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The Cdc2 protein kinase requires cyclin binding for activity and also binds to a small protein, Sucl. Charged-to-alanine scanning mutagenesis of Cdc2 was used previously to localize cyclin A-and B-and Sucl-binding sites (B. Ducommun, P. Brambilla, and G. Draetta, Mol. Cell. Biol. 11:6177-6184, 1991 Once phosphorylated, T-161 is predicted to play a major structural role in Cdc2, comparable to that of T-197 in cAPK, by assembling the active conformation required for peptide recognition. The inhibitory phosphorylation at Y-15 also comes close to the cleft interface and on the basis of this model would disrupt the cleft interface and the adjacent peptide recognition site rather than prevent ATP binding. In contrast to cyclin A, both lobes influence Sucl binding; however, the Sucl-binding sites are far from the active site. Several mutants map to the surface in cAPK, which is masked in part by the N-terminal 40 residues that lie outside the conserved catalytic core. The other Sucl-binding site maps to the large lobe near a 25-residue insert and includes R-215.

suc1 is an essential gene initially identified for its ability to rescue certain temperature-sensitive alleles of cdc2 in Schizosaccharomyces pombe. The role of suc1 in the regulation of the cdc2 kinase is not well understood. In our study, we have characterized the biochemical effect of loss of suc1 function on specific cdc2-cyclin complexes. We show that the cig1 cyclin is associated with cdc2 and that the cdc2-cig1 kinase is activated at mitosis, with kinetics similar to those of the cdc2-cdc13 kinase. We provide evidence that loss of suc1 function affects the kinase activity of the two distinct mitotic forms of the cdc2 kinase. We also show that a dramatic increase in the level of the cdc13 protein is associated with loss of suc1. These results suggest that mitosis cannot be properly completed in the absence of suc1, possibly because of an increase in the level of cdc2-cdc13 complex, and support the idea of a role for suc1 in the regulation of multiple forms of the cdc2 kinase.The cdc2 protein kinase is a key regulator of the eukaryotic cell cycle. In the yeasts Saccharomyces pombe and Saccharomyces cerevisiae, the cdc2/CDC28 gene is required for progression through the cell cycle at two transition points, in G 1 before Start and later in the cell cycle at the onset of mitosis (34,36). Increasing evidence indicates that in higher eukaryotes, cell cycle transitions are regulated by a family of cdc2-like kinases, the cyclin-dependent kinases (37,44). Cyclins are proteins which activate the cdk subunits at different phases of the cell cycle. The cdc2 kinase is activated at mitosis by B-type cyclins. In S. cerevisiae, four different cyclins (CLB1 to -4) which activate Cdc28 at mitosis have been identified. Two of these cyclins also function earlier in the cell cycle (18,39). Other cyclins which function in S phase (16,43) and in G 1 (20,40) are known. In fission yeast species, three different B-type cyclin genes have been reported, cdc13 (5, 21), cig1 (9), and cig2 (10). cdc13 is an essential gene, required for entry into mitosis (5). cig1 and cig2 are not essential and are required in late mitosis for nuclear separation (11). A role for cig1 and cig2 during the G 1 /S transition has also been suggested (9, 11; however, see reference 9 erratum). cdc2 also associates with a smaller subunit encoded by the suc1 gene (8, 12). suc1 was identified as a gene capable of rescuing some temperature-sensitive alleles of cdc2 in fission yeast species (23,24). Homologous genes in S. cerevisiae and higher eukaryotes have been identified (19,41). However, despite the experimental efforts, a clear picture has not emerged concerning the function of suc1. Experiments with Xenopus extracts have suggested an inhibitory role for suc1 in the tyrosine dephosphorylation of cdc2. Addition of 2 M suc1 to Xenopus oocyte extracts can effectively inhibit entry into M phase, as monitored by nuclear disassembly (15), cdc2 tyrosine dephosphorylation, and kinase activation (14). The suc1 inhibitory effect can be counteracted by the addition of c...

We have cloned a novel fission yeast gene, spd1, which causes G1 arrest when overexpressed. Deleting the gene results in cells being accelerated through G1 into S phase in certain circumstances when the G1–>S phase control is compromised. We have found that the encoded 14 kDa protein is cell cycle regulated, declining in level during S phase, and that p14spd1 physically associates with p34cdc2 in vivo when overexpressed, suggesting that p14spd1 may regulate S phase progression via an interaction with p34cdc2. We conclude that p14spd1 is a negative regulator of S phase, and that it may be part of the control ensuring an orderly onset of S phase or part of a G1–>S phase checkpoint control.

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