Cyclin-dependent kinases (CDKs) are essential for regulating key transitions in the cell cycle, including initiation of DNA replication, mitosis and prevention of re-replication. Mammalian CDC6, an essential regulator of initiation of DNA replication, is phosphorylated by CDKs. CDC6 interacts specifically with the active Cyclin A/CDK2 complex in vitro and in vivo, but not with Cyclin E or Cyclin B kinase complexes. The cyclin binding domain of CDC6 was mapped to an N-terminal Cy-motif that is similar to the cyclin binding regions in p21(WAF1/SDI1) and E2F-1. The in vivo phosphorylation of CDC6 is dependent on three N-terminal CDK consensus sites, and the phosphorylation of these sites has been shown to regulate the subcellular localization of CDC6. Consistent with this notion, it has been found that the subcellular localization of CDC6 is cell cycle regulated. In G1, CDC6 is nuclear and it relocalizes to the cytoplasm when Cyclin A/CDK2 is activated. In agreement with CDC6 phosphorylation being specifically mediated by Cyclin A/CDK2, ectopic expression of Cyclin A, but not of Cyclin E, leads to rapid relocalization of CDC6 from the nucleus to the cytoplasm. Based on these data it is suggested that the phosphorylation of CDC6 by Cyclin A/CDK2 is a negative regulatory event that could be implicated in preventing re-replication during S phase and G2 (Petersen, 1996).

In yeast, CDK activity is required both for entry into S phase of the cell cycle and for restricting replication to 'once and only once' per cell cycle. Since mammalian CDC6 is phosphorylated by Cyclin A/CDK2, and Cyclin A/CDK2 kinase activity is required for entry into the S phase of the cell cycle, it is conceivable that CDC6 is an essential substrate for Cyclin A/CDK2, and that the phosphorylation of CDC6 is required for cells to initiate DNA synthesis. If this were true, a non-phosphorylatable mutant of CDC6 that retains the biochemical activities of CDC6 should work as a dominant negative and prevent progression into S phase of the cell cycle. By several different assays, it is shown in this paper that two non-phosphorylatable mutants of hCDC6 are unable to prevent DNA replication in transfected or microinjected cells, suggesting that phosphorylation of CDC6 is not required for cells to enter S phase. Whether or not the hCDC6 mutants retain all their biochemical activities could not be tested; however, it is unlikely that the mutations destroy the structure of the proteins, since both mutants are capable of binding Cyclin A/CDK2. Consistent with the notion that CDC6 phosphorylation is not essential for S phase entry, it has been shown that phosphorylation of Cdc18p in S.pombe is not required for cells to enter S phase. These results strongly suggest that the phosphorylation of mammalian CDC6 by Cyclin A/CDK2 abolishes the ability of CDC6 to stimulate the formation of the pre-replication complex, which in higher eukaryotes has yet to be defined. An interesting question is whether phosphorylation and inactivation of CDC6 is a mechanism by which higher eukaryotes prevent re-replication of origins during S, G2 and M phases. In contrast with the ability of Cdc18p and the non-phosphorylatable mutant of Cdc18p to induce re-replication when overexpressed, no evidence is available that overexpression of CDC6 or the non-phosphorylatable mutants of CDC6 are sufficient by themselves to induce re-replication in mammalian cells. Interestingly, overexpression of S. cerevisiae Cdc6p is not sufficient to induce re-replication, but a dominant mutant of CDC6, cdc6-3, with point mutations within the conserved leucine zipper and a non-conserved region, is able to induce re-replication when overexpressed. Leucine zippers are known to mediate protein-protein interactions, and the destruction of the leucine zipper in cdc6-3 may suggest that this gain-of-function mutant cannot be inactivated due to the loss of specific protein-protein interactions. Unfortunately, the phosphorylation status of this mutant was not analysed, but re-initiation of DNA replication is not blocked by high CDK levels. This suggests that CDK inhibition of replication is mediated via Cdc6p, and that there is a functional interplay between the phosphorylation of Cdc6p and its ability to interact with other proteins through the leucine zipper. Future studies will be required to test whether the leucine zipper is implicated in regulation of mammalian CDC6 function, and if the phosphorylation of mammalian CDC6 is a mechanism by which re-replication is prevented. However, it is likely that there are other CDK targets than CDC6 implicated in preventing re-replication (Petersen, 1996).

In a number of cell lines (including HeLa cells, U-2 OS osteosarcoma cells and Hs 578Bst breast epithelial cells), ectopic expression of cyclin A increases hormone-dependent and hormone-independent transcriptional activation by the estrogen receptor. This effect can be further enhanced in HeLa cells by the concurrent expression of the cyclin-dependent kinase activator (cyclin H) and cdk7, and abolished by expression of the cdk inhibitor, p27(KIP1), or by the expression of a dominant negative catalytically inactive cdk2 mutant. Estrogen receptor is phosphorylated between amino acids 82 and 121 in vitro by the cyclin A/cdk2 complex; incorporation of phosphate into ER is stimulated by ectopic expression of cyclin A in vivo. Together, these results strongly suggest a direct role for the cyclin A/cdk2 complex in phosphorylating ER and regulating its transcriptional activity (Trowbridge, 1997).

Centrosomes nucleate microtubules and duplicate once per cell cycle. This duplication and subsequent segregation in mitosis results in maintenance of the one centrosome/cell ratio. Centrosome duplication occurs during the G1/S transition in somatic cells and must be coupled to the events of the nuclear cell cycle; failure to coordinate duplication and mitosis results in abnormal numbers of centrosomes and aberrant mitoses. Using both in vivo and in vitro assays, centrosome duplication in Xenopus laevis embryos has been shown to require cyclin/cdk2 kinase activity. Injection of the cdk (cyclin-dependent kinase) inhibitor p21 into one blastomere of a dividing embryo blocks centrosome duplication in that blastomere; the related cdk inhibitor p27 has a similar effect. An in vitro system using Xenopus extracts carries out separation of the paired centrioles within the centrosome. This centriole separation activity is dependent on cyclin/cdk2 activity; depletion of either cdk2 or of the two activating cyclins, cyclin A and cyclin E, eliminates centriole separation activity. In addition, centriole separation is inhibited by the mitotic state, suggesting a mechanism of linking the cell cycle to periodic duplication of the centrosome (Lacey, 1999).

Cell division is driven by cyclin-B-dependent kinase and anaphase-promoting complex (APC)-mediated proteolysis. Continuing transcription of E2F target genes beyond the G1/S transition is required for coordinating S-phase progression with cell division. Using an in vivo assay to measure protein stability in real time during the cell cycle, it has been shown that repression of E2F activity or inhibition of cyclin-A-dependent kinase in S phase triggers the destruction of cyclin B1 through the re-assembly of APC, the ubiquitin ligase that is essential for mitotic cyclin proteolysis, with its activatory subunit Cdh1. Phosphorylation-deficient mutant Cdh1 or immunodepletion of cyclin A results in assembly of active Cdh1-APC even in S-phase cells. These results implicate an E2F-dependent, cyclin A/Cdk2-mediated phosphorylation of Cdh1 in the timely accumulation of cyclin B1 and the coordination of cell-cycle progression during the G-1 phase post-restriction point period (Lukas, 1999).

A strong body of evidence indicates that cyclin-dependent protein kinases are required not only for the initiation of DNA replication but also for preventing over-replication in eukaryotic cells. Mcm proteins are one of the components of the replication licensing system that permits only a single round of DNA replication per cell cycle. It has been reported that Mcm proteins are phosphorylated by the cyclin-dependent kinases in vivo, suggesting that these two factors are cooperatively involved in the regulation of DNA replication. A 600-kDa Mcm4,6,7 complex has a DNA helicase activity that is probably necessary for the initiation of DNA replication. The in vitro phosphorylation of the Mcm complexes by cyclin A/Cdk2 has been studied to understand the interplay between Mcm proteins and cyclin-dependent kinases. The cyclin A/Cdk2 mainly phosphorylates the amino-terminal region of Mcm4 in the Mcm4,6,7 complex. The phosphorylation is associated with the inactivation of its DNA helicase activity. These results raise the possibility that the inactivation of Mcm4,6,7 helicase activity by Cdk2 is a part of the system for regulating DNA replication (Ishimi, 2000).

Cyclin A-mediated activation of cyclin-dependent kinases (CDKs) is essential for cell cycle transversal. Cyclin A activity is regulated on several levels and cyclin A elevation in a number of cancers suggests a role in tumorigenesis. In the present study, a modified DNA binding site selection and PCR amplification procedure was used to identify DNA binding proteins that are potential substrates of cyclin A-CDK. One of the sequences identified is the Sp1 transcription factor binding site. Co-immunoprecipitation experiments show that cyclin A and Sp1 can interact physically. In vitro and in vivo phosphorylation studies indicate that cyclin A-CDK complexes can phosphorylate Sp1. The phosphorylation site is located in the N-terminal region of the protein. Cells overexpressing cyclin A have elevated levels of Sp1 DNA binding activity, suggesting that cyclin A-CDK-mediated phosphorylation augments Sp1 DNA binding properties. In co-transfection studies, cyclin A expression stimulates transcription from an Sp1-regulated promoter. Mutation of the phosphorylation site abrogates cyclin A-CDK-dependent phosphorylation, augmentation of Sp1 transactivation function and DNA binding activity (Borja, 2001).

Centrosome duplication is a key requirement for bipolar spindle formation and correct segregation of chromosomes during cell division. In a manner highly reminiscent of DNA replication, the centrosome must be duplicated once, and only once, in each cell cycle. How centrosome duplication is regulated and coordinated with other cell-cycle functions remains poorly understood. A centrosome duplication assay has been established using mammalian somatic cells. Centrosome duplication is shown to require the activation of E2F transcription factors and Cdk2-Cyclin A activity (Meraldi, 1999).

The results clearly show that centrosome duplication in somatic mammalian cells requires the phosphorylation of Rb. This implies that both DNA replication and centrosome duplication are controlled through the same pathway. In normal proliferating cells, this would seem to provide an efficient means of ensuring the coordinate execution of these two key events. It also follows, however, that the loss of a functional Rb pathway, either through mutation or the action of viral oncogenes, might jeopardize the coordination between DNA replication and centrosome duplication and lead to genomic instability (Meraldi, 1999).

It is further shown that centrosome duplication requires the activity of E2F transcription factors. Rb has been reported to regulate the activities of several potential effector proteins, but overexpression of E2F is sufficient to induce centrosome duplication in cells expressing a non-phosphorylatable Rb mutant. This indicates that, among the Rb-binding proteins, E2F is the major downstream effector regulating centrosome duplication. Furthermore, the dependence of somatic cells on the transcriptional activity of E2F may explain why such cells need an intact nucleus and protein synthesis for centrosome duplication, whereas embryonic systems only require cytoplasmic components. In the case of DNA synthesis, E2F activity is required to synthesize several essential gene products, including key regulators of DNA replication such as Cdc6. By analogy, it seems plausible that E2F may activate the synthesis of gene products that are critical for centrosome duplication; these may include regulatory proteins as well as bona fide centrosomal components. In view of the results presented here, it is particularly intriguing that both the cyclin A and E genes are among the known target genes for E2F (Meraldi, 1999).

Centrosome duplication in somatic cells requires Cdk2 activity in addition to E2F. In contrast, no evidence could be obtained for involvement of Cdk1 or Cdk3, indicating that the Cdk2 requirement is specific. This conclusion is in excellent agreement with the results of two recent independent studies on centrosome duplication in cell-free systems based on Xenopus egg extracts. However, in Xenopus extracts cyclin E has been identified as the primary partner of Cdk2, whereas the current studies on somatic cells lead the authors to emphasize the role of cyclin A. This discrepancy cannot be explained but it is believed that it reflects a genuine difference in the regulation of centrosome duplication in the two systems. In fact, somatic cell cycles differ in several aspects from those of early embryonic cells. (1) They are characterized by the presence of prolonged G1 and G2 phases and they exhibit regulation at the transcriptional level, neither of which are seen in rapidly dividing embryonic cells. (2) The two systems differ markedly in the regulation of cyclins A and E; in somatic mammalian cells cyclin E levels peak sharply around G1/S, whereas they are almost constant during the cell cycles of early Xenopus embryos. Conversely, cyclin A is a prominent S-phase partner of Cdk2 in somatic mammalian cells, whereas in Xenopus embryos A-type cyclins do not associate with Cdk2 before the mid-blastula transition. In view of this, it is possible that cyclin E has a prominent role in centrosome duplication in early Xenopus embryos but that in somatic mammalian cells this function is performed primarily by cyclin A (Meraldi, 1999).

The experiments have revealed an unexpected but intriguing difference in the abilities of cyclins A and E to support Cdk2 activity with respect to either centrosome duplication or DNA replication. In particular, in cells in which E2F activation has been blocked by the expression of a non-phosphorylatable Rb mutant, only cyclin A is able to promote centrosome duplication; cyclin E is ineffective. Conversely, under very similar experimental conditions, cyclin E is more effective than cyclin A in inducing DNA replication. In somatic cells therefore, cyclin A may be the preferred partner of Cdk2 for centrosome duplication, whereas cyclin E may be primarily responsible for promoting the G1/S transition and initiating DNA replication. The temporal coincidence between centrosome duplication and cyclin A expression is consistent with such a model, but more work is required to substantiate or refute this hypothesis (Meraldi, 1999).

Cell-cycle transition at G2-M is controlled by MPF (M-phase-promoting factor), a complex consisting of the Cdc2 kinase and a B-type cyclin. In mice, targeted disruption of an A-type cyclin gene, cyclin A1, results in a block of spermatogenesis prior to the entry into metaphase I. The meiotic arrest is accompanied by a defect in Cdc2 kinase activation at the G2-M transition, raising the possibility that a cyclin A1-dependent process dictates the activation of MPF. Like Cdc2, the expression of B-type cyclins is retained in cyclin A1-deficient spermatocytes, while their associated kinases are kept at inactive states. Treatment of arrested germ cells with the protein phosphatase type-1 and -2A inhibitor okadaic acid restores the MPF activity and induces entry into M phase and the formation of normally condensed chromosome bivalents, concomitant with hyperphosphorylation of Cdc25 proteins. Conversely, inhibition of tyrosine phosphatases, including Cdc25s, by vanadate suppresses the okadaic acid-induced metaphase induction. The highest levels of Cdc25A and Cdc25C expression and their subcellular localization during meiotic prophase coincide with those of cyclin A1, and when overexpressed in HeLa cells, cyclin A1 coimmunoprecipitates with Cdc25A. Furthermore, the protein kinase complexes consisting of cyclin A1 and either Cdc2 or Cdk2 phosphorylate both Cdc25A and Cdc25C in vitro. These results suggest that in normal meiotic male germ cells, cyclin A1 participates in the regulation of other protein kinases or phosphatases critical for the G2-M transition. In particular, it the cyclin/cdk dimer be directly involved in the initial amplification of MPF through the activating phosphorylation on Cdc25 phosphatases (Liu, 2000).

Initiation of DNA replication is regulated by cyclin-dependent protein kinase 2 (Cdk2) in association with two different regulatory subunits, cyclin A and cyclin E. But why two different cyclins are required and why their order of activation is tightly regulated are unknown. Using a cell-free system for initiation of DNA replication that is based on G1 nuclei, G1 cytosol and recombinant proteins, it is found that cyclins E and A have specialized roles during the transition from G0 to S phase. Cyclin E stimulates replication complex assembly by cooperating with Cdc6, to make G1 nuclei competent to replicate in vitro. Cyclin A has two separable functions: it activates DNA synthesis by replication complexes that are already assembled, and it inhibits the assembly of new complexes. Thus, cyclin E opens a 'window of opportunity' for replication complex assembly that is closed by cyclin A. The dual functions of cyclin A ensure that the assembly phase (G1) ends before DNA synthesis (S) begins, thereby preventing re-initiation until the next cell cycle (Coverley, 2002).

Mitosis is thought to be triggered by the activation of Cdk-cyclin complexes 1 and 2. RNA interference (RNAi) was used to assess the roles of three mitotic cyclins, cyclins A2, B1, and B2, in the regulation of centrosome separation and nuclear-envelope breakdown (NEB) in HeLa cells. It was found that the timing of NEB was affected very little by knocking down cyclins B1 and B2 alone or in combination. However, knocking down cyclin A2 markedly delayed NEB, and knocking down both cyclins A2 and B1 delayed NEB further. The timing of cyclin B1-Cdk1 activation was normal in cyclin A2 knockdown cells, and there was no delay in centrosome separation, an event apparently controlled by the activation of cytoplasmic cyclin B1-Cdk1. However, nuclear accumulation of cyclin B1-Cdk1 was markedly delayed in cyclin A2 knockdown cells. Finally, a constitutively nuclear cyclin B1, but not wild-type cyclin B1, restored normal NEB timing in cyclin A2 knockdown cells. These findings show that cyclin A2 is required for timely NEB, whereas cyclins B1 and B2 are not. Nevertheless cyclin B1 translocates to the nucleus just prior to NEB in a cyclin A2-dependent fashion and is capable of supporting NEB if rendered constitutively nuclear (Gong, 2007).

Although the functional analysis of cyclins in animal cells stretches back nearly two decades, as yet no simple consensus has emerged on which cyclins are important for mitosis. In Xenopus egg extracts, cyclins B1 and B2 appear to redundantly drive NEB; it is less clear whether an A-type cyclin is required. In Drosophila embryos, cyclins B and B3 appear to play redundant roles in NEB. Cyclin A is required as well, although recent work suggests that this is because cyclin A is required for the inactivation of Cdh1 and the accumulation of cyclin B, cyclin B3, and Cdc25, rather than because of a direct role in mitosis. The situation may be different in HeLa cells, since cyclin B1 and B2 levels are not low in cyclin A2 knockdown cells, as would be expected if cyclin A2 were required to suppress Cdh1 activation in G2 phase or prophase. Nevertheless, these studies underscore the importance of cyclin A2 in NEB in HeLa cells, in part through regulating the localization of cyclin B1 and in part through cyclin B1/B2-independent effects (Gong, 2007 and references therein).