Functional Dynamics of Polo-Like Kinase 1 at the Centrosome

Article (PDF Available)inMolecular and Cellular Biology 29(11):3134-50 · April 2009with22 Reads
DOI: 10.1128/MCB.01663-08 · Source: PubMed
Abstract
Polo-like kinase 1 (Plk1) functions as a key regulator of mitotic events by phosphorylating substrate proteins on centrosomes, kinetochores, the mitotic spindle, and the midbody. Through mechanisms that are incompletely understood, Plk1 is released from and relocalizes to different mitotic structures as cells proceed through mitosis. We used fluorescence recovery after photobleaching to examine the kinetics of this process in more detail. We observed that Plk1 displayed a range of different recovery rates that differ at each mitotic substructure and depend on both the Polo-box domain and a functional kinase domain. Upon mitotic entry, centrosomal Plk1 becomes more dynamic, a process that is directly enhanced by Plk1 kinase activity. In contrast, Plk1 displays little dynamic exchange at the midbody, a process that again is modulated by the kinase activity of Plk1. Our findings suggest that the intrinsic kinase activity of Plk1 triggers its release from early mitotic structures and its relocalization to late mitotic structures. To assess the importance of Plk1 dynamic relocalization, Plk1 was persistently tethered to the centrosome. This resulted in a G(2) delay, followed by a prominent prometaphase arrest, as a consequence of defective spindle formation and activation of the spindle checkpoint. The dynamic release of Plk1 from early mitotic structures is thus crucial for mid- to late-stage mitotic events and demonstrates the importance of a fully dynamic Plk1 at the centrosome for proper cell cycle progression. This dependence on dynamic Plk1 was further observed during the mitotic reentry of cells after a DNA damage G(2) checkpoint, as this process was significantly delayed upon centrosomal tethering of Plk1. These results indicate that mitotic progression and control of mitotic reentry after DNA damage resides, at least in part, on the dynamic behavior of Plk1.

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    • "Prior work focused on the localization of PIk1 to centrosomes, mitotic kinetochores, the spindle midzone, and the midbody (Archambault and Glover, 2009). In addition, as reported by others (Arnaud et al., 1998; Kishi et al., 2009), we found that YFP-Plk1 localized to centromeres in GI, concurrent with Misl8a localization (Fig. S1A). We observed identical localization when we tagged the endogenous PLK1 locus with YFP using CRISPR/Cas-mediated genome editing (Plkl-YFP) (image. "
    [Show abstract] [Hide abstract] ABSTRACT: Each time a cell divides, the genome must be segregated equally between the two new daughter cells. To accomplish this, a specific region of each chromosome, termed the centromere, recruits the macromolecular kinetochore structure to mediate attachments to spindle microtubules. In vertebrates, each chromosome must establish a single site of microtubule attachment. The failure to maintain this site or the generation of multiple distinct microtubule attachment sites on a single chromosome can have profoundly deleterious effects on cell and organismal viability. My graduate work has used cell biological analyses in tissue culture cells and biochemical reconstitutions to define the molecular mechanisms by which human cells maintain one and only one site of microtubule attachment on each chromosome. First, I defined the regulatory paradigms that ensure the faithful propagation of the centromere. I identified Polo-like kinase 1 as a key player in controlling the deposition of the epigenetic mark that specifies the centromere, the CENP-A nucleosome. I defined the molecular basis for this control, as well as an additional level of control downstream of the cyclin-dependent kinases. By identifying and dissecting the molecular features of this two-step regulatory paradigm, I developed a strategy to bypass the control of CENP-A deposition, which resulted in severe mitotic defects. In my second project, I defined the architecture and properties of the sixteen-protein assembly that connects CENP-A to the other proteins of the kinetochore. I analyzed the genetic relationships between these proteins in human cells through a combination of inducible knockouts and inducible protein degradation. I then reconstituted the sixteen proteins in vitro as five sub-complexes and defined their interactions biochemically. These analyses revealed an intricate meshwork of direct interactions between the proteins at the centromere-kinetochore interface, which is critical for ensuring assembly of the kinetochore at the correct site on the chromosome. Together, these findings provide new insights into the molecular mechanisms of centromere propagation and kinetochore assembly.
    Thesis · Jan 2016 · Journal of Cell Science
    • "Active Plk1 controls entry into mitosis and also promotes centrosome maturation and separation (Bruinsma et al., 2012; Zitouni et al., 2014). Plk1 localization influences its activity and vice versa (Kishi et al., 2009). Upon Mio depletion, levels of total Plk1 and active Plk1 T210ph were reduced at centrosomes. "
    [Show abstract] [Hide abstract] ABSTRACT: Coordination of cell growth and proliferation in response to nutrient supply is mediated by mammalian target of rapamycin (mTOR) signaling. In this study, we report that Mio, a highly conserved member of the SEACAT/GATOR2 complex necessary for the activation of mTORC1 kinase, plays a critical role in mitotic spindle formation and subsequent chromosome segregation by regulating the proper concentration of active key mitotic kinases Plk1 and Aurora A at centrosomes and spindle poles. Mio-depleted cells showed reduced activation of Plk1 and Aurora A kinase at spindle poles and an impaired localization of MCAK and HURP, two key regulators of mitotic spindle formation and known substrates of Aurora A kinase, resulting in spindle assembly and cytokinesis defects. Our results indicate that a major function of Mio in mitosis is to regulate the activation/deactivation of Plk1 and Aurora A, possibly by linking them to mTOR signaling in a pathway to promote faithful mitotic progression. © 2015 by The Rockefeller University Press.
    Article · Jun 2015
    • "Consistent with this hypothesis, the centrosomal localization of Plk1 is highly dynamic from interphase to mitosis, and Plk1 is recruited to the PCM by PCM1 just before mitotic entry (Petronczki et al., 2008; Wang et al., 2013). Artificial and persistent tethering of Plk1 to the centrosome induces a delay in G2, prometaphase arrest and defective spindle formation (Kishi et al., 2009). Moreover, inhibition of the kinase activity of Plk1 results in structural defects of the centrosome even after its maturation, which can be rescued by restoring Plk1 activity (Mahen et al., 2011). "
    [Show abstract] [Hide abstract] ABSTRACT: The centrosome acts as the major microtubule-organizing center (MTOC) for cytoskeleton maintenance in interphase and mitotic spindle assembly in vertebrate cells. It duplicates only once per cell cycle in a highly spatiotemporally regulated manner. When the cell undergoes mitosis, the duplicated centrosomes separate to define spindle poles and monitor the assembly of the bipolar mitotic spindle for accurate chromosome separation and the maintenance of genomic stability. However, centrosome abnormalities occur frequently and often lead to monopolar or multipolar spindle formation, which results in chromosome instability and possibly tumorigenesis. A number of studies have begun to dissect the role of mitotic kinases, including NIMA-related kinases (Neks), cyclin-dependent kinases (CDKs), Polo-like kinases (Plks) and Aurora kinases, in regulating centrosome duplication, separation and maturation and subsequent mitotic spindle assembly during cell cycle progression. In this Commentary, we review the recent research progress on how these mitotic kinases are coordinated to couple the centrosome cycle with the cell cycle, thus ensuring bipolar mitotic spindle fidelity. Understanding this process will help to delineate the relationship between centrosomal abnormalities and spindle defects.
    Full-text · Article · Aug 2014
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