Polo-like Kinase 1 Creates the Tension-Sensing 3F3/2 Phosphoepitope and Modulates the Association of Spindle-Checkpoint Proteins at Kinetochores

Molecular, Cell and Developmental Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA.
Current Biology (Impact Factor: 9.57). 07/2005; 15(12):1078-89. DOI: 10.1016/j.cub.2005.05.026
Source: PubMed


In mitosis, a mechanochemical system recognizes tension that is generated by bipolar microtubule attachment to sister kinetochores. This is translated into multiple outputs including the stabilization of microtubule attachments, changes in kinetochore protein dynamics, and the silencing of the spindle checkpoint. How kinetochores sense tension and translate this into various signals represent critical unanswered questions. The kinetochores of chromosomes not under tension are specifically phosphorylated at an epitope recognized by the 3F3/2 monoclonal antibody. Determining the kinase that generates the 3F3/2 phosphoepitope at kinetochores should reveal an important component of this system that regulates mitotic progression.
We demonstrate that Polo-like kinase 1 (Plk1) creates the 3F3/2 phosphoepitope on mitotic kinetochores. In a permeabilized in vitro cell system, the depletion of Xenopus Plk1 from M phase extract leads to the loss of 3F3/2 kinase activity. Purified recombinant Plk1 is sufficient to generate the 3F3/2 phosphoepitope in this system. Using siRNA, we show that the reduction of Plk1 protein levels significantly diminishes 3F3/2 phosphoepitope expression at kinetochores. The consensus phosphorylation sites of Plk1 show strong similarity to the 3F3/2 phosphoepitope sequence determined by phosphopeptide mapping. The inhibition of Plk1 by siRNA alters the normal kinetochore association of Mad2, Cenp-E, Hec1/Ndc80, Spc24, and Cdc20 and induces a spindle-checkpoint-mediated mitotic arrest.
Plk1 generates the 3F3/2 phosphoepitope at kinetochores that are not under tension and contributes to the normal kinetochore association of several key proteins important in checkpoint signaling. Mechanical tension regulates Plk1 accumulation at kinetochores and possibly its kinase activity.

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    • "Because p27/p25 depletion does not affect spindle formation or dynein/dynactin integrity and recruitment to kinetochores, but does result in selective loss of Plk1 from this site, it represents a powerful new tool for analysing the functions of kinetochoreassociated Plk1 and its downstream targets in the processes of chromosome congression and execution of the spindle assembly checkpoint. Kinetochore-associated Plk1 has been implicated in kinetochore–MT engagement and checkpoint protein recruitment (Sumara et al, 2004; Ahonen et al, 2005; Wong and Fang, 2005; Elowe et al, 2007; Lenart et al, 2007; Hood et al, 2012; Liu et al, 2012; Maia et al, 2012). Our data suggest that full Plk1 activity is needed for establishment of proper kinetochore–MT attachments. "
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    ABSTRACT: Dynactin is a protein complex required for the in vivo function of cytoplasmic dynein, a microtubule (MT)-based motor. Dynactin binds both dynein and MTs via its p150Glued subunit, but little is known about the 'pointed-end complex' that includes the protein subunits Arp11, p62 and the p27/p25 heterodimer. Here, we show that the p27/p25 heterodimer undergoes mitotic phosphorylation by cyclin-dependent kinase 1 (Cdk1) at a single site, p27 Thr186, to generate an anchoring site for polo-like kinase 1 (Plk1) at kinetochores. Removal of p27/p25 from dynactin results in reduced levels of Plk1 and its phosphorylated substrates at kinetochores in prometaphase, which correlates with aberrant kinetochore-MT interactions, improper chromosome alignment and abbreviated mitosis. To investigate the structural implications of p27 phosphorylation, we determined the structure of human p27. This revealed an unusual left-handed β-helix domain, with the phosphorylation site located within a disordered, C-terminal segment. We conclude that dynactin plays a previously undescribed regulatory role in the spindle assembly checkpoint by recruiting Plk1 to kinetochores and facilitating phosphorylation of important downstream targets.
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    • "In addition, a phospho-specific epitope, recognized by monoclonal antibody 3 F3/2 [18], resides in kinetochore proteins. The epitope, generated at least partially by Plk1 kinase, is likely to be found at multiple centromere/kinetochore proteins [19,20]. Some proteins carrying 3 F3/2 epitopes have been determined (e.g. "
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    ABSTRACT: Proteins functioning in the same biological pathway tend to be transcriptionally co-regulated or form protein-protein interactions (PPI). Multiple spatially and temporally regulated events are coordinated during mitosis to achieve faithful chromosome segregation. The molecular players participating in mitosis regulation are still being unravelled experimentally or using in silico methods. An extensive literature review has led to a compilation of 196 human centromere/kinetochore proteins, all with experimental evidence supporting the subcellular localization. Sixty-four were designated as "core" centromere/kinetochore components based on peak expression and/or well-characterized functions during mitosis. By interrogating and integrating online resources, we have mined for genes/proteins that display transcriptional co-expression or PPI with the core centromere/kinetochore components. Top-ranked hubs in either co-expression or PPI network are not only enriched with known mitosis regulators, but also contain candidates whose mitotic functions are not yet established. Experimental validation found that KIAA1377 is a novel centrosomal protein that also associates with microtubules and midbody; while TRIP13 is a novel kinetochore protein and directly interacts with mitotic checkpoint silencing protein p31(comet). Transcriptional co-expression and PPI network analyses with known human centromere/kinetochore proteins as a query group help identify novel potential mitosis regulators.
    Full-text · Article · Jun 2012 · BMC Cell Biology
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    • "Plk1 plays a crucial role in stabilizing kinetochore– microtubule attachments, as evidenced by its role in generating the 3F3/2 phosphoepitope, which marks kinetochores that are not experiencing spindle-generated tension (Ahonen et al. 2005; Wong and Fang 2005 "
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