A Spindle Checkpoint Arrest and a Cytokinesis Failure by the Dominant-negative Polo-box Domain of Plk1 in U-2 OS Cells

Abstract

Polo kinases play critical roles for proper M-phase progression. They are characterized by the presence of two regions of homology in the C-terminal non-catalytic domain, termed polo-box 1 (PB1) and polo-box 2 (PB2). Here we demonstrate that both PB1 and PB2 are required for targeting the catalytic activity of Plk1 to centrosomes, midbody, and kinetochores. Expression of either kinase-inactive PLK1⁄K82M or the C-terminal plk1⌬N induced a pre-anaphase arrest with elevated Cdc2 and Plk1 activity. Prophase-arrested cells exhibited randomly oriented spindle structures, whereas metaphase cells exhibited aberrant bipolar spindles with Mad2 localization at kinetochores of misaligned chromosomes. Microtubule nucleation activity of centrosomes was not compromised. In vivo time-lapse studies revealed that expression of plk1⌬N resulted in repeated cycles of bipolar spindle formation and disruption, suggestive of a defect in spindle stability. A prolonged arrest frequently led to the generation of micronucleated cells in the absence of sisterchromatid separation and centrosome duplication, indicating that micronucleation is not a result of accumulated cytokinesis failures. Interestingly, bypass of the mitotic arrest by dominantnegative spindle checkpoint components led to a failure in completion of cytokinesis. We propose that, in mammalian cells, the polo-box-dependent Plk1 activity is required for proper metaphase⁄anaphase transition and for cytokinesis.

Phosphorylation of the cytokinesis regulator ECT2 at G2/M phase stimulates association of the mitotic kinase Plk1 and accumulation of GTP-bound RhoA

“…Another mitotic kinase, Plk1, also localizes to the midbody and plays an important role in mitosis and cytokinesis (Seong et al, 2002). Thus, we tested whether ECT2 is a substrate of Plk1.…”

“…The role of phosphorylation of ECT2 by Plk1 might be to stimulate ECT2 at a spatio-temporal manner. Plk1 localizes to the centrosomes at metaphase, to the spindle midzone in anaphase, and to the midbody during cytokinesis (Seong et al, 2002). ECT2 is also localized at the spindle midzone and midbody in HeLa cells, where Plk1 might interact with ECT2.…”

“…ECT2 is also localized at the spindle midzone and midbody in HeLa cells, where Plk1 might interact with ECT2. Since Plk1 is known to regulate cytokinesis as well as other mitotic functions (Song and Lee, 2001;Seong et al, 2002), it is possible that Plk1 regulates cytokinesis through phosphorylation of ECT2. Further studies on identification of the sites phosphorylated by Plk1 and mutational analyses of these sites should clarify the physiological role of ECT2 phosphorylation by Plk1 for cytokinesis.…”

“…The full-length fragment was digested with BamHI and EcoRI, and cloned into pcDNA3.1-Myc (Invitrogen) to express ECT2 as a Myc epitope-fused protein. Flag-tagged WT Plk1 and pGEX-4T-1-Plk1 (residues 326-603) have been described (Seong et al, 2002). ECT2 mutants (ECT2-F T412A, ECT2-F-T412D) were prepared with the Quickchange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA) according to the manufacturer's instructions.…”

“…The serine/threonine protein kinase Plk1 contributes to normal mitotic phase progression, and localizes at mitotic spindles in metaphase and then at the midbody in cytokinesis (Nigg et al, 1996;Yuan et al, 2002). The polo-box domain (PBD) of Plk1 can function as a dominant-negative mutant, and induces pre-anaphase arrest and cytokinesis failure (Seong et al, 2002). Plk1 PBD serves as a specific phospho-Ser-or phospho-Thr-binding domain, which recognizes a subset of substrates phosphorylated by proline-directed protein kinases (Elia et al, 2003a).…”