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

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 G2 phase, when checkpoints determine whether a cell enters mitosis. The cdk that drives the G2/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 G2 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. It has also been suggested that the rate of phosphorylation of Cdc2 on tyrosine is increased when DNA replication is incomplete (52). The other Cdc25C regulatory mechanism is implicated in the G2 DNA damage checkpoint and involves activation of the kinase Chk1 to phosphorylate Cdc25C at a specific site, an event that promotes the binding of 14-3-3 proteins (10, 47, 51). Although this binding does not directly inhibit the phosphatase activity of Cdc25C in vitro, the proximity of the 14-3-3 binding site to a nuclear localization signal suggests that it may affect the colocalization of Cdc25C with its cdk substrate in the nucleus (10, 47, 51). To better understand the DNA replication checkpoint, it is necessary to define the phosphorylation pathway by which Cdc25C becomes activated at the G2/M transition. Cyclin B-Cdc2 itself is able to phosphorylate Cdc25C at the activating sites, forming a positive feedback loop that contributes to the abrupt transition from G2 into M phase (23). However, a variety of evidence indicates that initial phosphorylation of Cdc25C at the G2/M transition occurs prior to cyclin B-Cdc2 activation, and full phosphorylation and activation of Cdc25C can be obtained in microcystin-treated egg extracts devoid of Cdc2 and Cdk2 (24). This has focused attention on the identification of other protein kinases that might function as “trigger” kinases for Cdc25C activation and might be subject to inhibition when the DNA replication checkpoint is activated. Recently, it was reported that the Xenopus polo-like kinase (plk), Plx1, can phosphorylate and activate Cdc25C in vitro (30). However, it is not known if Plx1 activity increases during the Xenopus cell cycle or if its activity is required for Cdc25C activation in vivo. Genetic studies with Drosophila melanogaster, Saccharomyces cerevisiae, and Schizosaccharomyces pombe implicate plk activity in centrosome functions, spindle assembly, and cytokinesis events, and mutations in plks usually correlate with defects in spindles or septum formation (39, 44; reviewed in references 14 and 33). These phenotypes are more consistent with a kinase that functions late in mitosis rather than during the initial stages of mitosis at the G2/M transition, and indeed, in D. melanogaster, polo activity peaks at the late-anaphase/telophase border, later than cyclin B-Cdc2 activity (8). In contrast, in mammalian cells, Plk1 activity peaks in concert with that of cyclin B-Cdc2 at the onset of mitosis (15, 17, 36). In these cells, Plk1 colocalizes and interacts with various components of the mitotic spindle apparatus, and moreover, microinjection of an antibody to Plk1 blocks cells in G2 or in a psuedomitotic state, with monopolar spindles (15, 32, 36). These considerations make it imperative to determine whether Plx1 activity is regulated during the Xenopus cell cycle and whether it is required for Cdc25C activation and spindle assembly events during mitosis.