Regulation of the cell cycle timing of mitosis

Two classes of mutants have been used to investigate mitotic regulation in fission yeast. The first class are the cdc (cell division cycle) type, which are conditionally defective in functions required for mitosis (Nurse et al. 1976). These can be heat- or cold-sensitive, and on incubation at the restrictive temperature become arrested in cell cycle progress either in late G₂ or during the process of mitosis. The second class are of the wee type which result in mitosis being initiated earlier in the cell cycle than normal (Nurse, 1975). These mutants undergo mitosis normally but prematurely, and generate cells that divide at a small size, accounting for the name wee. It is this second class of mutants that provided the key for unravelling mitotic regulation. Only gene functions which influence the cell cycle timing of mitosis can mutate to generate the wee phenotype. This can be best understood if it is imagined that the events leading to mitotic initiation are variable in length; those events which take a long time to complete contribute most to determining the cell cycle timing of mitosis (Nurse, 1981). If such major rate-limiting steps are speeded up by mutation then initiation of mitosis will be advanced. Because growth rate remains normal the cell initiates mitosis before it grows to the usual cell size and this results in premature division at a reduced size. In contrast, events which are completed rapidly contribute little to the cell cycle timing of mitosis, and so cannot be speeded up significantly by mutation. Therefore the genes required for these events cannot generate the wee mutant phenotype. Genes involved in both rate-limiting and non-rate-limiting events can be mutated so the events take longer to be completed. If the event is never completed then cells become arrested in cell cycle progress and generate the cdc mutant phenotype.

Four genes have been identified which can be mutated to the wee phenotype. The first to be described was weel⁺ (Nurse, 1975); classical genetic analysis indicated that loss of weel⁺ function led to mitotic advancement (Nurse and Thuriaux, 1980) and this conclusion was later confirmed by molecular genetic analysis (Russell and Nurse, 1987a). The second gene identified was cdc2⁺, which could be mutated to yield both cdc and wee mutant phenotypes (Nurse and Thuriaux, 1980). Analysis demonstrated that loss of cdc2⁺ function prevented mitotic initiation whilst altered cdc2⁺ function led to mitotic advancement. Two further genes, cdc25⁺ and niml ⁺, were found to generate the wee phenotype if present in several copies in the cell (Russell and Nurse, 1986, 19876). This led to them being described as gene dosagedependent inducers of mitosis, in contrast to weel ⁺, which behaved as a gene dosage-dependent inhibitor. A series of experiments carried out using combinations of these mutants led to the proposal that the four gene functions were organised in a regulatory network (Fantes, 1979; Russell and Nurse, 1986, 1987a,b). The most important feature of this model is that activation of the cdc2⁺ gene function leads to mitotic initiation. Activation is regulated by two pathways, one consisting of cdc25⁺and the second consisting of vueel ⁺ and niml ⁺. The weel⁺ /niml⁺ pathway inhibits cdc2⁺ function, whilst cdc25⁺ acts positively and as a consequence leads to cdc2⁺activation. All four genes operating together determine the cell cycle timing of mitosis.

The cloning of these genes by complementation of the appropriate mutants enabled the molecular mechanisms underlying mitotic initiation to be investigated. Sequencing of cdc2⁺, weel⁺ and niml ⁺ established that they encode protein kinase homologues (Hindley and Phear, 1984; Russell and Nurse, 1987a,b). Use of antibodies raised against the cdc2⁺ gene product demonstrated that cdc2⁺ did indeed encode a 34K (K=10³M_r) protein kinase whose activity was correlated with cell proliferation (Simanis and Nurse, 1986). Assays of p34^cdc2 protein kinase activity through the cell cycle using Hl histone as the in vitro substrate have shown that it is present at low levels during interphase and increases about four- or five-fold on entry into mitosis (Moreno et al. 1989). Activity is high during metaphase and then drops during anaphase-telophase to a low level as cells enter interphase. The cdc25⁺ gene product is required for the activation which occurs on entry into mitosis. p34^cdc2 protein kinase activity remains low in cdc25⁺ mutants but immediately increases when cdc25⁺ function is restored to the cells. Therefore cdc25⁺ appears to regulate the activation of p34^cdc2 protein kinase, although the precise molecular mechanism of activation remains unclear. The weel⁺/niml⁺ pathway is likely to involve an inhibitory phosphorylation signal mediated by the putative weel ⁺ protein kinase. We imagine that weel⁺ inhibits p34^cdc2 function at mitosis by phosphorylation, and a reduction in p34^cdc2 phosphorylation has recently been reported to take place at mitosis (K. Gould, personal communication).

Two further gene products play a role in regulating p34^cdc2 protein kinase activity during mitosis. The first of these is sucl ⁺, a gene identified initially because over expression of sucl⁺ gene transcripts can suppress the deleterious effects of certain cdc2^ts alleles (Hayles et al. 1986a,b; Hindley et al. 1987). These observations led to the proposal that the sucl⁺ and cdc2⁺ gene products interact, a suggestion subsequently confirmed when it was shown that pl3^sucl physically associates with p34^cdc2 (Brizuela et al. 1987). Deletion of the sucl⁺ gene leads to an interesting terminal phenotype (Moreno et al. 1989). Cells arrest with condensed chromosomes and an elongated spindle characteristic of the anaphase-telophase transition. This result indicates that pl3^sucl is required at a late stage in mitosis. The p34^cdc2 protein kinase activity also remains at a high level in these cells. Because of the close interactions between the two proteins we have suggested that pl3^sucl plays a role in the inactivation of the p34^cdc2 kinase at the end of mitosis. In the absence of pl3^sucl the p34^cdc2 kinase is not inactivated and cells cannot leave mitosis. This view also explains why pl3^sucl causes delay of mitotic initiation as has been observed in cells containing elevated levels of pl3^sucl. An enhanced level of the p34^cdc2 kinase inactivation mechanism could compete with the activation process in late G2 leading to mitotic delay.

The second gene involved in regulating p34^cdc2 protein kinase activity is cdc13⁺(Booher and Beach, 1988; Hagan et al. 1988). This gene product was shown to interact in some way with p34^cdc2 by the demonstration that over expression of p34^cdc2 allows a cdcl3^t!i mutant to grow at its restrictive temperature (Booher and Beach, 1987). The gene is also interesting because the temperature sensitive allele cdcl3-\VI arrests with a terminal phenotype characteristic of both G2 and mitosis. Cells arrest with condensed chromosomes like cells in metaphase but with an interphase cytosplasmic array of microtubules typical of G2 (Hagan et al. 1988). Analysis has suggested that cdc13⁺ is required at two stages during mitosis. When cdc13⁺ is deleted from cells they block in late G2 with low p34^cdc2 protein kinase activity. In cdcl3-ÏVl there is only a partial loss of function and cells can activate the p34^cdc2 protein kinase and initiate mitosis. But entry into mitosis is only partial in this mutant; chromosomes become condensed but the mitotic spindle cannot be generated and the interphase cytoplasmic array of microtubules persists. Thus cdcl3⁺ is required for p34^cdc2 protein kinase activation and then again at a later stage after activation, leading to generation of the mitotic spindle.

These studies with fission yeast indicate that the cell cycle timing of mitosis is regulated by p34^cdc2 protein kinase activation via an upstream regulatory gene network involving cdc25⁺. We presume that a number of substrates are phosphorylated by this kinase which brings about the two major pathways of mitosis, chromosome condensation and reorganisation of the microtubular cytoskeleton into a mitotic spindle. The cdcl3⁺ gene product plays a role in both activating p34^cdc2 kinase activity and in its later function. The molecular mechanisms involved are not known but possibly the cdc13⁺ gene product is required to facilitate interaction with both the activating process at mitotic initiation and then with its various substrates later during mitosis. Exit from mitosis and re-entry into interphase requires p34^cdc2 kinase inactivation brought about by pl3^sucl. This view of p34^cdc2 regulation of mitosis is shown in Fig. 1.

Basic elements in this mitotic control system are conserved in all eukaryotic cells. This was first indicated when the human homologue was cloned by complementation of a defective cdc2^ts mutant (Lee and Nurse, 1987). The human CZ)C2Hs gene can completely substitute for the fission yeast cdc2⁺ gene. The predicted amino acid sequence of the human gene product was found to be 63 % identical with the fission yeast product. A separate study using p34^cdc2 and pl3^sueI antibodies demonstrated that these two proteins were present in human cells and that p34^cdc2 had protein kinase activity (Draetta et al. 1987; Draetta and Beach, 1988). Expression of the CDC2Hs gene was also correlated with cell proliferation (Lee et al. 1988). In addition to cdc2⁺ and sucl ⁺, genes sharing sequence similarities with cdc;⁺ and cdc25⁺ have also been identified (Goebl and Byers, 1988; Solomon et al. 1988; Hagan et al. 1988; Russell et al. 1989).

These genes are important for regulating entry into M phase in multicellular eukaryotic organisms. A factor called maturation-promoting factor (MPF) has been described in both Xenopus and starfish which induces M phase when injected into oocytes arrested just before meiosis. MPF activity oscillates during mitotic cell cycles peaking in level just as cells enter mitosis. MPF has been highly purified and activity found to be associated with two proteins fromXenopus and one protein from starfish (Lohka et al. 1988; Labbe et al. 1988a). Both purified preparations contain a protein equivalent to p34^cdc2 that is recognised by anti-p34^cdc2 antibodies (Gautier et al. 1988; Labbé et al. 19886). Thus MPF contains the p34^cdc2 protein kinase. It should be noted that M phase-inducing activity is the biochemical analogue of the wee mutant phenotype defined as genetics. In both situations the treatment leads to advance cells into mitosis. Further support for the notion that MPF contains p34^cdc2 has been obtained by passing Xenopus and starfish extracts through a pl3^sucl column. This depletes the extracts of p34^cdc2 and also removes the MPF activity (Dunphy et al. 1988; Arion et al. 1988).

A second class of components clearly implicated in M phase control in multicellular eukaryotes are the cyclins (Evans et al. 1983; Swenson et al. 1986; Standart et al. 1987; Minshull et al. 1989). These are proteins which oscillate in level, peaking at M phase. Injection of cyclin message into oocytes results in their entry into M phase, similar to the effects induced by MPF. Cyclins have been identified in a number of organisms and all share sequence similarities over a central region of 150 amino acids. The cdcI3⁺ gene is also about 50 % identical over the same region indicating that it may be a functional homologue of cyclins (Goebl and Byers, 1988; Solomon et al. 1988; Hagan et al. 1988), just as cdc2⁺ is a functional homologue of the 34K component of purified MPF. Since cdcl3⁺ is required for activation of the p34^cdc2 protein kinase, injection of cyclin mRNA may induce M phase in oocytes by allowing p34^cdc2 protein kinase activation.

A third gene implicated in M phase control in multicellular eukaryotes is the Drosophila gene string, which shares some similarities in sequence with cdc25⁺(Edgar and O’Farrell, 1989). Deletion of this gene leads to arrest of cell division in Drosophila embryos, a phenotype similar to that observed when the cdc25⁺ function is blocked in fission yeast.

Biochemical investigation of p34^cdc2 function in multicellular eukaryotes indicates that the molecular mechanism of mitotic regulation may also be very similar to that found in fission yeast. The p34^cdc2 kinase activity is periodic, peaking in level at M phase in mammalian cells, starfish and Xenopus oocytes and eggs (Draetta and Beach, 1988; Arion et al. 1988; Labbé et al. 1989). Regulation involves phosphorylation; in both starfish and Xenopus, p34^cdc2 is phosphorylated during interphase and upon entry into M phase the protein becomes dephosphorylated co-incident with activation of protein kinase activity (Labbé et al. 1989). This is consistent with the suggestion in fission yeast that the weel⁺ putative protein kinase inhibits p34^cdc2 function by phosphorylation. In all these organisms the preferred substrate for the p34^cdc2 kinase activity is Hl histone. The major Hl histone kinase in the mammalian and Physarum cells is the so-called growth-associated Hl histone kinase. Over 15 years ago this activity was described to vary in level during the cell cycle, peaking at M phase co-incident with hyperphosphorylation of Hl histones in vivo during mitosis (Bradbury et al. 1974). The authors of this earlier work went on to add partially purified Hl histone protein kinase to Physarum and demonstrated some advancement of mitosis. This result led to the prophetic suggestion that the cell cycle timing of mitosis might be regulated by the H1 histone kinase bringing about entry into mitosis. Although this early work was not pursued because of problems in obtaining purified kinase, it accurately anticipated what has now been established.

The experiments described above and elsewhere in this volume have not only established links between the controls operative in multicellular eukaryotes and unicellular eukaryotes like fission yeast but in addition they open up new avenues for future analysis. It should also be possible to exploit fission yeast for carrying out genetic studies on the mammalian genes. The functionally equivalent mammalian genes can be used to replace the fission yeast homologue and its interactions with other yeast genes such as cdcl3⁺, cdc25⁺, sucl ⁺ and weel ⁺ can be described. These experiments should be informative about which of these interactions can be expected to be conserved in mammalian cells. They will also allow dominant acting mutants of mammalian genes to be isolated in yeast which can then be transferred back to mammalian cells to test if the effects are similar, in the two organisms. Isolation of dominant negative mutants may even allow investigators to knock out functions in mammalian cells. Xenopus and starfish eggs are ideal for biochemical analysis and for the development of in vitro systems. The precise biochemical mechanisms involved will now be extensively studied in these organisms and it should be possible to construct the regulatory systems occurring during entry into and passage through mitosis using the various in vitro mitotic extracts now available. This combined genetic and biochemical approach should be synergistic and allow a much better description of mitotic regulation than would be possible with either approach alone.