Mitotic effects of a constitutively active mutant of the Xenopus polo-like kinase Plx1.

Progression through the eucaryotic cell cycle is controlled through the periodic activation or inactivation of various cyclin-dependent protein kinases (cdk’s) at specific points in the cycle (26). The fidelity of events in a given cell cycle phase is monitored by checkpoints, which control a signaling system that can delay cell cycle progression and changes in cdk activity (5, 9). One of the best-understood checkpoints blocks activation of the cyclin B-Cdc2 complex in G2 phase if DNA replication is incomplete (see reference 27 for a review). This block to mitotic entry requires that the phosphatase Cdc25C remain inactive. Throughout late S and early G2 phases, cyclin B is synthesized and immediately complexes with Cdc2, which is kept catalytically inactive by phosphorylation of Thr-14 and Tyr-15 in the ATP-binding site. This phosphorylation and inactivation are catalyzed by the protein kinases Wee1 and Myt1 (22, 29), and dephosphorylation and activation of cyclin B-Cdc2 are catalyzed by the phosphatase Cdc25C (6, 14, 21). Activation of Cdc25C requires phosphorylation on specific serine and threonine sites, which fails to occur if DNA synthesis is incomplete and the replication checkpoint is activated (13, 16). These considerations have focused attention on the phosphorylation pathway by which Cdc25C becomes activated at the G2/M transition. Cyclin B-Cdc2 can phosphorylate and activate Cdc25C, forming a positive feedback loop that contributes to the abrupt transition from G2 into M phase (10, 12). However, a variety of evidence indicates that in Xenopus initial phosphorylation and activation of Cdc25C result from activation of the polo-like kinase (plk) Plx1. Plx1 can phosphorylate and activate Cdc25C in vitro (15), and in vivo activation of Plx1 is concurrent with the activation of Cdc25C (30). Moreover, inhibition of Plx1 delays the activation of Cdc25C, and an elevated level of Plx1 accelerates the rate of Cdc25C activation (30). Plx1 is also activated by phosphorylation, and the newly identified Xenopus polo-like kinase kinase 1 (xPlkk1) is able to phosphorylate and activate Plx1 in vitro (31). The xPlkk1 protein itself is also activated by phosphorylation, and in vivo activation of xPlkk1 coincides with the activation of Plx1. Moreover, an elevated level of xPlkk1 accelerates the timing of activation of Plx1 and the transition from the G2 to the M phase of the cell cycle (31). Both Plx1 and xPlkk1 might be subject to inhibition when the DNA replication checkpoint is activated. In addition to the role for plk’s at the G2/M transition, in a variety of organisms, including Xenopus, plk’s also have other roles in mitosis. One important role in yeast, Drosophila, Xenopus, and mammalian cells is the requirement of plk function for bipolar spindle formation (7, 8, 17, 28, 30, 36). In the absence of plk function, monopolar spindles form, and localization studies show that plk’s can be found on centrosomes and kinetochores at metaphase (30, 35). plk’s appear to continue to function in mitosis even after the metaphase/anaphase transition has been triggered. In Xenopus egg extracts, addition of a catalytically inactive (kinase-dead) form of Plx1 blocks the degradation of B-type cyclins, and the system remains in M phase with high histone H1 kinase activity (4). In budding yeast cells the plk homolog Cdc5p is normally degraded by the anaphase-promoting complex (APC) (3, 33), and in Xenopus Plx1 activity decreases late in mitosis (30). Overexpression of Cdc5p results in increased degradation of certain Clbs, suggesting that degradation of Cdc5p is required to turn off the degradation of B-type cyclins by the APC (3). In both Saccharomyces cerevisiae and Drosophila, plk’s are localized to the midbody and plk function appears to be required for cytokinesis (2, 19, 20). Most of the information obtained to date about the function of plk’s has come from studies with loss-of-function mutants or inhibitory antibodies. To learn more about the function of plk’s it was imperative to generate a constitutively active mutant of Plx1 and determine its effects on mitosis, as reported here.