Activated Polo-Like Kinase Plx1 Is Required at Multiple Points during Mitosis in Xenopus laevis

Abstract

Entry into mitosis depends upon activation of the dual-specificity phosphatase Cdc25C, which dephosphorylates and activates the cyclin B-Cdc2 complex. Previous work has shown that the Xenopus polo-like kinase Plx1 can phosphorylate and activate Cdc25C in vitro. In the work presented here, we demonstrate that Plx1 is activated in vivo during oocyte maturation with the same kinetics as Cdc25C. Microinjection of wild-type Plx1 into Xenopus oocytes accelerated the rate of activation of Cdc25C and cyclin B-Cdc2. Conversely, microinjection of either an antibody against Plx1 or kinase-dead Plx1 significantly inhibited the activation of Cdc25C and cyclin B-Cdc2. This effect could be reversed by injection of active Cdc25C, indicating that Plx1 is upstream of Cdc25C. However, injection of Cdc25C, which directly activates cyclin B-Cdc2, also caused activation of Plx1, suggesting that a positive feedback loop exists in the Plx1 activation pathway. Other experiments show that injection of Plx1 antibody into early embryos, which do not require Cdc25C for the activation of cyclin B-Cdc2, resulted in an arrest of cleavage that was associated with monopolar spindles. These results demonstrate that in Xenopus laevis, Plx1 plays important roles both in the activation of Cdc25C at the initiation of mitosis and in spindle assembly at late stages of mitosis.Progression through the eucaryotic cell cycle is controlled by the activation of several cyclin-dependent kinases (cdks) at specific points in the cycle. To maintain genomic stability, surveillance mechanisms known as checkpoints monitor the completion of essential events to prevent cell cycle progression if DNA is damaged, unreplicated, or improperly assembled on the mitotic spindle (reviewed in references 6 and 20). The biochemistry of cell cycle-dependent checkpoints is best characterized during G 2 phase, when checkpoints determine whether a cell enters mitosis. The cdk that drives the G 2 /M transition, cyclin B-Cdc2, is not activated on schedule if DNA is damaged or if DNA replication is incomplete (2; reviewed in reference 7). Both the DNA damage and DNA replication checkpoints regulate cyclin B-Cdc2 activation in part through the phosphatase Cdc25C. Throughout late S and early G 2 phases, cyclin B is synthesized and immediately complexes with Cdc2, which is kept catalytically inactive by phosphorylation of Tyr15 and Thr14 in the ATP-binding site (12,16,29). This phosphorylation and inactivation is catalyzed by the protein kinases Wee1 and Myt1 (42,45), and dephosphorylation and activation of cyclin B-Cdc2 is catalyzed by the phosphatase Cdc25C (4, 13, 37).Studies on vertebrate Cdc25C have shown that its ability to dephosphorylate cyclin B-Cdc2 and initiate mitosis is regulated by two distinct mechanisms. Activation of Cdc25C requires phosphorylation on specific serine and threonine sites (21,25,31), and this phosphorylation fails to occur if DNA synthesis is incomplete and the replication checkpoint is activated.

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

“…Cdk1 (-Cyclin B) and Plx1 activities are sustained within the Cdc25 amplification loop, which reaches a peak in mitosis (Hoffmann et al, 1993;Kumagai and Dunphy 1996;Abrieu et al,1998;Qian et al, 1998). As we established by monitoring Cdk1 and Plx1, both kinase activities were unaffected by ATM and ATR activation.…”

“…In Xenopus, active Plx1 has been shown to participate in the Cdk1-Cyclin B amplification loop by activating Cdc25 (Kumagai and Dunphy, 1996). At the onset of mitosis, Cdk1-Cyclin B activates Plk1, which suggests Cdk1 and Plk1 mutually sustain one another through the Cdk1-Cyclin B/Cdc25 positive feedback amplification loop (Abrieu et al, 1998;Qian et al, 1998). In addition, both active Cdk1 and Plk1 kinases have been shown to promote Wee1 degradation, thus depleting Cdk1 inhibitory phosphorylation at Thr14 (Watanabe et al, 2004).…”

“…Cdk1-Cyclin B (MPF) functions in mitosis onset and mitotic events, whilst Cdk1 inactivation promotes mitotic exit Nurse et al, 1990;Verde et al, 1990;Fourest-Lieuvin et al, 2006;Glotzer et al, 1991;Sullivan and Morgan, 2007). Plx1 kinase also displays multiple functions in entry, progression and exit from mitosis (Qian et al, 1998;van Yugt and Medema, 2005;Liu et al, 2005). Plx1 and Cdk1-Cyclin B sustain one another through a Cdc25 amplification feedback loop (Hoffmann et al, 1993;Kumagai and Dunphy 1996;Abrieu et al, 1998;Qian et al, 1998).…”

“…Plx1 kinase also displays multiple functions in entry, progression and exit from mitosis (Qian et al, 1998;van Yugt and Medema, 2005;Liu et al, 2005). Plx1 and Cdk1-Cyclin B sustain one another through a Cdc25 amplification feedback loop (Hoffmann et al, 1993;Kumagai and Dunphy 1996;Abrieu et al, 1998;Qian et al, 1998). Cdc25 phosphatase, is essential in initiating the transition from G2 phase to M phase, which is down-regulated by ATM and ATR checkpoint targeting .…”

Ca2+ signaling differentiation during oocyte maturation

“…Cdc25 activation in Xenopus oocytes depends on polo-like kinase (Plx1) through an unknown upstream signal ( Fig. 1; Qian et al, 1998;Nebreda and Ferby, 2000). Interestingly, cdc25 activation during Xenopus oocyte maturation depends on transition metal homeostasis (Sun et al, 2007).…”