Identification of the nuclear localization signal in Xenopus cyclin E and analysis of its role in replication and mitosis.
Cyclin-dependent kinase (Cdk)2/cyclin E is imported into nuclei assembled in Xenopus egg extracts by a pathway that requires importin-alpha and -beta. Here, we identify a basic nuclear localization sequence (NLS) in the N-terminus of Xenopus cyclin E. Mutation of the NLS eliminated nuclear accumulation of both cyclin E and Cdk2, and such versions of cyclin E were unable to trigger DNA replication. Addition of a heterologous NLS from SV40 large T antigen restored both nuclear targeting of Cdk2/cyclin E and DNA replication. We present evidence indicating that Cdk2/cyclin E complexes must become highly concentrated within nuclei to support replication and find that cyclin A can trigger replication at much lower intranuclear concentrations. We confirmed that depletion of endogenous cyclin E increases the concentration of cyclin B necessary to promote entry into mitosis. In contrast to its inability to promote DNA replication, cyclin E lacking its NLS was able to cooperate with cyclin B in promoting mitotic entry.
Moore
Jonathan D
JD
Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, Herts, EN6 3LD, United Kingdom.
Kornbluth
Sally
S
Hunt
Tim
T
D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D000818:Animals; D042846:CDC2-CDC28 Kinases; D002467:Cell Nucleus / Q000378:metabolism; D019927:Cyclin E / Q000235:genetics* / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004261:DNA Replication; D004305:Dose-Response Relationship, Drug; D005982:Glutathione Transferase / Q000378:metabolism; D008938:Mitosis; D008969:Molecular Sequence Data; D009154:Mutation; D019913:Nuclear Localization Signals / Q000096:biosynthesis*; D010957:Plasmids / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D014981:Xenopus / Q000378:metabolism*; D029867:Xenopus Proteins
D019927:Cyclin E; D019913:Nuclear Localization Signals; D029867:Xenopus Proteins; D005982:Glutathione Transferase; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495891:Cdk2 protein, Xenopus; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases