Characterization of the yeast Cdc7p/Dbf4p complex purified from insect cells. Its protein kinase activity is regulated by Rad53p

The yeast Saccharomyces cerevisiae Cdc7p/Dbf4p protein kinase complex was purified to near homogeneity from insect cells. The complex efficiently phosphorylated yeast Mcm2p and less efficiently the remaining Mcm proteins or other replication proteins. Significantly, when pretreated with alkaline pho...

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Bibliographic Details
Published in:The Journal of biological chemistry 2000-11, Vol.275 (45), p.35051-35062
Main Authors: Kihara, M, Nakai, W, Asano, S, Suzuki, A, Kitada, K, Kawasaki, Y, Johnston, L H, Sugino, A
Format: Article
Language:English
Subjects:
Alkaline Phosphatase - pharmacology
Animals
Cdc7 protein
Cell Cycle Proteins - chemistry
Cell Cycle Proteins - genetics
Cell Cycle Proteins - isolation & purification
Cell Line
Checkpoint Kinase 2
Chromosomal Proteins, Non-Histone
Dbf4 gene
Dbf4 protein
Dose-Response Relationship, Drug
Electrophoresis, Polyacrylamide Gel
Enzyme Activation
Fungal Proteins - chemistry
Fungal Proteins - genetics
Fungal Proteins - isolation & purification
Fungal Proteins - metabolism
Insecta
Kinetics
Mcm2 protein
Mutagenesis
Phosphorylation
Plasmids - metabolism
Protein Binding
Protein Conformation
Protein Kinases - metabolism
Protein Kinases - pharmacology
Protein Structure, Tertiary
Protein-Serine-Threonine Kinases - chemistry
Protein-Serine-Threonine Kinases - genetics
Protein-Serine-Threonine Kinases - isolation & purification
Rad53 protein
Recombinant Proteins - chemistry
Recombinant Proteins - metabolism
S Phase
Saccharomyces cerevisiae
Saccharomyces cerevisiae - chemistry
Saccharomyces cerevisiae Proteins
Sodium Chloride - pharmacology
Temperature
Time Factors
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Summary:The yeast Saccharomyces cerevisiae Cdc7p/Dbf4p protein kinase complex was purified to near homogeneity from insect cells. The complex efficiently phosphorylated yeast Mcm2p and less efficiently the remaining Mcm proteins or other replication proteins. Significantly, when pretreated with alkaline phosphatase, Mcm2p became completely inactive as a substrate, suggesting that it must be phosphorylated by other protein kinase(s) to be a substrate for the Cdc7p/Dbf4p complex. Mutant Cdc7p/Dbf4p complexes containing either Cdc7-1p or Dbf4-1 approximately 5p were also partially purified from insect cells and characterized in vitro. Furthermore, the autonomously replicating sequence binding activity of various dbf4 mutants was also analyzed. These studies suggest that the autonomously replicating sequence-binding and Cdc7p protein kinase activation domains of Dbf4p collaborate to form an active Cdc7p/Dbf4p complex and function during S phase in S. cerevisiae. It is shown that Rad53p phosphorylates the Cdc7p/Dbf4p complex in vitro and that this phosphorylation greatly inhibits the kinase activity of Cdc7p/Dbf4p. This result suggests that Rad53p controls the initiation of chromosomal DNA replication by regulating the protein kinase activity associated with the Cdc7p/Dbf4p complex.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M003491200