Active cyclin B1–Cdk1 first appears on centrosomes in prophase

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.

An ATM- and ATR-dependent checkpoint inactivates spindle assembly by targeting CEP63

“…An increasing number of reports also discuss the potential compartmentalisation of proteins in the context of DNA damage responses Löffer et al, 2006). Centrosomes are becoming key structures of interest, containing an abundance of mitotic control and DNA damage response factors (Takada et al, 2003;Löffer et al, 2006;Jackman et al, 2003;Golsteyn et al, 1995;Tsvetkov et al, 2003). This thesis shows evidence correlating with previous investigations in other systems, in which centrosomes were inactivated as the end-result of DNA damage induced pathways in mitosis (Sibon et al, 2000;Sibon, 2003;Takada et al, 2003;Löffer et al, 2006).…”

“…Furthermore, this proposed model for inducing mitotic arrest provides a possible explanation to why Plk1, Cdk1-Cyclin B and other mitosis regulation factors are also found at centrosomes during mitosis (Jackman et al, 2003;Golsteyn et al, 1995;Tsvetkov et al, 2003). The two posited mitotic arrests pathways orchestrated by centrosomal checkpoint signalling in response to DNA damage are depicted below in figure 1.8.…”

“…Whereas, other non-centrosomal proteins involved in spindle assembly associate only temporary to centrosomes during mitosis, such as Nuclear Mitotic Apparatus protein (NuMA), which has multiple functions in spindle assembly including cross-linking microtubules (bundling) into the correct organization (Sun and Schatten, 2006). Particularly noteworthy is the presence of Cdk1-Cyclin B, Plk1 (Polo-like kinase-1) and Aurora A mitotic cell cycle regulatory proteins within mitotic centrosomes (Jackman et al, 2003;Golsteyn et al, 1995;Tsvetkov et al, 2003;Barr and Gergely 2007). These centrosomal factors play important roles in spindle formation.…”

“…Centrosomal Cdk1-Cyclin B activation has a role in the process of centrosome separation (Blangy et al, 1997;Jackman et al, 2003;Lindqvist et al, 2005). Interestingly, Kramer et al, determined that Cdk1 is prevented from premature activation by Chk1 at centrosomes (Kramer et al, 2004).…”

Polo-like kinase isoform expression is a prognostic factor in ovarian carcinoma

“…This isoenzyme is able to activate CDC25c which in turn activates the CDC2/Cyclin B1 complex leading to the import of Cyclin B1 into the nucleus (Toyoshima-Morimoto et al, 2002). Moreover, a recent study suggests that PLK1 is able to phosphorylate Cyclin B1 (Jackman et al, 2003) directly, and that specific phosphorylation cooperated with other signals in the nuclear import of Cyclin protein, a crucial checkpoint for the initiation of mitosis (Yuan et al, 2002). Later in mitosis, PLK1 has been implicated in the regulation of the anaphasepromoting complex (Kotani et al, 1998;Golan et al, 2002), centrosome maturation and destructions of cohesins (Sumara et al, 2002).…”