Phosphorylation of Mcm2 by Cdc7 promotes pre-replication complex assembly during cell-cycle re-entry.

Cyclin E has been shown to have a role in pre-replication complex (Pre-RC) assembly in cells re-entering the cell cycle from quiescence. The assembly of the pre-RC, which involves the loading of six MCM subunits (Mcm2-7), is a prerequisite for DNA replication. We found that cyclin E, through activation of Cdk2, promotes Mcm2 loading onto chromatin. This function is mediated in part by promoting the accumulation of Cdc7 messenger RNA and protein, which then phosphorylates Mcm2. Consistent with this, a phosphomimetic mutant of Mcm2 can bypass the requirement for Cdc7 in terms of Mcm2 loading. Furthermore, ectopic expression of both Cdc6 and Cdc7 can rescue the MCM loading defect associated with expression of dominant-negative Cdk2. These results are consistent with a role for cyclin E-Cdk2 in promoting the accumulation of Cdc6 and Cdc7, which is required for Mcm2 loading when cells re-enter the cell cycle from quiescence.

The E3 ubiquitin ligase skp2 regulates neural differentiation independent from the cell cycle.

BACKGROUND: The SCFskp2 complex is an E3 ubiquitin ligase that is known to target a number of cell cycle regulators, including cyclin-dependent kinase inhibitors, for proteolysis. While its role in regulation of cell division has been well documented, additional functions in differentiation, including in the nervous system, have not been investigated. RESULTS: Using Xenopus as a model system, here we demonstrate that skp2 has an additional role in regulation of differentiation of primary neurons, the first neurons to differentiate in the neural plate. Xenopus skp2 shows a dynamic expression pattern in early embryonic neural tissue and depletion of skp2 results in generation of extra primary neurons. In contrast, over-expression of skp2 inhibits neurogenesis in a manner dependent on its ability to act as part of the SCFskp2 complex. Moreover, inhibition of neurogenesis by skp2 occurs upstream of the proneural gene encoding NeuroD and prior to cell cycle exit. We have previously demonstrated that the Xenopus cyclin dependent kinase inhibitor Xic1 is essential for primary neurogenesis at an early stage, and before these cells exit the cell cycle. We show that SCFskp2 degrades Xic1 in embryos and this contributes to the ability of skp2 to regulate neurogenesis. CONCLUSION: We conclude that the SCFskp2 complex has functions in the control of neuronal differentiation additional to its role in cell cycle regulation. Thus, it is well placed to be a co-ordinating factor regulating both cell proliferation and cell differentiation directly.

Cyclin E overexpression impairs progression through mitosis by inhibiting APC(Cdh1).

Overexpression of cyclin E, an activator of cyclin-dependent kinase 2, has been linked to human cancer. In cell culture models, the forced expression of cyclin E leads to aneuploidy and polyploidy, which is consistent with a direct role of cyclin E overexpression in tumorigenesis. In this study, we show that the overexpression of cyclin E has a direct effect on progression through the latter stages of mitotic prometaphase before the complete alignment of chromosomes at the metaphase plate. In some cases, such cells fail to divide chromosomes, resulting in polyploidy. In others, cells proceed to anaphase without the complete alignment of chromosomes. These phenotypes can be explained by an ability of overexpressed cyclin E to inhibit residual anaphase-promoting complex (APC(Cdh1)) activity that persists as cells progress up to and through the early stages of mitosis, resulting in the abnormal accumulation of APC(Cdh1) substrates as cells enter mitosis. We further show that the accumulation of securin and cyclin B1 can account for the cyclin E-mediated mitotic phenotype.

Ubiquitination of cyclin-dependent kinase inhibitor, Xic1, is mediated by the Xenopus F-box protein xSkp2.

In the frog, Xenopus laevis, the Cip/Kip-type cyclin-dependent kinase (CDK) inhibitor, Xic1, inhibits DNA replication in interphase egg extracts through the binding of CDK2-cyclins and Proliferating Cell Nuclear Antigen (PCNA). During DNA polymerase switching in the replicating Xenopus egg extract, Xic1 is targeted for ubiquitination and degradation when localized to chromatin through its binding to PCNA. To date, the machinery responsible for Xic1 ubiquitination is unknown and although it is predicted that the E3 called SCF may mediate Xic1 ubiquitination, characterization of the SCF in Xenopus is lacking. In this study, we describe the identification and characterization of Xenopus Skp2 (xSkp2) and the role of xSkp2 in the ubiquitination of Xic1. Our results indicate that the expression of xSkp2 appears to be developmentally regulated with low protein levels found in the egg and increased levels found in the developing embryo. We also demonstrate that when ectopically expressed, a xSkp2 F-box deletion mutant inhibits the initiation of DNA replication suggesting a role for the SCF in the onset of S phase in Xenopus egg extracts. We further show that xSkp2 binds to C-terminal residues of Xic1 and when coexpressed with Skp1, promotes the proteolysis of Xic1 in the egg extract. Moreover, the xSkp2 F-box deletion mutant inhibits the DNA-dependent ubiquitination and proteolysis of Xic1 when added to the interphase egg extract. Importantly, our studies demonstrate that SCF(xSkp2) supports the ubiquitination of Xic1 in a reconstituted in vitro ubiquitination assay and that this Xic1 ubiquitination does not require either CDK2-cyclins or Cks1. These studies provide the first characterization of the SCF in Xenopus and its role in the ubiquitination of CDK inhibitor, Xic1, during DNA replication initiation.

The C-terminal domain of the Xenopus cyclin-dependent kinase inhibitor, p27Xic1, is both necessary and sufficient for phosphorylation-independent proteolysis.

Cell cycle progression is regulated by cyclin-dependent kinases (CDKs), cyclins, and CDK inhibitors. In the frog, Xenopus laevis, the CDK inhibitor p27(Xic1) (Xic1) inhibits DNA synthesis by negatively regulating CDK2-cyclin E. Using the frog egg extract as a model system for the study of Xic1, studies have demonstrated that Xic1 protein levels are regulated by nuclear ubiquitination and proteolysis. To characterize the molecular mechanism that regulates Xic1 turnover, we have identified the minimal sequences of Xic1 that are necessary and sufficient for its nuclear ubiquitination and degradation. Using deletion mutagenesis, our studies indicated that the C-terminal 50 amino acids of Xic1 are critical for its proteolysis beyond a role in nuclear transport. Replacement of the Xic1 C terminus with the SV40 nuclear localization sequence resulted in the nuclear localization of Xic1 but not its ubiquitination or degradation. Our deletion studies also indicated that the CDK2-cyclin binding domain of Xic1 is important for its efficient retention in the nucleus. Further deletion analyses identified at least 3 lysine residues within the Xic1 C terminus that are targeted for specific ubiquitination. Importantly, our studies demonstrated that the Xic1 C-terminal 50 amino acids can serve as a nuclear degradation signal when fused to a stable heterologous nuclear protein. Moreover, a 30-amino-acid region within the C terminus of Xic1 can serve as a nuclear ubiquitination signal. To address the role of phosphorylation on Xic1 turnover, all the potential phosphorylation sites within the C-terminal 50 amino acids of Xic1 were mutated to alanine to prevent possible phosphorylation. This resulted in a Xic1 protein that was nevertheless degraded in a manner similar to wild-type Xic1, suggesting that phosphorylation of Xic1 is not critical for its nuclear ubiquitination or proteolysis.

Proliferating cell nuclear antigen recruits cyclin-dependent kinase inhibitor Xic1 to DNA and couples its proteolysis to DNA polymerase switching.

The Xenopus cyclin-dependent kinase (CDK) inhibitor, p27(Xic1) (Xic1), binds to CDK2-cyclins and proliferating cell nuclear antigen (PCNA), inhibits DNA synthesis in Xenopus extracts, and is targeted for ubiquitin-mediated proteolysis. Previous studies suggest that Xic1 ubiquitination and degradation are coupled to the initiation of DNA replication, but the precise timing and molecular mechanism of Xic1 proteolysis has not been determined. Here we demonstrate that Xic1 proteolysis is temporally restricted to late replication initiation following the requirements for DNA polymerase alpha-primase, replication factor C, and PCNA. Our studies also indicate that Xic1 degradation is absolutely dependent upon the binding of Xic1 to PCNA in both Xenopus egg and gastrulation stage extracts. Additionally, extracts depleted of PCNA do not support Xic1 proteolysis. Importantly, while the addition of recombinant wild-type PCNA alone restores Xic1 degradation, the addition of a PCNA mutant defective for trimer formation does not restore Xic1 proteolysis in PCNA-depleted extracts, suggesting Xic1 proteolysis requires both PCNA binding to Xic1 and the ability of PCNA to be loaded onto primed DNA by replication factor C. Taken together, our studies suggest that Xic1 is targeted for ubiquitination and degradation during DNA polymerase switching through its interaction with PCNA at a site of initiation.