Mitotic control by metaphase-promoting factor and Cdc proteins

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Abstract
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 & Nurse, 1986a; Lee & Nurse, 1988). Some temperature-sensitive mutants of cdc2 arrest in either G1 or G2 when cells are incubated at non-permissive temperatures. Others are dominant and cause cells to advance into mitosis precociously, with a reduced cell size and shortened G2 period. These phenotypes indicate that the cdc2 gene product is involved in the mechanisms that control the timing of cell division.The CDC28 gene of the budding yeast, Saccharomyces cerevisiae, also encodes a 34 ×103Mr protein kinase required during G1 (Reed et al. 1985) and possibly again during G2 (Piggott et al. 1982). CDC28 is functionally equivalent to cdc2 in that it can complement mutants of 5. pombe deficient in cdc2 function (Beach et al. 1982), permitting growth at restrictive temperatures. A similar complementation assay has been used to isolate a homologue of cdc2, cdc2(Hs), from a human cDNA library (Lee & Nurse, 1987). The proteins encoded by cdc2(Sp), cdc2(Hs) and CDC28 share approximately 60% amino acid sequence identity between any two of them, including a stretch of 14 amino acids (EGVPSTAIRELLKE), referred to as the PSTAIR sequence, that is perfectly conserved in all. Antisera raised against cdc2(Sp) (Draetta et al. 1987) or against peptides containing the PSTAIR sequence (Lee & Nurse, 1987), or the aminoterminal sequence of CDC28 (Mendenhall et al. 1987), recognize 34 ×103Mr proteins in cells of yeast, humans and a variety of other eukaryotes (see below). Since genes that are functionally equivalent to cdc2 are conserved from yeast to man, their products are likely to play a fundamental role in the regulation of the eukaryotic cell cycle.The exact role of p34 in the initiation of cell division is not yet understood. Entry into mitosis is not triggered by an accumulation of p34 during G2, since its levels do not change appreciably during the cell cycle of S. pombe (Simanis & Nurse, 1986), .S’. cerevisiae (Wittenberg & Reed, 1988) or HeLa cells (Draetta & Beach, 1988). In contrast, the protein kinase activity of p34CDC28and p34cdc2(Hs)Qscillates with the cell cycle, reaching highest levels in G1 for S. cerevisiae (Wittenberg & Reed, 1988) and in late G2 and mitosis for HeLa cells (Draetta & Beach, 1988). Coincident with the increase in protein kinase activity in both species is the formation of a high molecular weight complex of p34 and other proteins (see below). Similar cell cycle-dependent changes are not observed in S. pombe but, instead, the p34 protein kinase activity changes in response to nutritional conditions (Simanis & Nurse, 1986).The onset of mitosis is marked by an increase in the phosphorylation of a large number of intracellular proteins. Although most of the phosphoproteins remain unidentified, lamins (Ottaviano & Gerace, 1985), vimentin (Evans & Fink, 1982), histone Hl (Bradbury et al. 1973, 1974) and histone H3 (Gurley et al. 1974) are known to become highly phosphorylated. The phosphorylation of histone Hl has long been known to accompany cell proliferation, and the protein kinase responsible is called the ‘growth-associated Hl kinase’ (GAK) (Lake & Salzman, 1972). Specific sites in the carboxyl- and amino-terminal regions of the Hl molecule, but not in the globular central region, are phosphorylated by GAK (Langan, 1978). Hl phosphorylation begins in G1 continues through S and G2, and rises sharply in mitosis (Lake & Salzman, 1972; 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...