The Polo Box Is Required for Multiple Functions of Plx1 in Mitosis

Abstract

Polo-like kinases comprise a family of evolutionarily conserved serine/threonine protein kinases that play multiple roles in cell cycle regulation. In addition to the N-terminal catalytic domain, polo-like kinases have one or two highly conserved C-terminal non-catalytic regions, termed polo boxes. These motifs are required for targeting these kinases to subcellular mitotic structures. Here we report that kinase-dead Xenopus pololike kinase (Plx1NA) functions as a competitor of endogenous Plx1 for polo box binding site(s) and inhibits the activation of Cdc25C and the G 2 -M transition in vivo. However, kinase-dead Plx1 with a point mutation in the polo box region (Plx1NAWF) did not have inhibitory effects. The ability of Plx1NA to block activation of the anaphase-promoting complex/cyclosome also requires an intact polo box. Microinjection of Plx1NA but not Plx1NAWF mRNA into Xenopus embryos caused cleavage arrest and formation of monopolar spindles, an effect previously seen in embryos injected with anti-Plx1 antibody. Spindle assembly experiments in vitro also showed that only monopolar spindles formed in Xenopus egg extracts supplemented with recombinant Plx1NA and that the spindle assembly process was delayed. Taken together, these results indicate that the polo box is required for Plx1 function in both the G 2 -M and the metaphase/anaphase transitions during the cell cycle.

Xenopus Polo-like kinase Plx1: a multifunctional mitotic kinase

“…In supporting this direct model, the fission yeast Polo-like kinase, Plo1, has been demonstrated to interact directly with Cut23 (Apc8), a subunit of the APC/C (May et al, 2002). Moreover, Plx1 coimmunoprecipitates with the Xenopus APC/C in a PBD-dependent manner, although a direct interaction has not been established (Liu et al, 2004).…”

“…This highly synchronized oocyte maturation process provides an ideal system to study Cdc25C activation. It was found that upon PG treatment, Plx1 activation is concurrent with Cdc25C and Cdc2/cyclin B activation, and injection of Plx1 facilitated the activation of both Cdc25C and Cdc2/ cyclin B compared to that in buffer-injected oocytes, whereas injection of kinase-dead Plx1 (N172) or affinitypurified anti-Plx1 antibody significantly delayed their activation (Qian et al, 1998a;Liu et al, 2004). The inhibitory effect of Plx1 antibody was overcome by injection of wild-type, but not phosphatase-inactive Cdc25C, indicating that Plx1 is upstream of Cdc25C (Qian et al, 1998a).…”

“…Concomitantly, the proteolytic destruction of several targets of the APC/ C, including cyclin B, and the inactivation of the Cdc2 protein kinase were prevented. Subsequent work showed that the metaphase to anaphase transition could be blocked by immunodepletion of Plx1, and was restored upon the addition of recombinant Plx1 (Qian et al, 1999;Liu et al, 2004). These results demonstrate that the metaphase/anaphase transition requires active Plx1, suggesting an important role for Plx1 in activation of the proteolytic machinery that controls the exit from mitosis.…”

Plk1 Regulates Activation of the Anaphase Promoting Complex by Phosphorylating and Triggering SCFβTrCP-dependent Destruction of the APC Inhibitor Emi1

“…Fittingly, Emi1 is required to maintain the meiotic arrest in Xenopus egg extracts , suggesting a plausible explanation for the involvement of Plx1 in release from this arrest. Recent work by Liu et al (2004) showed that inhibition of meiotic exit by dominant negative Plx1 can be almost entirely relieved by addition of excess Cdc20 -the same treatment that relieves the inhibition of meiotic exit by excess Emi1. This again suggests that Plk1 does not significantly regulate the intrinsic activity of the APC, but instead regulates some upstream aspect of Cdc20 function.…”

Polo-like kinases, an introduction

“…Topics covered in this issue include functions of Plks in yeast (Lee et al), Drosophila (Glover), Xenopus (Liu and Maller), and mammals (Seeburg et al; Swallow et al; Myer et al). Since Plks possess the unique Polo box domain, the structure of which is critical for their function (Elia et al, 2003b;Liu et al, 2004), a specific chapter is devoted to the structure and function of Plks (Lowery et al, 2004). In more highly evolved animals, Plks have been shown to play an essential role in controlling cell proliferation, genomic stability, and oncogenesis.…”