Phosphorylation of MCM4 at sites inactivating DNA helicase activity of the MCM4-MCM6-MCM7 complex during Epstein-Barr virus productive replication.
Induction of Epstein-Barr virus (EBV) lytic replication blocks chromosomal DNA replication notwithstanding an S-phase-like cellular environment with high cyclin-dependent kinase (CDK) activity. We report here that the phosphorylated form of MCM4, a subunit of the MCM complex essential for chromosomal DNA replication, increases with progression of lytic replication, Thr-19 and Thr-110 being CDK2/CDK1 targets whose phosphorylation inactivates MCM4-MCM6-MCM7 (MCM4-6-7) complex-associated DNA helicase. Expression of EBV-encoded protein kinase (EBV-PK) in HeLa cells caused phosphorylation of these sites on MCM4, leading to cell growth arrest. In vitro, the sites of MCM4 of the MCM4-6-7 hexamer were confirmed to be phosphorylated with EBV-PK, with the same loss of helicase activity as with CDK2/cyclin A. Introducing mutations in the N-terminal six Ser and Thr residues of MCM4 reduced the inhibition by CDK2/cyclin A, while EBV-PK inhibited the helicase activities of both wild-type and mutant MCM4-6-7 hexamers, probably since EBV-PK can phosphorylate MCM6 and another site(s) of MCM4 in addition to the N-terminal residues. Therefore, phosphorylation of the MCM complex by redundant actions of CDK and EBV-PK during lytic replication might provide one mechanism to block chromosomal DNA replication in the infected cells through inactivation of DNA unwinding by the MCM4-6-7 complex.
Kudoh
Ayumi
A
Division of Virology, Aichi Cancer Center Research Institute, 1-1 Kanokoden, Chikusa-ku, Nagoya 464-8681, Japan.
Daikoku
Tohru
T
Ishimi
Yukio
Y
Kawaguchi
Yasushi
Y
Shirata
Noriko
N
Iwahori
Satoko
S
Isomura
Hiroki
H
Tsurumi
Tatsuya
T
D019943:Amino Acid Substitution; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002455:Cell Division; D019925:Cyclin A / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2 / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004265:DNA Helicases / Q000037:antagonists & inhibitors*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006367:HeLa Cells; D004854:Herpesvirus 4, Human / Q000502:physiology*; D006801:Humans; D064148:Minichromosome Maintenance Complex Component 4; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D013912:Threonine / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication / Q000502:physiology*
D018797:Cell Cycle Proteins; D019925:Cyclin A; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D014764:Viral Proteins; D013912:Threonine; D004247:DNA; D011494:Protein Kinases; D051357:Cyclin-Dependent Kinase 2; D004265:DNA Helicases; C099243:MCM4 protein, human; D064148:Minichromosome Maintenance Complex Component 4