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.

We describe a cell-free system from HeLa cells that initiates DNA replication under cell cycle control. G1 but not G2 phase nuclei initiate replication when coincubated with S phase nuclei in cytosolic extracts from S phase but not from G1 or G2 phase HeLa cells. S phase nuclei or an S phase nuclear extract are required for the initiation of semiconservative DNA replication in G1 nuclei but not for elongation in S phase nuclei. S phase nuclear extract could be replaced by recombinant human cyclins A and E complexed to Cdk2 but not by Cdk2 alone or by human cyclin B1 complexed to Cdc2. In S phase cytosol, cyclins A/Cdk2 and E/Cdk2 triggered initiation synergistically.