If not by checkpoints or surveillance mechanisms, how is the ordering of the cell cycle achieved? An important hint came with the discovery of G1 cyclins in budding yeast, Cln1–Cln3, that are required for entry into the cell cycle at the G1–S transition [32
], followed by the identification of distinct budding yeast cyclins required for mitosis, Clb1–Clb4, more closely related to the previously known metazoan mitotic cyclins [16
]. A third class of cyclins (Clb5 and Clb6) appear at, and are required for, the timely onset of DNA replication in S phase [36
]. This suggested that different cyclins in the same organism might act at different times to promote sequential cell cycle events. Investigation of the transcriptional control of cyclin expression provided a compelling model for how the cell cycle switches from a G1 stage, dominated by G1 cyclins that maintain their own synthesis and promote expression of S-phase and mitotic cyclins, towards mitosis when mitotic cyclins repress G1 cyclins () [38
]. Can the thereby generated alternating cyclin waves explain the ordering of the cell cycle? i.e. can only G1 cyclins prime Cdk to phosphorylate proteins that trigger the Start transition and do S-phase cyclins target molecules that initiate DNA replication? Do mitotic cyclins in turn provide specificity for entry into mitosis?
Figure 2. A model for how cyclin specificity orders S phase and mitosis in budding yeast. In this model, G1 and S-phase cyclins (Cln1,2 and Clb5,6) promote S phase, while mitosis is triggered by the mitotic cyclins Clb1,2. G1 cyclins and mitotic cyclins maintain (more ...)
Cyclin subunits are essential adaptors of Cdks that activate the kinase and target it to its substrates. Cyclins are evolutionarily derived from a common ancestor, but G1 cyclins appear sufficiently diverged to make it plausible that they convey a different substrate specificity from S-phase or mitotic cyclins. This idea has been biochemically confirmed in the case of a few G1 cyclin substrates [39
]. The understanding of substrate recognition received a boost from crystal structures of the human S-phase cyclin A–Cdk2 complex, bound to the stoichiometric Cdk inhibitor p27 or a fragment of its substrate p107 [40
]. These structures not only explained the Cdk consensus S/T-P-x-K/R recognition motif. Together with biochemical analyses, they also identified an RxL peptide motif on p27 and Cdk substrates, that is recognized by a hydrophobic patch on the S-phase cyclin [42
]. A similar patch is found on G1 cyclins, but is not present in the same shape on mitotic cyclins [43
]. Following the large-scale identification of Cdk substrates in budding yeast [46
], the role of the hydrophobic patch in providing S-phase Cdk substrate recognition was strikingly confirmed [47
]. This suggests the RxL motif as a means by which S-phase cyclins recognize specific substrates.
With a rationale for cyclin-specific substrate recognition in hand, is it true that S-phase cyclins are required to trigger S phase? We now know the two crucial Cdk targets whose phosphorylation initiates DNA replication in budding yeast, Sld2 and Sld3 [48
]. Sld2 is indeed a preferred substrate for Clb5/Cdk, but Sld3 is equally well phosphorylated by the mitotic Clb2/Cdk [47
]. A more rigorous test for the importance of S-phase cyclins comes from analyses of cells lacking them. Budding yeast deleted for clb5
, and its close paralogue clb6
, are viable but show a delay in S phase [37
]. Advanced expression of mitotic Clb2 under control of the Clb5 promoter cannot rescue the delay, which has been taken as an indication that timely DNA replication requires specific substrate phosphorylation by S-phase cyclins [50
]. On closer inspection, deletion of the Cdk inhibitory kinase Swe1 in the Clb5 promoter-Clb2 strain was found to restore normal S-phase timing [51
]. The reason why Clb2 was slow in triggering S phase therefore turned out to be the stronger negative regulation of Clb2/Cdk, compared with Clb5/Cdk, by Swe1. Once this is corrected, the mitotic cyclin Clb2 is proficient in promoting S phase, and indeed in phosphorylating Sld2 in vivo
, with normal kinetics [51
]. A similar conclusion was reached from experiments replacing Xenopus
S-phase cyclin A with the mitotic cyclin B in a cell-free extract system that recapitulates cell cycle progression. Cyclin B is normally excluded from interphase nuclei, but removal of its nuclear export signal allowed nuclear accumulation. This change was sufficient for cyclin B to initiate S phase as efficiently as cyclin A would have achieved [52
]. Therefore, the ability to trigger S phase is not restricted to S-phase cyclins. Mitotic cyclins are capable of initiating both S phase as well as mitosis, and they do so in the correct order. A more detailed analysis of DNA replication without S-phase cyclins is warranted to discern the possible advantages of the substrate specificity endowed to S-phase cyclins by its RxL recognition motif.
Numerous experiments to delete or replace individual cyclins, or combinations thereof, have been meanwhile performed in various organisms. A few key findings are summarized in [65
]. This shows that the function of most G1 and S-phase cyclins is dispensable for ordered cell cycle progression, or can be made dispensable by compensatory changes in the cell cycle machinery. In contrast, mitotic cyclins are essential. This suggests that the function of G1 and S-phase cyclins can be taken over by mitotic cyclins, but not the other way around. S-phase cyclins cannot substitute for mitotic cyclins even at elevated levels [55
]. Mitotic cyclins thus appear to be the more generic Cdk activators, with G1 and S-phase cyclins having taken on more specific roles. This is also supported by phylogenetic analyses of cyclins, which place mitotic cyclins at the root of the cyclin tree with S-phase and G1 cyclins being younger derivatives [5
]. An example to illustrate this relationship are the budding yeast G1 cyclins. At least one of the three G1 cyclins (Cln1–Cln3) is usually required for cell proliferation [33
]. However, they all become dispensable in cells expressing an ectopic source of S-phase cyclins, or if the stoichiometric Cdk inhibitor Sic1 is removed [36
]. Sic1 is part of the mechanism by which mitotic Cdk is downregulated during exit from mitosis. In contrast, Sic1 is a poor inhibitor of G1 cyclins [55
]. G1 cyclins are, therefore, destined to overcome Sic1 and turn on the expression of S-phase cyclins during entry into the next cell cycle. In the absence of Sic1, or if S-phase cyclins are expressed from an independent source, G1 cyclins are no longer required. Cells lacking all G1 cyclins and Sic1 are viable, suggesting that G1 cyclin specificity is not essential to achieve ordering of cell cycle progression. Cells lacking Sic1, however, are compromised in maintaining a stable G1 arrest, e.g. in preparation for mating. Thus, cell cycle exit, as part of cellular differentiation, which is promoted by Cdk inhibitors, created a requirement for G1 cyclins that overcome these Cdk inhibitors.
Cyclin gene deletions and their phenotypes. MEFs, mouse embryonic fibroblasts.
In mouse models, numerous cyclins can be deleted with only mild consequences on organismal development and only the major mitotic cyclin B1 is essential for early embryonic cell divisions (). Again, most cyclins appear dispensable for the ordering of cell cycle progression. Instead, some of them have taken on specific roles at particular developmental stages. For example, the S-phase cyclins E1 and E2 together are essential for embryonic development. This is not because of a requirement for cell cycle progression, as cells derived from embryos lacking both cyclins proliferate well in culture. Rather, cyclins E1 and E2 are required to promote the endoreduplication cycles leading to the highly polyploid giant trophoblast nuclei in the placenta. Chimaeric embryos—in which the extra-embryonal cells that give rise to the placenta are wild-type, while the embryo proper lacks both theses cyclins—can be derived. Development is strikingly restored in these cyclin E1- and E2-deficient embryos by the wild-type placenta, with residual cardiovascular abnormalities [60
In addition to distinct cyclins, vertebrates also encode a number of different catalytic Cdk subunits, including Cdk1, Cdk2, Cdk3, Cdk4 and Cdk6. Depending on their time of activation by the Cdk-activating kinase (CAK), they preferentially associate with the cyclins present at the respective cell cycle stage [67
]. Ablation of any of the Cdks, other than the major mitotic Cdk1, has little effect on cell cycle progression and mouse embryonic development. Even simultaneous deletion of Cdk2, Cdk3, Cdk4 and Cdk6, leaving behind only Cdk1, causes only mild delays to cell cycle progression. Cdk1 can substitute for all the other Cdks in these cells and even supports apparently normal mouse embryonic development until mid-gestation. Only after that, a specific requirement of Cdk4 and 6 for haematopoiesis leads to embryonic death owing to anaemia [68
]. Taken together, one Cdk subunit is sufficient for setting up orderly cell cycle progression, although individual Cdks have evolved specific functions that they fulfil during certain developmental processes. The molecular nature of these specific functions remains to be elucidated.