Abstract
Polo-like kinases and kinesin-like motor proteins are among the many proteins implicated in the execution of cytokinesis. Polo-like-kinase 1 (Plk1) interacts with the mitotic kinesin-like motor protein CHO1/MKLP-1 during anaphase and telophase, and CHO1/MKLP-1 is a Plk1 substrate in vitro. Here, we explore the molecular interactions of these two key contributors to mitosis and cytokinesis. Using the transient transfection approach, we show that the C-terminus of Plk1 binds CHO1/MKLP-1 in a Polo-box-dependent manner and that the stalk domain of CHO1/MKLP-1 is responsible for its binding to Plk1. The stalk domain was found to localize with Plk1 to the mid-body, and Plk1 appears to be mislocalized in CHO1/MKLP-1-depleted cells during late mitosis. We showed that Ser904 and Ser905 are two major Plk1 phosphorylation sites. Using the vector-based RNA interference approach, we showed that depletion of CHO1/MKLP-1 causes the formation of multinucleate cells with more centrosomes, probably because of a defect in the early phase of cytokinesis. Overexpression of a non-Plk1-phosphorylatable CHO1 mutant caused cytokinesis defects, presumably because of dominant negative effect of the construct. Finally, CHO1-depletion-induced multinucleation could be partially rescued by co-transfection of a non-degradable hamster wild-type CHO1 construct, but not an unphosphorylatable mutant. These data provide more detailed information about the interaction between Plk1 and CHO1/MKLP-1, and the significance of this is discussed.
Highlights
The Polo-like kinase family has emerged as a key player in many cell-cycle-related events
Interference approach, we showed that depletion of CHO1/mitotic kinesin-like protein 1 (MKLP-1) causes the formation of multinucleate cells with more centrosomes, probably because of a defect in the early phase of cytokinesis
C-terminal Polo-box domain of Polo-like-kinase 1 (Plk1) is responsible for CHO1/MKLP-1 binding
Summary
The Polo-like kinase (plk) family has emerged as a key player in many cell-cycle-related events. In addition to the N-terminal kinase domain, all plk family members (including mammalian Plk, Snk and Fnk/Prk, Xenopus laevis Plx, Drosophila Polo, fission yeast Plo and budding yeast Cdc5) have a distinctive highly conserved region in the C-terminal non-catalytic domain, denoted the Polo box (Clay et al, 1993; Glover et al, 1998). Plx was proposed to be a trigger kinase for cells to enter mitosis (Kumagai and Dunphy, 1996). Both Drosophila polo and fission yeast Plo mutants show the phenotype of monopolar spindles, indicating a role for plks in centrosome assembly and separation during the formation of bipolar mitotic spindles (Ohkura et al, 1995; Sunkel and Glover, 1998). It was recently demonstrated that the centrosomal localization of Plk results from the recognition and interaction of the Polo-box domain with phosphorylated peptides (Elia et al, 2003a; Elia et al, 2003b)
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