Mitotic control by metaphase-promoting factor and Cdc proteins

Entry into mitosis brings about a dramatic reorganization of both nuclear and cytoplasmic structures in preparation for cell division. In general, three approaches have been taken to study the mechanisms controlling the onset of this reorganization: (1) genetic analysis of mutants, mostly of yeast and other fungi, that are defective in the cell division cycle (cdc mutants); (2) biochemical assays of protein kinases and other enzymes whose activities oscillate with the cell cycle and peak during mitosis; (3) the use of biological assays to test for mitotic inducers in dividing cells. The underlying premise of these approaches, which utilize a variety of different cell types, is that at least some of the mechanisms regulating the transition from G2 to mitosis will prove to be similar in all eukaryotes. Indeed, this premise seems to be correct. Recently published results from each of the approaches have implicated the same proteins, the products of the yeast cdc2/CDC28 genes and their homologues, in the regulation of early mitotic events.In Schizosaccharomyces pombe the cdc2 gene (cdc2(Sp)) encodes a 34 × 103Mr protein kinase (p34) that is required at two points in the cell cycle, at the transitions from G1 to S and from G2 to mitosis (for more detailed reviews, see Nurse, 1985; Hayles Lee Bradbury et al. 1974). The rapid increase in Hl phosphorylation during mitosis has led to the suggestion that GAK activity might be involved in mitotic chromosome condensation (Bradbury et al. 1973), although no conclusive evidence for such a role has been obtained.An Hl kinase, which has peak levels of activity in mitosis, has been purified from starfish eggs (Labbe et al. 1988; Arion et al. 1988). This Hl kinase activity copurifies with a 34 ×103Mr protein and is both immunoprecipitated and recognized on immunoblots by antibodies to p34cdc2, indicating that the mitotic kinase is a starfish homologue of p34cdc2. Conclusive evidence that p34CDC28 is functionally identical to mammalian GAK has recently been obtained (Langan et al. 1988). Lysates of wild-type S. cerevisiae have an Hl kinase activity whose levels are elevated in mitosis, and which phosphorylates histone Hl on the same sites as GAK purified from Novikoff rat hepatoma cells. This Hl kinase is temperature-sensitive in extracts prepared from CDC28 mutants. Moreover, the PSTAIR antibody reacts with a 32–34 (×103) Mr protein in the purified mammalian GAK preparation. Taken together these results indicate that the GAK from starfish, mammals and probably a variety of other cells are homologues of the yeast cdc2 gene product, and are likely to be a part of a conserved mechanism that is involved in the regulation of cell division.Proteins that induce entry into mitosis can be assayed by microinjection into frog or starfish oocytes. Fully grown oocytes of these species are physiologically arrested in prophase I of meiosis (G2) and when treated with the appropriate hormone, such as progesterone for frogs or 1-methyladenine for starfish, resume cell division as they mature into unfertilized eggs (for reviews, see Masui Meüer Smith Kishimoto Kishimoto, 1988). In all of these cells, activity is highest during metaphase and decreases precipitously to undetectable levels at other times. MPF can induce GVBD in the absence of new protein synthesis, suggesting that it is a fundamental component of the mechanism that regulates metaphase in both meiotic and mitotic cells.MPF activity can also be detected by another recently developed method, which utilizes two types of extracts from Xenopus laevis eggs that support in vitro the same nuclear behaviour that occurs in cells from which the extracts are prepared (for review, see Lohka Miake-Lye Adlakha et al. 1985; Nguyen-Gia et al. 1986; Kishimoto Russell Lee Goebl & Byers, 1988), it is probable that homologues of other yeast regulatory genes are also present in all eukaryotes. Analysis of cell cycle mutants in Aspergillus has identified other genes, unrelated to known yeast cdc genes, that act as positive and negative regulators of mitosis (Osani et al. 1988a,b). Whether homologues of these genes exist in other eukaryotes is not known.Another family of proteins that undoubtedly plays a key role in the regulation of mitotic entry is the cyclins. Cyclins are characterized by their steady synthesis and accumulation during the cell cycle until metaphase when, after reaching their highest levels, they are rapidly and specifically degraded as cells enter anaphase (Evans et al. 1983). Injection of cyclin mRNA into immature oocytes results in GVBD (Swenson et al. 1986), and its addition to cell-free extracts induces mitotic entry (Murray et al. 1988). The high degree of sequence conservation between cyclins and cdclJ argues that cyclins, like the product of cdc!3, may interact with p34”/r2 to regulate cell division. The hypothesis proposed by Solomon et al. (1988) that proteins associated with p34, such as p62 in HeLa cells, are related to cyclins is attractive in that it places many of the components thought to be involved in mitotic control in a single multimeric regulatory complex.In conclusion, gene products that function in the control of mitosis in yeast have counterparts in many eukaryotes, including man. One of these proteins, p34 cdc2/CDC28 is funtionally equivalent to the growth-associated Hl kinase activated during mitosis in proliferating cells. Active p34cdc2/CDC28 protein kinase, which is associated with other proteins as part of a high molecular weight complex, may be a component of MPF, although the smallest number of proteins necessary to induce metaphase in each of the MPF assays has not been determined. Whereas these recent results strongly indicate that homologues of yeast cdc proteins function in a highly conserved mechanism for mitotic control, their targets, which are more directly involved in nuclear envelope disassembly, chromosome condensation, spindle formation and other mitotic processes, remain unknown.