The polo box is required for multiple functions of P1x1 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 polo-like kinase (P1x1NA) functions as a competitor of endogenous P1x1 for polo box binding site(s) and inhibits the activation of Cdc25C and the G2-M transition in vivo. However, kinase-dead P1x1 with a point mutation in the polo box region (P1x1NAWF) did not have inhibitory effects. The ability of P1x1NA to block activation of the anaphase-promoting complex/cyclosome also requires an intact polo box. Microinjection of P1x1NA but not P1x1NAWF mRNA into Xenopus embryos caused cleavage arrest and formation of monopolar spindles, an effect previously seen in embryos injected with anti-P1x1 antibody. Spindle assembly experiments in vitro also showed that only monopolar spindles formed in Xenopus egg extracts supplemented with recombinant P1x1NA and that the spindle assembly process was delayed. Taken together, these results indicate that the polo box is required for P1x1 function in both the G2-M and the metaphase/anaphase transitions during the cell cycle.