p53(-/-) cells.