Interaction of the S phase regulator cdc18 with cyclin-dependent kinase in fission yeast.
The fission yeast gene cdc18(+) is required for entry into S phase and for coupling mitosis to the successful completion of S phase. Cdc18 is a highly unstable protein that is expressed only once per cell cycle at the G1/S boundary. Overexpression of Cdc18 causes a mitotic delay and reinitiation of DNA replication, suggesting that the inactivation of Cdc18 plays a role in preventing rereplication within a given cell cycle. In this paper, we present evidence that Cdc18 is associated with active cyclin-dependent kinase in vivo. We have expressed Cdc18 as a glutathione S-transferase fusion in fission yeast and demonstrated that the fusion protein is functional in vivo. We find that the Cdc18 fusion protein copurifies with a kinase activity capable of phosphorylating histone H1 and Cdc18. The activity was identified by a variety of methods as the cyclin-dependent kinase containing the product of the cdc2(+) gene. The amino terminus of Cdc18 is required for association with cyclin-dependent kinase, but the association does not require the consensus cyclin-dependent kinase phosphorylation sites in this region. Additionally, both G1/S and mitotic forms of cyclin-dependent kinase phosphorylate and interact with Cdc18. These interactions between Cdc18 and cyclin-dependent kinases suggest mechanisms by which cyclin-dependent kinases could activate the initiation of DNA replication and could prevent rereplication.
Brown
G W
GW
Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Jallepalli
P V
PV
Huneycutt
B J
BJ
Kelly
T J
TJ
D000595:Amino Acid Sequence; D001665:Binding Sites; D016203:CDC2 Protein Kinase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016384:Consensus Sequence; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D005656:Fungal Proteins / Q000378:metabolism; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D016196:S Phase; D012568:Schizosaccharomyces / Q000166:cytology / Q000502:physiology*; D029702:Schizosaccharomyces pombe Proteins
D018797:Cell Cycle Proteins; D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D029702:Schizosaccharomyces pombe Proteins; C082462:cdc18 protein, S pombe; D016203:CDC2 Protein Kinase; D018844:Cyclin-Dependent Kinases