Cyclin A-CDK activity during G1 phase impairs MCM chromatin loading and inhibits DNA synthesis in mammalian cells

Progression through the mammalian cell division cycle is regulated by the sequential activation of cyclin-dependent kinases, CDKs, at specific phases of the cell cycle. Cyclin A-CDK2 and cyclin A-CDK1 phosphorylate nuclear substrates during S and G2 phases, respectfully. However, the DNA helicase complex, MCM2-7, is loaded onto the origin of replications in G1, prior to the normally scheduled induction of cyclin A. It has previously been shown that cyclin A-CDKs phosphorylate MCM2 and MCM4 in vitro, thereby diminishing helicase activity. Thus, in this study we hypothesize that, in vivo, cyclin A-CDK activity during G1 would result in an inhibition of progression into the S phase. To test this, we establish an in vivo method of inducing cyclin A-CDK activity in G1 phase and observe that activation of cyclin A-CDK, but not cyclin E-CDK complexes, inhibit DNA synthesis without affecting other G1 events such as cyclin D synthesis, E2F activation and cdc6 loading onto chromatin. We further report that the mechanism of this S phase inhibition occurs, at least in part, through impaired loading of MCM onto chromatin, presumably due to decreased levels of cdt1 and premature phosphorylation of MCM by cyclin A-CDK. In addition to providing in vivo confirmation of in vitro predictions regarding cyclin A-CDK phosphorylation of the MCM complex, our results provide insight into the cellular effects of unscheduled cyclin A-CDK activity in mammalian cells.     

Phosphorylation of MCM3 protein by cyclin E/cyclin-dependent kinase 2 (Cdk2) regulates its function in cell cycle

MCM2-7 proteins form a stable heterohexamer with DNA helicase activity functioning in the DNA replication of eukaryotic cells. The MCM2-7 complex is loaded onto chromatin in a cell cycle-dependent manner. The phosphorylation of MCM2-7 proteins contributes to the formation of the MCM2-7 complex. However, the regulation of specific MCM phosphorylation still needs to be elucidated. In this study, we demonstrate that MCM3 is a substrate of cyclin E/Cdk2 and can be phosphorylated by cyclin E/Cdk2 at Thr-722. We find that the MCM3 T722A mutant binds chromatin much less efficiently when compared with wild type MCM3, suggesting that this phosphorylation site is involved in MCM3 loading onto chromatin. Interestingly, overexpression of MCM3, but not MCM3 T722A mutant, inhibits the S phase entry, whereas it does not affect the exit from mitosis. Knockdown of MCM3 does not affect S phase entry and progression, indicating that a small fraction of MCM3 is sufficient for normal S phase completion. These results suggest that excess accumulation of MCM3 protein onto chromatin may inhibit DNA replication. Other studies indicate that excess of MCM3 up-regulates the phosphorylation of CHK1 Ser-345 and CDK2 Thr-14. These data reveal that the phosphorylation of MCM3 contributes to its function in controlling the S phase checkpoint of cell cycle in addition to the regulation of formation of the MCM2-7 complex.     

Cyclin E ablation in the mouse

E type cyclins (E1 and E2) are believed to drive cell entry into the S phase. It is widely assumed that the two E type cyclins are critically required for proliferation of all cell types. Here, we demonstrate that E type cyclins are largely dispensable for mouse development. However, endoreplication of trophoblast giant cells and megakaryocytes is severely impaired in the absence of cyclin E. Cyclin E-deficient cells proliferate actively under conditions of continuous cell cycling but are unable to reenter the cell cycle from the quiescent G(0) state. Molecular analyses revealed that cells lacking cyclin E fail to normally incorporate MCM proteins into DNA replication origins during G(0)-->S progression. We also found that cyclin E-deficient cells are relatively resistant to oncogenic transformation. These findings define a molecular function for E type cyclins in cell cycle reentry and reveal a differential requirement for cyclin E in normal versus oncogenic proliferation.     

Human TopBP1 participates in cyclin E/CDK2 activation and preinitiation complex assembly during G1/S transition

Human TopBP1 with eight BRCA1 C terminus domains has been mainly reported to be involved in DNA damage response pathways. Here we show that TopBP1 is also required for G(1) to S progression in a normal cell cycle. TopBP1 deficiency inhibited cells from entering S phase by up-regulating p21 and p27, resulting in down-regulation of cyclin E/CDK2. Although co-depletion of p21 and p27 with TopBP1 restored the cyclin E/CDK2 kinase activity, however, cells remained arrested at the G(1)/S boundary, showing defective chromatin-loading of replication components. Based on these results, we suggest a dual role of TopBP1 necessary for the G(1)/S transition: one for activating cyclin E/CDK2 kinase and the other for loading replication components onto chromatin to initiate DNA synthesis.     

The consequences of differential origin licensing dynamics in distinct chromatin environments

Eukaryotic chromosomes contain regions of varying accessibility, yet DNA replication factors must access all regions. The first replication step is loading MCM complexes to license replication origins during the G1 cell cycle phase. It is not yet known how mammalian MCM complexes are adequately distributed to both accessible euchromatin regions and less accessible heterochromatin regions. To address this question, we combined time-lapse live-cell imaging with immunofluorescence imaging of single human cells to quantify the relative rates of MCM loading in euchromatin and heterochromatin throughout G1. We report here that MCM loading in euchromatin is faster than that in heterochromatin in early G1, but surprisingly, heterochromatin loading accelerates relative to euchromatin loading in middle and late G1. This differential acceleration allows both chromatin types to begin S phase with similar concentrations of loaded MCM. The different loading dynamics require ORCA-dependent differences in origin recognition complex distribution. A consequence of heterochromatin licensing dynamics is that cells experiencing a truncated G1 phase from premature cyclin E expression enter S phase with underlicensed heterochromatin, and DNA damage accumulates preferentially in heterochromatin in the subsequent S/G2 phase. Thus, G1 length is critical for sufficient MCM loading, particularly in heterochromatin, to ensure complete genome duplication and to maintain genome stability.     

Cyclin-dependent kinases and cell-cycle transitions: does one fit all?

Cell-cycle transitions in higher eukaryotes are regulated by different cyclin-dependent kinases (CDKs) and their activating cyclin subunits. Based on pioneering findings that a dominant-negative mutation of CDK1 blocks the cell cycle at G2-M phase, whereas dominant-negative CDK2 inhibits the transition into S phase, a model of cell-cycle control has emerged in which each transition is regulated by a specific subset of CDKs and cyclins. Recent work with gene-targeted mice has led to a revision of this model. We discuss cell-cycle control in light of overlapping and essential functions of the different CDKs and cyclins.     

CDK1-dependent Inhibition of the E3 Ubiquitin Ligase CRL4CDT2 Ensures Robust Transition from S Phase to Mitosis

Replication-coupled destruction of a cohort of cell cycle proteins ensures efficient and precise genome duplication. Three proteins destroyed during replication via the CRL4^CDT2 ubiquitin E3 ligase, CDT1, p21, and SET8 (PR-SET7), are also essential or important during mitosis, making their reaccumulation after S phase a critical cell cycle event. During early and mid-S phase and during DNA repair, proliferating cell nuclear antigen (PCNA) loading onto DNA (PCNA^DNA) triggers the interaction between CRL4^CDT2 and its substrates, resulting in their degradation. We have discovered that, beginning in late S phase, PCNA^DNA is no longer sufficient to trigger CRL4^CDT2-mediated degradation. A CDK1-dependent mechanism that blocks CRL4^CDT2 activity by interfering with CDT2 recruitment to chromatin actively protects CRL4^CDT2 substrates. We postulate that deliberate override of replication-coupled destruction allows anticipatory accumulation in late S phase. We further show that (as for CDT1) de novo SET8 reaccumulation is important for normal mitotic progression. In this manner, CDK1-dependent CRL4^CDT2 inactivation contributes to efficient transition from S phase to mitosis.     

The cyclin D1-dependent kinase associates with the pre-replication complex and modulates RB.MCM7 binding

The capacity of the cyclin D-dependent kinase to promote G(1) progression through modulation of RB.E2F is well documented. We now demonstrate that the cyclin D1/CDK4 kinase binds to components of the MCM complex. MCM7 and MCM3 were identified as cyclin D1-binding proteins. Catalytically active cyclin D1/CDK4 complexes were incorporated into chromatin-bound protein complexes with the same kinetics as MCM7 and MCM3, where they associated specifically with MCM7. Although the cyclin D1-dependent kinase did not phosphorylate MCM7, active cyclin D1/CDK4, but not cyclin E/CDK2, did catalyze the dissociation of an RB.MCM7 complex. Finally, expression of an active D1/CDK4 kinase but not cyclin E/CDK2 promoted the removal of RB from chromatin-bound protein complexes. Our data suggest that D1/CDK4 complexes play a direct role in altering an inhibitory RB.MCM7 complex possibly allowing for setting of the origin in preparation for DNA replication.     

Expression of cyclin E renders cyclin D-CDK4 dispensable for inactivation of the retinoblastoma tumor suppressor protein, activation of E2F, and G1-S phase progression

The activation of CDK2-cyclin E in late G1 phase has been shown to play a critical role in retinoblastoma protein (pRb) inactivation and G1-S phase progression of the cell cycle. The phosphatidylinositol 3-OH-kinase inhibitor LY294002 has been shown to block cyclin D1 accumulation, CDK4 activity and, thus, G1 progression in alpha-thrombin-stimulated IIC9 cells (Chinese hamster embryonic fibroblasts). Our previous results show that expression of cyclin E rescues S phase progression in alpha-thrombin-stimulated IIC9 cells treated with LY294002, arguing that cyclin E renders CDK4 activity dispensable for G1 progression. In this work we investigate the ability of alpha-thrombin-induced CDK2-cyclin E activity to inactivate pRb in the absence of prior CDK4-cyclin D1 activity. We report that in the absence of CDK4-cyclin D1 activity, CDK2-cyclin E phosphorylates pRb in vivo on at least one residue and abolishes pRb binding to E2F response elements. We also find that expression of cyclin E rescues E2F activation and cyclin A expression in cyclin D kinase-inhibited, alpha-thrombin-stimulated cells. Furthermore, the rescue of E2F activity, cyclin A expression, and DNA synthesis by expression of E can be blocked by the expression of either CDK2(D145N) or RbDeltaCDK, a constitutively active mutant of pRb. However, restoring four known cyclin E-CDK2 phosphorylation sites to RbDeltaCDK renders it susceptible to inactivation in late G1, as assayed by E2F activation, cyclin A expression, and S phase progression. These data indicate that CDK2-cyclin E, without prior CDK4-cyclin D activity, can phosphorylate and inactivate pRb, activate E2F, and induce DNA synthesis.     

Differential susceptibility of yeast S and M phase CDK complexes to inhibitory tyrosine phosphorylation

BACKGROUND: Several checkpoint pathways employ Wee1-mediated inhibitory tyrosine phosphorylation of cyclin-dependent kinases (CDKs) to restrain cell-cycle progression. Whereas in vertebrates this strategy can delay both DNA replication and mitosis, in yeast cells only mitosis is delayed. This is particularly surprising because yeasts, unlike vertebrates, employ a single family of cyclins (B type) and the same CDK to promote both S phase and mitosis. The G2-specific arrest could be explained in two fundamentally different ways: tyrosine phosphorylation of cyclin/CDK complexes could leave sufficient residual activity to promote S phase, or S phase-promoting cyclin/CDK complexes could somehow be protected from checkpoint-induced tyrosine phosphorylation. RESULTS: We demonstrate that in Saccharomyces cerevisiae, several cyclin/CDK complexes are protected from inhibitory tyrosine phosphorylation, allowing Clb5,6p to promote DNA replication and Clb3,4p to promote spindle assembly, even under checkpoint-inducing conditions that block nuclear division. In vivo, S phase-promoting Clb5p/Cdc28p complexes were phosphorylated more slowly and dephosphorylated more effectively than were mitosis-promoting Clb2p/Cdc28p complexes. Moreover, we show that the CDK inhibitor (CKI) Sic1p protects bound Clb5p/Cdc28p complexes from tyrosine phosphorylation, allowing the accumulation of unphosphorylated complexes that are unleashed when Sic1p is degraded to promote S phase. The vertebrate CKI p27(Kip1) similarly protects Cyclin A/Cdk2 complexes from Wee1, suggesting that the antagonism between CKIs and Wee1 is evolutionarily conserved. CONCLUSIONS: In yeast cells, the combination of CKI binding and preferential phosphorylation/dephosphorylation of different B cyclin/CDK complexes renders S phase progression immune from checkpoints acting via CDK tyrosine phosphorylation.     

Cyclin E2, the cycle continues

The eukaryotic cell cycle is regulated by a family of serine/threonine protein kinases known as cyclin-dependent kinases (CDKs). The activation of a CDK is dependent on its association with a cyclin regulatory subunit. The formation of distinct cyclin-CDK complexes controls the progression through the first gap phase (G(1)) and initiation of DNA synthesis (S phase). These complexes are in turn regulated by protein phosphorylation and cyclin-dependent kinase inhibitors (CKIs). Cyclin E2 has emerged as the second member of the E-type cyclin family. Cyclin E2-associated kinase activity is regulated in a cell cycle dependent manner with peak activity at the G(1) to S transition. Ectopic expression of cyclin E2 in human cells accelerates G(1), suggesting that cyclin E2 is rate limiting for G(1) progression. Although the pattern and level of cyclin E2 expression in some primary tumor and normal tissue RNAs are distinct from cyclin E1, both E-type cyclins appear to have inherent functional redundancies. This functional redundancy has facilitated the rapid characterization of cyclin E2 and uncovered unique features associated with each E-type cyclin.     

CDK2 regulation through PI3K and CDK4 is necessary for cell cycle progression of primary rat hepatocytes

INTRODUCTION/OBJECTIVES: Cell cycle progression is driven by the coordinated regulation of cyclin-dependent kinases (CDKs). In response to mitogenic stimuli, CDK4 and CDK2 form complexes with cyclins D and E, respectively, and translocate to the nucleus in the late G(1) phase. It is an on-going discussion whether mammalian cells need both CDK4 and CDK2 kinase activities for induction of S phase. METHODS AND RESULTS: In this study, we have explored the role of CDK4 activity during G(1) progression of primary rat hepatocytes. We found that CDK4 activity was restricted by either inhibiting growth factor induced cyclin D1-induction with the PI3K inhibitor LY294002, or by transient transfection with a dominant negative CDK4 mutant. In both cases, we observed reduced CDK2 nuclear translocation and reduced CDK2-Thr160 phosphorylation. Furthermore, reduced pRb hyperphosphorylation and reduced cellular proliferation were observed. Ectopic expression of cyclin D1 alone was not sufficient to induce CDK4 nuclear translocation, CDK2 activity or cell proliferation. CONCLUSIONS: Thus, epidermal growth factor-induced CDK4 activity was necessary for CDK2 activation and for hepatocyte proliferation. These results also suggest that, in addition to regulating cyclin D1 expression, PI3K is involved in regulation of nuclear shuttling of cyclin-CDK complexes in G(1) phase.     

Amniotic membrane-derived cells inhibit proliferation of cancer cell lines by inducing cell cycle arrest

Cells derived from the amniotic foetal membrane of human term placenta have drawn particular attention mainly for their plasticity and immunological properties, which render them interesting for stem-cell research and cell-based therapeutic applications. In particular, we have previously demonstrated that amniotic mesenchymal tissue cells (AMTC) inhibit lymphocyte proliferation in vitro and suppress the generation and maturation of monocyte-derived dendritic cells. Here, we show that AMTC also significantly reduce the proliferation of cancer cell lines of haematopoietic and non-haematopoietic origin, in both cell-cell contact and transwell co-cultures, therefore suggesting the involvement of yet-unknown inhibitory soluble factor(s) in this 'cell growth restraint'. Importantly, we provide evidence that the anti-proliferative effect of AMTC is associated with induction of cell cycle arrest in G0/G1 phase. Gene expression analyses demonstrate that AMTC can down-regulate cancer cells' mRNA expression of genes associated with cell cycle progression, such as cyclins (cyclin D2, cyclin E1, cyclin H) and cyclin-dependent kinase (CDK4, CDK6 and CDK2), whilst they up-regulate cell cycle negative regulator such as p15 and p21, consistent with a block in G0/G1 phase with no progression to S phase. Taken together, these findings warrant further studies to investigate the applicability of these cells for controlling cancer cell proliferation in vivo.     

Acceleration of the G1/S phase transition by expression of cyclins D1 and E with an inducible system.

Conditional overexpression of human cyclins B1, D1, and E was accomplished by using a synthetic cDNA expression system based on the Escherichia coli tetracycline repressor. After induction of these cyclins in asynchronous Rat-1 fibroblasts, a decrease in the length of the G1 interval was observed for cyclins D1 and E, consistent with an acceleration of the G1/S phase transition. We observed, in addition, a compensatory lengthening of S phase and G2 so that the mean cell cycle length in populations constitutively expressing these cyclins was unchanged relative to those of their uninduced counterparts. We found that expression of cyclin B1 had no effect on cell cycle dynamics, despite elevated levels of cyclin B-associated histone H1 kinase activity. Induction of cyclins D1 and E also accelerated entry into S phase for synchronized cultures emerging from quiescence. However, whereas cyclin E exerted a greater effect than cyclin D1 in asynchronous cycling cells, cyclin D1 conferred a greater effect upon stimulation from quiescence, suggesting a specific role for cyclin D1 in the G0-to-G1 transition. Overexpression of cyclins did not prevent cells from entering into quiescence upon serum starvation, although a slight delay in attainment of quiescence was observed for cells expressing either cyclin D1 or cyclin E. These results suggest that cyclins D1 and E are rate-limiting activators of the G1-to-S phase transition and that cyclin D1 might play a specialized role in facilitating emergence from quiescence.     

The tumour suppressor gene product APC blocks cell cycle progression from G0/G1 to S phase.

The APC gene is mutated in familial adenomatous polyposis (FAP) as well as in sporadic colorectal tumours. The product of the APC gene is a 300 kDa cytoplasmic protein associated with the adherence junction protein catenin. Here we show that overexpression of APC blocks serum-induced cell cycle progression from G0/G1 to the S phase. Mutant APCs identified in FAP and/or colorectal tumours were less inhibitory and partially obstructed the activity of the normal APC. The cell-cycle blocking activity of APC was alleviated by the overexpression of cyclin E/CDK2 or cyclin D1/CDK4. Consistent with this result, kinase activity of CDK2 was significantly down-regulated in cells overexpressing APC although its synthesis remained unchanged, while CDK4 activity was barely affected. These results suggest that APC may play a role in the regulation of the cell cycle by negatively modulating the activity of cyclin-CDK complexes.     

Multi-step Loading of Human Minichromosome Maintenance Proteins in Live Human Cells

Once-per-cell cycle replication is regulated through the assembly onto chromatin of multisubunit protein complexes that license DNA for a further round of replication. Licensing consists of the loading of the hexameric MCM2–7 complex onto chromatin during G₁ phase and is dependent on the licensing factor Cdt1. In vitro experiments have suggested a two-step binding mode for minichromosome maintenance (MCM) proteins, with transient initial interactions converted to stable chromatin loading. Here, we assess MCM loading in live human cells using an in vivo licensing assay on the basis of fluorescence recovery after photobleaching of GFP-tagged MCM protein subunits through the cell cycle. We show that, in telophase, MCM2 and MCM4 maintain transient interactions with chromatin, exhibiting kinetics similar to Cdt1. These are converted to stable interactions from early G₁ phase. The immobile fraction of MCM2 and MCM4 increases during G₁ phase, suggestive of reiterative licensing. In late G₁ phase, a large fraction of MCM proteins are loaded onto chromatin, with maximal licensing observed just prior to S phase onset. Fluorescence loss in photobleaching experiments show subnuclear concentrations of MCM-chromatin interactions that differ as G₁ phase progresses and do not colocalize with sites of DNA synthesis in S phase.     

Coordinated Activation of the Origin Licensing Factor CDC6 and CDK2 in Resting Human Fibroblasts Expressing SV40 Small T Antigen and Cyclin E

We have previously shown that SV40 small t antigen (st) cooperates with deregulated cyclin E to activate CDK2 and bypass quiescence in normal human fibroblasts (NHF). Here we show that st expression in serum-starved and density-arrested NHF specifically induces up-regulation and loading of CDC6 onto chromatin. Coexpression of cyclin E results in further accumulation of CDC6 onto chromatin concomitantly with phosphorylation of CDK2 on Thr-160 and CDC6 on Ser-54. Investigation of the mechanism leading to CDC6 accumulation and chromatin loading indicates that st is a potent inducer of cdc6 mRNA expression and increases CDC6 protein stability. We also show that CDC6 expression in quiescent NHF efficiently promotes cyclin E loading onto chromatin, but it is not sufficient to activate CDK2. Moreover, we show that CDC6 expression is linked to phosphorylation of the activating T loop of CDK2 in serum-starved NHF stimulated with mitogens or ectopically expressing cyclin E and st. Our data suggest a model where the combination of st and deregulated cyclin E result in cooperative and coordinated activation of both an essential origin licensing factor, CDC6, and an activity required for origin firing, CDK2, resulting in progression from quiescence to S phase.Upon mitogenic stimulation mammalian G1 CDKs⁴ trigger passage through the restriction point and the transition into DNA replication. In particular, cyclin E/CDK2 is activated in mid to late G1 and phosphorylates a variety of substrates that play critical roles in these processes. CDK2 cooperates with D-type cyclin/CDKs to inactivate E2F/pocket protein repressor complexes inducing the expression of DNA synthesis factors and other cell cycle regulators (reviewed in Refs. 1 and 2). CDK2 also phosphorylates DNA replication factors facilitating prereplication complex assembly and origin firing and plays additional roles in centrosome duplication and histone synthesis (reviewed in Ref. 1). In particular, it has been proposed that CDK2 phosphorylates the essential origin licensing factor CDC6 promoting its stabilization prior to inactivation of the APC^Cdh1 ubiquitin ligase (3). This is thought to ensure that CDC6 accumulation precedes accumulation of other APC substrates that inhibit origin licensing. Moreover, CDK2-independent cyclin E functions have also been reported to be important for prereplication complex assembly in cells in transit from G0 into G1 (4, 5). In keeping with its role as positive regulator of major G1 transitions, deregulation of the cyclin E via gene amplification or defective protein turnover is commonly seen in primary tumors and is associated with poor prognosis (6–8). In normal fibroblasts, ectopic expression of cyclin E has been associated with shortening of the G1 phase of the cell cycle (9, 10), and with induction of DNA damage (reviewed in Ref. 8). Cyclin E deregulation in certain human tumor cell lines and immortalized rat fibroblasts is associated with mitogen-independent cell cycle entry and progression through the cell cycle (11). However, when cyclin E is ectopically expressed in quiescent normal human fibroblasts (NHF), cells remain in G0 (12).We have recently reported that coexpression of SV40 small t antigen (st) in quiescent NHF with deregulated cyclin E expression is sufficient to trigger mitogen-independent cell cycle progression, proliferation beyond cell confluence, and foci formation. The bypass of quiescence induced by the expression of st and cyclin E is dependent on CDK2 activation (12). Thus, contrary to what is seen in normal murine cells (13), CDK2 activity appears essential for cell cycle progression when it is oncogenically driven by cyclin E and st expression (12). Because st is known to target pathways uniquely required for the transformation of human cells (14, 15), tumor cells with altered pathways that mimic st/cyclin E expression could predictably be sensitive to selective inhibition of CDK2 activity.Given the critical role of CDK2 activity in cyclin E and st cooperation in inducing cell proliferation and transformation of NHF, we sought to determine the factors and mechanisms by which st modulates CDK2 activation. In this report we have identified the CDC6 replication licensing factor as a cellular target of st. We also uncover CDC6 as a participant in the events leading to chromatin association of cyclin E and CDK2 and in phosphorylation of CDK2 on its activating T loop both in response to mitogenic stimulation, as well as expression of cyclin E and st in NHF.