Polo-like Kinase1 Is Required for Recruitment of Dynein to Kinetochores during Mitosis

Abstract

Kinetochore dynein has been implicated in microtubule capture, correcting inappropriate microtubule attachments, chromosome movement, and checkpoint silencing. It remains unclear how dynein coordinates this diverse set of functions. Phosphorylation is responsible for some dynein heterogeneity (Whyte, J., Bader, J. R., Tauhata, S. B., Raycroft, M., Hornick, J., Pfister, K. K., Lane, W. S., Chan, G. K., Hinchcliffe, E. H., Vaughan, P. S., and Vaughan, K. T. (2008) J. Cell Biol. 183, 819 -834), and phosphorylated and dephosphorylated forms of dynein coexist at prometaphase kinetochores. In this study, we measured the impact of inhibiting polo-like kinase 1 (Plk1) on both dynein populations. Phosphorylated dynein was ablated at kinetochores after inhibiting Plk1 with a small molecule inhibitor (5-Cyano-7-nitro-2-(benzothiazolo-Noxide)-carboxamide) or chemical genetic approaches. The total complement of kinetochore dynein was also reduced but not eliminated, reflecting the presence of some dephosphorylated dynein after Plk1 inhibition. Although Plk1 inhibition had a profound effect on dynein, kinetochore populations of dynactin, spindly, and zw10 were not reduced. Plk1-independent dynein was reduced after p150Glued depletion, consistent with the binding of dephosphorylated dynein to dynactin. Plk1 phosphorylated dynein intermediate chains at Thr-89 in vitro and generated the phospho-Thr-89 phosphoepitope on recombinant dynein intermediate chains. Finally, inhibition of Plk1 induced defects in microtubule capture and persistent microtubule attachment, suggesting a role for phosphorylated dynein in these functions during prometaphase. These findings suggest that Plk1 is a dynein kinase required for recruitment of phosphorylated dynein to kinetochores.Cytoplasmic dynein plays important roles at spindle poles, the cell cortex, and kinetochores during mitosis. The mechanisms that allow dynein to associate specifically with each site remain unknown. One possibility is suggested by the identification of a novel mitotic phosphorylation site in the dynein intermediate chains (ICs) 2 (1). This phosphorylated form of dynein (phospho-Thr-89, pT89) is specific for kinetochores from nuclear envelope breakdown (NEB) to metaphase. Previous work suggests that pT89 dynein is recruited to kinetochores through a direct interaction with zw10 but undergoes dephosphorylation in response to bioriented microtubule attachment and chromosome alignment. This dephosphorylation of dynein shifts binding to dynactin and stimulates poleward transport of checkpoint proteins; a transition required for the silencing of the spindle assembly checkpoint at metaphase. Defects in this process induce metaphase arrest/delay.One prediction of this model is that dynein could interact with multiple proteins at kinetochores, depending on the degree of microtubule attachment, microtubule occupancy, and chromosome alignment. Consistent with this scenario, several mitotic proteins have been implicated in interacting with or anchoring kinetochore dynein, including dy...

Identification of Pyrroloformamide as a Cytokinesis Modulator

“…[23] The inhibition of Plk1 leads to mitotic entry delay followed by prometaphase arrest. [24] Recently, teams led by Kapoor and Straight have examined the effects of Plk1 inhibitors on metaphase, [25] and observed failure during contractile ring assembly and in the cleavage furrow as well as defects in RhoA localisation; these observations compare favourably with our confocal studies. In fact, the observed features suggested that the effects of 1 included mitotic arrest (Figure 5 Actually, many reports have shown that Plk1 inhibition impairs RhoA binding to other proteins, such as endothelial cell transforming complex 2 (Ect-2), which are involved in the latter stages of cytokinesis.…”

Integrated Proteomics Identified Novel Activation of Dynein IC2-GR-COX-1 Signaling in Neurofibromatosis Type I (NF1) Disease Model Cells

“…The enzyme responsible for phosphorylation in this system has not been identified; however, polo-like kinase (25) and casein kinase (26,27) have been reported to phosphorylate dynein IC in vitro and in Xenopus melanophores (27), and also activations of MAPK, CDK5, GSK3, and ROCK in NF1-KD PC12 cells were found in our previous study (7). Thus, these enzymes may be involved in the regulation of neurite outgrowth in NF1-KD cells.…”

Cdk1 Activates Pre-mitotic Nuclear Envelope Dynein Recruitment and Apical Nuclear Migration in Neural Stem Cells

“…Very similar results were observed using RO-3306 or the Cdk1/Cdk2 inhibitor III (Gavet and Pines, 2010) (Figure 2A and 2E). The Aurora A inhibitor VX680 (Harrington et al, 2004) had no significant effect, but a mild effect was observed with the Plk1 inhibitor BTO-1 (Bader et al, 2011) (Figure 2E). This could reflect a role for Plk1 in Cdk1 activation, though a more direct role in dynein recruitment is also possible (Li et al, 2010).…”

“…For better visualization of NE dynein and dynein partners, cells were incubated for 1 hr in Nocodazol (10 μM) prior to fixation (Bolhy et al, 2011; Hu et al, 2013; Raaijmakers et al, 2013; Splinter et al, 2012). The following drugs were used for protein kinase inhibition: 55 μM Roscovitine (Meijer et al, 1997) (Selleck Chemicals); 0.9 μM Cdk1/2 inhibitor III (Gavet and Pines, 2010) (Calbiochem); 28 μM RO-3306 (Deibler and Kirschner, 2010; Vassilev et al, 2006) (Tocris Bioscience); 0.5 μM PD166285 (Potapova et al, 2009) (Tocris Bioscience); 0.3 μM VX-680 (Harrington et al, 2004) (Selleck Chemicals); 22 μM BTO-1 (Bader et al, 2011) (EMD Millipore).…”