Abstract. Cyclin-dependent kinase complexes that contain the same catalytic subunit are able to induce different events at different times during the cell cycle, but the mechanisms by which they do so remain largely unknown. To address this problem, we have used affinity chromatography to identify proteins that bind specifically to mitotic cyclins, with the goal of finding proteins that interact with mitotic cyclins to carry out the events of mitosis. This approach has led to the identification of a 60-kD protein called NAP1 that interacts specifically with members of the cyclin B family. This interaction has been highly conserved during evolution: NAP1 in the Xenopus embryo interacts with cyclins B1 and B2, but not with cyclin A, and the S. cerevisiae homolog of NAP1 interacts with Clb2 but not with Clb3.Genetic experiments in budding yeast indicate that NAP1 plays an important role in the function of Clb2, while biochemical experiments demonstrate that purified NAP1 can be phosphorylated by cyclin B/p34 ~c2 kinase complexes, but not by cyclin A/p34 cd~2 kinase complexes. These results suggest that NAP1 is a protein involved in the specific functions of cyclin B/p34 cdc2 kinase complexes. In addition to NAP1, we found a 43-kD protein in Xenopus that is homologous to NAP1 and also interacts specifically with B-type cyclins. This protein is the Xenopus homolog of the human SET protein, which was previously identified as part of a putative oncogenic fusion protein (Von Lindern et al., 1992).
pASSAgE through the eukaryotic cell cycle is controlled by the activity of protein kinase complexes that are composed of an activating subunit (a cyclin) and a catalytic subunit (a cyclin-dependent kinase [Cdk]l; for reviews see Murray and Hunt, 1993;Norbury and Nurse, 1992). Cyclins were originally identified as proteins that fluctuate in abundance during the cell cycle (Evans et al., 1983), while cyclin-dependent kinases were identified genetically as the products of genes required for cell cycle progression in yeasts (the CDC28 gene in S. cerevisiae and the homologous cdc2 gene in S. pombe) (Hartwell et al., 1974;Nurse and Thuriaux, 1980). An active cyclin-dependent kinase complex was first identified biochemically as purified maturation promoting factor (MPF), an activity from Xenopus eggs that can drive cells into mitosis and meiosis (Dunphy et al., 1988;Gautier et al., 1988;Lohka et al., 1988 As of August 1995, the address of D. R. Kellogg will be the Department of Biology, Sinsheimer Laboratories, University of California, Santa Cruz, CA 95064.
Abbreviations used in this paper:Cdk, cyclin-dependent kinase; GST, glutathione-S-transferase; LB, Luria-Beltrani media; NAP1, nucleosome assembly protein 1.The cell cycle of the budding yeast S. cerevisiae is driven by a single cyclin-dependent kinase called p34 Cotes, which is activated during G1 by association with members of the G1 class of cyclins (encoded by three CLN genes), and during S phase and mitosis by association with the B-type cyclins (encoded by 6 CLB genes) (Ep...