Abstract
Cyclin B1–Cdk1 is the key initiator of mitosis, but when and where activation occurs has not been precisely determined in mammalian cells. Activation may occur in the nucleus or cytoplasm, as just before nuclear envelope breakdown, Polo-like kinase1 (Plk1) is proposed to phosphorylate cyclin B1 in its nuclear export sequence (NES), to trigger rapid nuclear import. We raised phospho-specific antibodies against cyclin B1 that primarily recognise the active form of the complex. We show that cyclin B1 is initially phosphorylated on centrosomes in prophase and that Plk1 phosphorylates cyclin B1, but not in the NES. Furthermore, phosphorylation by Plk1 does not cause cyclin B1 to move into the nucleus. We conclude that cyclin B1–Cdk1 is first activated in the cytoplasm and that centrosomes may function as sites of integration for the proteins that trigger mitosis.
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Acknowledgements
We are indebted to C. Broad for help with the cell culture. We are grateful to S. Kornbluth for discussing results before publication, to J. Gannon and T. Hunt for anti-Cdk1 antibodies, and K. Lee and R. Erikson (Havard, USA) for the Plk1 baculovirus. We thank E. Nishida (Kyoto, Japan) for sending us anti-phospho-Ser 147 serum. This work was supported by a Wellcome Trust Training Fellowship to CL, by the Association for International Cancer Research [AICR], and by a programme grant to JP from Cancer Research UK (CRC).
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Supplementary Figures
Figure S1 S126-P antibody staining focuses at centrosomes. (PDF 470 kb)
Figure S2 PLK1 preferentially phosphorylates S133 and not S147 in cyclin B1
Figure S3 Plk1 accumulates in the nucleus at prophase before cyclin B1.
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Jackman, M., Lindon, C., Nigg, E. et al. Active cyclin B1–Cdk1 first appears on centrosomes in prophase. Nat Cell Biol 5, 143–148 (2003). https://doi.org/10.1038/ncb918
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DOI: https://doi.org/10.1038/ncb918
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