Self-regulated mechanism of Plk1 localization to kinetochores: lessons from the Plk1-PBIP1 interaction

Mammalian polo-like kinase 1 (Plk1) has been studied extensively as a critical element in regulating various mitotic events during M-phase progression. Plk1 function is spatially regulated through the targeting activity of the conserved polo-box domain (PBD) present in the C-terminal non-catalytic region. Recent progress in our understanding of Plk1 localization to the centromeres shows that Plk1 self-regulates its initial recruitment by phosphorylating a centromeric component PBIP1 and generating its own PBD-binding site. Paradoxically, Plk1 also induces PBIP1 delocalization and degradation from the mitotic kinetochores late in the cell cycle, consequently permitting itself to bind to other kinetochore components. Thus, PBIP1-dependent self-recruitment of Plk1 to the interphase centromeres serves as a prelude to the efficient delivery of Plk1 itself to other kinetochore components whose interactions with Plk1 are vital for proper mitotic progression.     

Phosphorylation of Tara by Plk1 is essential for faithful chromosome segregation in mitosis

Trio-associated repeat on actin (Tara) is an F-actin binding protein and regulates actin cytoskeletal organization. In our previous study, we have found that Tara associates with telomeric repeat binding factor 1 (TRF1) and mediates the function of TRF1 in mitotic regulation. We also found that overexpression HECTD3, a member of HECT E3 ubiquitin ligases, enhances the ubiquitination of Tara in vivo and promotes the degradation of Tara, and such degradation of Tara facilitates cell cycle progression. However, less is known about the post-translational modification of Tara in mitosis. Here we show that Tara is a novel Polo-like kinase 1 (Plk1) target protein. Plk1 interacts with and phosphorylates Tara in vivo and in vitro. Actually, the Thr-457 in Tara was a bona fide in vivo phosphorylation site for Plk1. Interestingly, we found that the centrosomal localization of Tara depended on the Thr-457 phosphorylation and the kinase activity of Plk1. Furthermore, overexpression of non-phosphorylatable mutant of Tara caused aberrant mitosis delay in HeLa cells. Our study demonstrated that Plk1-mediated phospho-dependent centrosomal localization of Tara is important for faithful chromosome segregation, and provided novel insights into understanding on the role of Plk1 in cooperation with Tara in mitotic progression.     

TMAP/CKAP2 is essential for proper chromosome segregation

Tumor-associated microtubule-associated protein (TMAP), also known as cytoskeleton associated protein 2 (CKAP2), is a novel mitotic spindle-associated protein which is frequently up-regulated in various malignances. However, its cellular functions remain unknown. Previous reports suggested that the cellular functions of TMAP/CKAP2 pertain to regulation of the dynamics and assembly of the mitotic spindle. To investigate its role in mitosis, we studied the effects of siRNA-mediated depletion of TMAP/CKAP2 in cultured mammalian cells. Unexpectedly, TMAP/CKAP2 knockdown did not result in significant alterations of the spindle apparatus. However, TMAP/CKAP2-depleted cells often exhibited abnormal nuclear morphologies, which were accompanied by abnormal organization of the nuclear lamina, and chromatin bridge formation between two daughter cell nuclei. Time lapse video microscopy revealed that the changes in nuclear morphology and chromatin bridge formations observed in TMAP/CKAP2-depleted cells are the result of defects in chromosome segregation. Consistent with this, the spindle checkpoint activity was significantly reduced in TMAP/CKAP2-depleted cells. Moreover, chromosome missegregation induced by depletion of TMAP/CKAP2 ultimately resulted in reduced cell viability and increased chromosomal instability. Our present findings demonstrate that TMAP/CKAP2 is essential for proper chromosome segregation and for maintaining genomic stability.     

PP2A is required for centromeric localization of Sgo1 and proper chromosome segregation

Loss of sister-chromatid cohesion triggers chromosome segregation in mitosis and occurs through two mechanisms in vertebrate cells: (1) phosphorylation and removal of cohesin from chromosome arms by mitotic kinases, including Plk1, during prophase, and (2) cleavage of centromeric cohesin by separase at the metaphase-anaphase transition. Bub1 and the MEI-S332/Shugoshin (Sgo1) family of proteins protect centromeric cohesin from mitotic kinases during prophase. We show that human Sgo1 binds to protein phosphatase 2A (PP2A). PP2A localizes to centromeres in a Bub1-dependent manner. The Sgo1-PP2A interaction is required for centromeric localization of Sgo1 and proper chromosome segregation in human cells. Depletion of Plk1 by RNA interference (RNAi) restores centromeric localization of Sgo1 and prevents chromosome missegregation in cells depleted of PP2A_Aalpha. Our findings suggest that Bub1 targets PP2A to centromeres, which in turn maintains Sgo1 at centromeres by counteracting Plk1-mediated chromosome removal of Sgo1.     

NudC is required for Plk1 targeting to the kinetochore and chromosome congression

The equal distribution of chromosomes during mitosis is critical for maintaining the integrity of the genome. Essential to this process are the capture of spindle microtubules by kinetochores and the congression of chromosomes to the metaphase plate . Polo-like kinase 1 (Plk1) is a mitotic kinase that has been implicated in microtubule-kinetochore attachment, tension generation at kinetochores, tension-responsive signal transduction, and chromosome congression . The tension-sensitive substrates of Plk1 at the kinetochore are unknown. Here, we demonstrate that human Nuclear distribution protein C (NudC), a 42 kDa protein initially identified in Aspergillus nidulans and shown to be phosphorylated by Plk1 , plays a significant role in regulating kinetochore function. Plk1-phosphorylated NudC colocalizes with Plk1 at the outer plate of the kinetochore. Depletion of NudC reduced end-on microtubule attachments at kinetochores and resulted in defects in chromosome congression at the metaphase plate. Importantly, NudC-deficient cells exhibited mislocalization of Plk1 and the Kinesin-7 motor CENP-E from prometaphase kinetochores. Ectopic expression of wild-type NudC, but not NudC containing mutations in the Plk1 phosphorylation sites, recovered Plk1 localization at the kinetochore and rescued chromosome congression. Thus, NudC functions as both a substrate and a spatial regulator of Plk1 at the kinetochore to promote chromosome congression.     

Polo-like kinase1 is required for recruitment of dynein to kinetochores during mitosis

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-N-oxide)-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 p150(Glued) 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 phospho-epitope 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.     

Cyclin B1 is localized to unattached kinetochores and contributes to efficient microtubule attachment and proper chromosome alignment during mitosis

Cyclin B1 is a key regulatory protein controlling cell cycle progression in vertebrates. Cyclin B1 binds CDK1, a cy-clin-dependent kinase catalytic subunit, forming a complex that orchestrates mitosis through phosphorylation of key proteins. Cyclin B1 regulates both the activation of CDK1 and its subcellular localization, which may be critical for substrate selection. Here, we demonstrate that cyclin B1 is concentrated on the outer plate of the kinetochore during prometaphase. This localization requires the cyclin box region of the protein. Cyclin B1 is displaced from individual kinetochores to the spindle poles by microtubule attachment to the kinetochores, and this displacement is dependent on the dynein/dynactin complex. Depletion of cyclin B1 by vector-based siRNA causes inefficient attachment between kinetochores and microtubules, and chromosome alignment defects, and delays the onset of anaphase. We conclude that cyclin B1 accumulates at kinetochores during prometaphase, where it contributes to the correct attachment of mi- crotubules to kinetochores and efficient alignment of the chromosomes, most likely through localized phosphorylation of specific substrates by cyclin B1-CDK1. Cyclin B1 is then transported from each kinetochore as microtubule attachment is completed, and this relocalization may redirect the activity of cyclin B1-CDK1 and contribute to inactivation of the spindle assembly checkpoint.     

Bub1 targeting to centromeres is sufficient for Sgo1 recruitment in the absence of kinetochores

Centromeric chromatin containing the histone H3 variant centromere protein A (CENP-A) directs kinetochore assembly through a hierarchical binding of CENPs, starting with CENP-C and CENP-T. Centromeres are also the chromosomal regions where cohesion, mediated by cohesin, is most prominently maintained in mitosis. While most cohesin dissociates from chromosome arms in prophase, Shugoshin 1 (Sgo1) prevents this process at centromeres. Centromeric localization of Sgo1 depends on histone H2A phosphorylation by the kinase Bub1, but whether additional interactions with kinetochore components are required for Sgo1 recruitment is unclear. Using the Xenopus egg cell-free system, we here show that both CENP-C and CENP-T can independently drive centromeric accumulation of Sgo1 through recruitment of Bub1 to the KNL1, MIS12, NDC80 (KMN) network. The spindle assembly checkpoint (SAC) kinase Mps1 is also required for this pathway even in the absence of checkpoint signaling. Sgo1 recruitment is abolished in chromosomes lacking kinetochore components other than CENP-A. However, forced targeting of Bub1 to centromeres is sufficient to restore Sgo1 localization under this condition.     

Plk1 regulates the kinesin-13 protein Kif2b to promote faithful chromosome segregation

Solid tumors are frequently aneuploid, and many display high rates of ongoing chromosome missegregation in a phenomenon called chromosomal instability (CIN). The most common cause of CIN is the persistence of aberrant kinetochore-microtubule (k-MT) attachments, which manifest as lagging chromosomes in anaphase. k-MT attachment errors form during prometaphase due to stochastic interactions between kinetochores and microtubules. The kinesin-13 protein Kif2b promotes the correction of k-MT attachment errors in prometaphase, but the mechanism restricting this activity to prometaphase remains unknown. Using mass spectrometry, we identified multiple phosphorylation sites on Kif2b, some of which are acutely sensitive to inhibition of Polo-like kinase 1 (Plk1). We show that Plk1 directly phosphorylates Kif2b at threonine 125 (T125) and serine 204 (S204), and that these two sites differentially regulate Kif2b function. Phosphorylation of S204 is required for the kinetochore localization and activity of Kif2b in prometaphase, and phosphorylation of T125 is required for Kif2b activity in the correction of k-MT attachment errors. These data demonstrate that Plk1 regulates both the localization and activity of Kif2b during mitosis to promote the correction of k-MT attachment errors to ensure mitotic fidelity.     

Plk1 phosphorylates sgt1 at the kinetochores to promote timely kinetochore-microtubule attachment

Accurate chromosome segregation during cell division maintains genomic integrity and requires the proper establishment of kinetochore-microtubule attachment in mitosis. As a key regulator of mitosis, Polo-like kinase 1 (Plk1) is essential for this attachment process, but the molecular mechanism remains elusive. Here we identify Sgt1, a cochaperone for Hsp90, as a novel Plk1 substrate during mitosis. We show that Sgt1 dynamically localizes at the kinetochores, which lack microtubule attachments during prometaphase. Plk1 is required for the kinetochore localization of Sgt1 and phosphorylates serine 331 of Sgt1 at the kinetochores. This phosphorylation event enhances the association of the Hsp90-Sgt1 chaperone with the MIS12 complex to stabilize this complex at the kinetochores and thus coordinates the recruitment of the NDC80 complex to form efficient microtubule-binding sites. Disruption of Sgt1 phosphorylation reduces the MIS12 and NDC80 complexes at the kinetochores, impairs stable microtubule attachment, and eventually results in chromosome misalignment to delay the anaphase onset. Our results demonstrate a mechanism for Plk1 in promoting kinetochore-microtubule attachment to ensure chromosome stability.     

Nsk1 ensures accurate chromosome segregation by promoting association of kinetochores to spindle poles during anaphase B

Type 1 phosphatase (PP1) antagonizes Aurora B kinase to stabilize kinetochore-microtubule attachments and to silence the spindle checkpoint. We screened for factors that exacerbate the growth defect of Δdis2 cells, which lack one of two catalytic subunits of PP1 in fission yeast, and identified Nsk1, a novel protein required for accurate chromosome segregation. During interphase, Nsk1 resides in the nucleolus but spreads throughout the nucleoplasm as cells enter mitosis. Following dephosphorylation by Clp1 (Cdc14-like) phosphatase and at least one other phosphatase, Nsk1 localizes to the interface between kinetochores and the inner face of the spindle pole body during anaphase. In the absence of Nsk1, some kinetochores become detached from spindle poles during anaphase B. If this occurs late in anaphase B, then the sister chromatids of unclustered kinetochores segregate to the correct daughter cell. These unclustered kinetochores are efficiently captured, retrieved, bioriented, and segregated during the following mitosis, as long as Dis2 is present. However, if kinetochores are detached from a spindle pole early in anaphase B, then these sister chromatids become missegregated. These data suggest Nsk1 ensures accurate chromosome segregation by promoting the tethering of kinetochores to spindle poles during anaphase B.     

Usp16 regulates kinetochore localization of Plk1 to promote proper chromosome alignment in mitosis

During the G2 to M phase transition, a portion of mitotic regulator Plk1 localizes to the kinetochores and regulates the initiation of kinetochore–microtubule attachments for proper chromosome alignment. Once kinetochore–microtubule attachment is achieved, this portion of Plk1 is removed from the kinetochores as a result of ubiquitination. However, the crucial molecular mechanism that promotes the localization and the maintenance of Plk1 on the kinetochores until metaphase is still unclear. We report that ubiquitin-specific peptidase 16 (Usp16) plays a key role during this process. Usp16 deubiquitinates Plk1, resulting in an enhanced interaction with kinetochore-localized proteins such as BubR1, and thereby retains Plk1 on the kinetochores to promote proper chromosome alignment in early mitosis. Down-regulation of Usp16 causes increased ubiquitination and decreased kinetochore localization of Plk1. Thus, our data unveil a unique mechanism by which Usp16 promotes the localization and maintenance of Plk1 on the kinetochores for proper chromosome alignment.     

The Lrs4-Csm1 monopolin complex associates with kinetochores during anaphase and is required for accurate chromosome segregation

Lrs4 and Csm1, components of the monopolin complex, localize to the rDNA where they regulate rDNA maintenance and segregation. During meiosis, the complex also associates with kinetochores to bring about sister kinetochore co-orientation, an essential aspect of meiosis I chromosome segregation. We show here that the Lrs4-Csm1 complex associates with kinetochores during mitosis. This kinetochore localization is observed during anaphase and depends on the on the Mitotic Exit Network, a signaling cascade essential for the completion of mitosis. Furthermore, we find that Lrs4 and Csm1 are important for chromosome segregation fidelity. Our results reveal a previously unanticipated function for Lrs4-Csm1 in mitotic chromosome segregation.Key words: mitosis, monopolin, Lrs4, Csm1, kinetochore, Mitotic Exit Network, chromosome segregation     

The CENP-F-like proteins HCP-1 and HCP-2 target CLASP to kinetochores to mediate chromosome segregation

During chromosome segregation, kinetochores form dynamic connections with spindle microtubules. In vertebrates, these attachments require the activities of a number of outer kinetochore proteins, including CENP-F [1, 2] and the widely conserved microtubule-associated protein CLASP [3]. Here, we investigate the functional relationship between HCP-1/2, two redundant CENP-F-like proteins, and CLASP(CLS-2) in Caenorhabditis elegans. HCP-1/2 and CLASP(CLS-2) localize transiently to mitotic C. elegans kinetochores with nearly identical kinetic profiles, and biochemical purifications demonstrate that they also associate physically. In embryos depleted of HCP-1/2, CLASP(CLS-2) no longer localizes to chromosomes, whereas CLASP(CLS-2) depletion does not prevent HCP-1/2 targeting. Consistent with the localization dependency and biochemical association, depletion of HCP-1/2 or CLASP(CLS-2) resulted in virtually identical defects in mitotic chromosome segregation characterized by a failure of sister-chromatid biorientation. This phenotype could be partially suppressed by disrupting the astral forces that pull spindle poles apart in the 1 cell embryo, indicating that CLASP(CLS-2) is required for biorientation when chromosome-spindle attachments are subjected to poleward force. Our results establish that the key role of HCP-1/2 is to target CLASP(CLS-2) to kinetochores, and they support the recently proposed model that CLASP functions to promote the polymerization of kinetochore bound microtubules [4].     

Penicillin-binding protein-related factor A is required for proper chromosome segregation in Bacillus subtilis

Previous work has shown that the ponA gene, encoding penicillin-binding protein 1 (PBP1), is in a two-gene operon with prfA (PBP-related factor A) (also called recU), which encodes a putative 206-residue basic protein (pI = 10.1) with no significant sequence homology to proteins with known functions. Inactivation of prfA results in cells that grow slower and vary significantly in length relative to wild-type cells. We now show that prfA mutant cells have a defect in chromosome segregation resulting in the production of approximately 0.9 to 3% anucleate cells in prfA cultures grown at 30 or 37 degrees C in rich medium and that the lack of PrfA exacerbates the chromosome segregation defect in smc and spoOJ mutant cells. In addition, overexpression of prfA was found to be toxic for and cause nucleoid condensation in Escherichia coli.     

Phosphorylation of histone H3 is required for proper chromosome condensation and segregation

Phosphorylation of histone H3 at serine 10 occurs during mitosis in diverse eukaryotes and correlates closely with mitotic and meiotic chromosome condensation. To better understand the function of H3 phosphorylation in vivo, we created strains of Tetrahymena in which a mutant H3 gene (S10A) was the only gene encoding the major H3 protein. Although both micronuclei and macronuclei contain H3 in typical nucleosomal structures, defects in nuclear divisions were restricted to mitotically dividing micronuclei; macronuclei, which are amitotic, showed no defects. Strains lacking phosphorylated H3 showed abnormal chromosome segregation, resulting in extensive chromosome loss during mitosis. During meiosis, micronuclei underwent abnormal chromosome condensation and failed to faithfully transmit chromosomes. These results demonstrate that H3 serine 10 phosphorylation is causally linked to chromosome condensation and segregation in vivo and is required for proper chromosome dynamics.     

The genomic stability regulator PTIP is required for proper chromosome segregation in mitosis

The centrosome is a multifunctional organelle that is known primarily for its microtubule organising function. Centrosomal defects caused by changes in centrosomal structure or number have been associated with human diseases ranging from congenital defects to cancer. We are only beginning to appreciate how the non-microtubule organising roles of the centrosome are related to these clinical conditions. In this review, we will discuss the historical evidence that led to the proposal that the centrosome participates in cell cycle regulation. We then summarize the body of work that describes the involvement of the mammalian centrosome in triggering cell cycle progression and checkpoint signalling. Then we will highlight work from the fission yeast model organism, revealing the molecular details that explain how the spindle pole body (SPB, the yeast functional equivalent of the centrosome), participates in these cell cycle transitions. Importantly, we will discuss some of the emerging questions from recent discoveries related to the role of the centrosome as a cell cycle regulator.

     

CENP-B is not critical for meiotic chromosome segregation in male mice

Centromere protein B (CENP-B) is a constitutive protein that binds to a highly conserved 17 bp motif located at most mammalian centromeres. To determine whether disruption of this gene affects chromosome segregation in male germ cells, we evaluated the frequencies of disomic and diploid sperm in CENP-B heterozygous and homozygous null mice using the mouse epididymal sperm aneuploidy (m-ESA) assay, a multicolor FISH method with probes for chromosomes X, Y and 8. The specificity and sensitivity of the m-ESA assay was demonstrated using Robertsonian (2.8) translocation heterozygotes as positive controls for sperm aneuploidy. Our results show that the frequencies of disomic and diploid sperm did not differ significantly between CENP-B heterozygous and homozygous null mice (P≥0.5) or from 129/Swiss isogenic mice (P≥0.5) and B6C3F1 mice (P≥0.2). These findings indicate that CENP-B does not have an essential role during chromosome segregation in male meiosis.     

The human Mis12 complex is required for kinetochore assembly and proper chromosome segregation

During cell division, kinetochores form the primary chromosomal attachment sites for spindle microtubules. We previously identified a network of 10 interacting kinetochore proteins conserved between Caenorhabditis elegans and humans. In this study, we investigate three proteins in the human network (hDsn1Q9H410, hNnf1PMF1, and hNsl1DC31). Using coexpression in bacteria and fractionation of mitotic extracts, we demonstrate that these proteins form a stable complex with the conserved kinetochore component hMis12. Human or chicken cells depleted of Mis12 complex subunits are delayed in mitosis with misaligned chromosomes and defects in chromosome biorientation. Aligned chromosomes exhibited reduced centromere stretch and diminished kinetochore microtubule bundles. Consistent with this, localization of the outer plate constituent Ndc80HEC1 was severely reduced. The checkpoint protein BubR1, the fibrous corona component centromere protein (CENP) E, and the inner kinetochore proteins CENP-A and CENP-H also failed to accumulate to wild-type levels in depleted cells. These results indicate that a four-subunit Mis12 complex plays an essential role in chromosome segregation in vertebrates and contributes to mitotic kinetochore assembly.