Phosphorylation- and polo-box-dependent binding of Plk1 to Bub1 is required for the kinetochore localization of Plk1

Polo-like kinase 1 (Plk1) is required for the generation of the tension-sensing 3F3/2 kinetochore epitope and facilitates kinetochore localization of Mad2 and other spindle checkpoint proteins. Here, we investigate the mechanism by which Plk1 itself is recruited to kinetochores. We show that Plk1 binds to budding uninhibited by benzimidazole 1 (Bub1) in mitotic human cells. The Plk1-Bub1 interaction requires the polo-box domain (PBD) of Plk1 and is enhanced by cyclin-dependent kinase 1 (Cdk1)-mediated phosphorylation of Bub1 at T609. The PBD-dependent binding of Plk1 to Bub1 facilitates phosphorylation of Bub1 by Plk1 in vitro. Depletion of Bub1 in HeLa cells by RNA interference (RNAi) diminishes the kinetochore localization of Plk1. Ectopic expression of the wild-type Bub1, but not the Bub1-T609A mutant, in Bub1-RNAi cells restores the kinetochore localization of Plk1. Our results suggest that phosphorylation of Bub1 at T609 by Cdk1 creates a docking site for the PBD of Plk1 and facilitates the kinetochore recruitment of Plk1.     

Kinetochore microtubule dynamics and the metaphase-anaphase transition

We have quantitatively studied the dynamic behavior of kinetochore fiber microtubules (kMTs); both turnover and poleward transport (flux) in metaphase and anaphase mammalian cells by fluorescence photoactivation. Tubulin derivatized with photoactivatable fluorescein was microinjected into prometaphase LLC-PK and PtK1 cells and allowed to incorporate to steady-state. A fluorescent bar was generated across the MTs in a half-spindle of the mitotic cells using laser irradiation and the kinetics of fluorescence redistribution were determined in terms of a double exponential decay process. The movement of the activated zone was also measured along with chromosome movement and spindle elongation. To investigate the possible regulation of MT transport at the metaphase-anaphase transition, we performed double photoactivation analyses on the same spindles as the cell advanced from metaphase to anaphase. We determined values for the turnover of kMTs (t1/2 = 7.1 +/- 2.4 min at 30 degrees C) and demonstrated that the turnover of kMTs in metaphase is approximately an order of magnitude slower than that for non-kMTs. In anaphase, kMTs become dramatically more stable as evidenced by a fivefold increase in the fluorescence redistribution half-time (t1/2 = 37.5 +/- 8.5 min at 30 degrees C). Our results also indicate that MT transport slows abruptly at anaphase onset to one-half the metaphase value. In early anaphase, MT depolymerization at the kinetochore accounted, on average, for 84% of the rate of chromosome movement toward the pole whereas the relative contribution of MT transport and depolymerization at the pole contributed 16%. These properties reflect a dramatic shift in the dynamic behavior of kMTs at the metaphase-anaphase transition. A release-capture model is presented in which the stability of kMTs is increased at the onset of anaphase through a reduction in the probability of MT release from the kinetochore. The reduction in MT transport at the metaphase-anaphase transition suggests that motor activity and/or subunit dynamics at the centrosome are subject to modulation at this key cell cycle point.     

CCT chaperonin complex is required for the biogenesis of functional Plk1

Experiments from several different organisms have demonstrated that polo-like kinases are involved in many aspects of mitosis and cytokinesis. Here, we provide evidence to show that Plk1 associates with chaperonin-containing TCP1 complex (CCT) both in vitro and in vivo. Silencing of CCT by use of RNA interference (RNAi) in mammalian cells inhibits cell proliferation, decreases cell viability, causes cell cycle arrest with 4N DNA content, and leads to apoptosis. Depletion of CCT in well-synchronized HeLa cells causes cell cycle arrest at G(2), as demonstrated by a low mitotic index and Cdc2 activity. Complete depletion of Plk1 in well-synchronized cells also leads to G(2) block, suggesting that misfolded Plk1 might be responsible for the failure of CCT-depleted cells to enter mitosis. Moreover, partial depletion of CCT or Plk1 leads to mitotic arrest. Finally, the CCT-depleted cells reenter the cell cycle upon reintroduction of the purified constitutively active form of Plk1, indicating that Plk1 might be a CCT substrate.     

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.     

MISP is a novel Plk1 substrate required for proper spindle orientation and mitotic progression

Precise positioning of the mitotic spindle determines the correct cell division axis and is crucial for organism development. Spindle positioning is mediated through a cortical machinery by capturing astral microtubules, thereby generating pushing/pulling forces at the cell cortex. However, the molecular link between these two structures remains elusive. Here we describe a previously uncharacterized protein, MISP (C19orf21), as a substrate of Plk1 that is required for correct mitotic spindle positioning. MISP is an actin-associated protein throughout the cell cycle. MISP depletion led to an impaired metaphase-to-anaphase transition, which depended on phosphorylation by Plk1. Loss of MISP induced mitotic defects including spindle misorientation accompanied by shortened astral microtubules. Furthermore, we find that MISP formed a complex with and regulated the cortical distribution of the +TIP binding protein p150^glued, a subunit of the dynein–dynactin complex. We propose that Plk1 phosphorylates MISP, thus stabilizing cortical and astral microtubule attachments required for proper mitotic spindle positioning.     

Detection of protein kinase activity specifically activated at metaphase-anaphase transition

We have previously reported that Ser13 and Ser34 on glial fibrillary acidic protein (GFAP) in the cleavage furrow of glioma cells are phosphorylated during late mitotic phase (Matsuoka, Y., K. Nishizawa, T. Yano, M. Shibata, S. Ando, T. Takahashi, and M. Inagaki. 1992, EMBO (Eur. Mol. Biol. Organ.) J. 11:2895-2902). This observation implies a possibility that there is a protein kinase specifically activated at metaphase-anaphase transition. To further analyze the cell cycle- dependent GFAP phosphorylation, we prepared monoclonal antibodies KT13 and KT34 which recognize the phosphorylation of GFAP at Ser13 and Ser34, respectively. Immunocytochemical studies with KT13 and KT34 revealed that the GFAP phosphorylation in the cleavage furrow during late mitotic phase occurred not only in glioma cells but also in human SW-13 and mouse Ltk- cells in which GFAP was ectopically expressed, thus the phosphorylation can be monitored in a wide range of cell types. Furthermore, we detected kinase activity which phosphorylates GFAP at Ser13 and Ser34 in the lysates of late mitotic cells but not in those of interphase cells or early mitotic cells. These results suggest that there exists a protein kinase which is specifically activated at the transition of metaphase to anaphase not only in GFAP-expressing cells but also in cells without GFAP.     

Cep152 interacts with Plk4 and is required for centriole duplication

Centrioles are microtubule-based structures that organize the centrosome and nucleate cilia. Centrioles duplicate once per cell cycle, and duplication requires Plk4, a member of the Polo-like kinase family; however, the mechanism linking Plk4 activity and centriole formation is unknown. In this study, we show in human and frog cells that Plk4 interacts with the centrosome protein Cep152, the orthologue of Drosophila melanogaster Asterless. The interaction requires the N-terminal 217 residues of Cep152 and the crypto Polo-box of Plk4. Cep152 and Plk4 colocalize at the centriole throughout the cell cycle. Overexpression of Cep152 (1-217) mislocalizes Plk4, but both Cep152 and Plk4 are able to localize to the centriole independently of the other. Depletion of Cep152 prevents both normal centriole duplication and Plk4-induced centriole amplification and results in a failure to localize Sas6 to the centriole, an early step in duplication. Cep152 can be phosphorylated by Plk4 in vitro, suggesting that Cep152 acts with Plk4 to initiate centriole formation.     

A polo match for Plk1

New work by Elia et al. in this issue of Cell reveals the molecular basis of phosphopeptide recognition by the polo domain and the domain's dual function to promote substrate recognition by targeting the kinase to subcellular structures and to autoregulate the adjacent protein kinase catalytic domain.     

Plk1 negatively regulates PRC1 to prevent premature midzone formation before cytokinesis

To achieve mitosis and cytokinesis, microtubules must assemble into distinct structures at different stages of cell division-mitotic spindles to segregate the chromosomes before anaphase and midzones to keep sister genomes apart and guide the cleavage furrow after anaphase. This temporal regulation is believed to involve Cdk1 kinase, which is inactivated in a switch-like way after anaphase. We found that inhibiting Plk1 caused premature assembly of midzones in cells still in metaphase, breaking the temporal regulation of microtubules. The antiparallel microtubule-bundling protein PRC1 plays a key role in organizing the midzone complex. We found that Plk1 negatively regulates PRC1 through phosphorylation of a single site, Thr-602, near the C-terminus of PRC1. We also found that microtubules stimulated Thr-602 phosphorylation by Plk1. This creates a potential negative feedback loop controlling PRC1 activity. It also made the extent of Thr-602 phosphorylation during mitotic arrest dependent on the mechanism of the arresting drug. Unexpectedly, we could not detect a preanaphase regulatory role for Cdk1 sites on PRC1. We suggest that PRC1 is regulated by Plk1, rather than Cdk1 as previously proposed, because its activity must be spatiotemporally regulated both preanaphase and postanaphase, and Cdk1 activity is too binary for this purpose.     

OsAM1 is required for leptotene-zygotene transition in rice

The events occurring at the onset of meiosis have not been fully elucidated. In the present study, OsAM1 was identified in rice (Oryza sativa L.) by map-based cloning. OsAM1, a homolog of Arabidopsis SWI1 and maize AM1, encodes a protein with a coiled-coil domain in its central region. In the Osam1 mutant, pollen mother cells are arrested at leptotene, showing that OsAM1 is required for the leptotene-zygotene transition. Immunocytological analysis revealed that OsAM1 exists as foci in early prophase I meiocytes. Very faint OsREC8 foci persisted in the Osam1 mutant, indicating that OsAM1 is not required for the initial meiotic recruitment of OsREC8. In the absence of OsAM1, many other critical meiotic components, including PAIR2, ZEP1 and OsMER3, could not be correctly installed onto chromosomes. In contrast, in pair2, Osmer3 and zep1 mutants, OsAM1 could be loaded normally, suggesting that OsAM1 plays a fundamental role in building the proper chromosome structure at the beginning of meiosis.     

Coronin-1 function is required for phagosome formation

Coronin-1 is an actin-associated protein whose function in actin dynamics has remained obscure. All coronin proteins have a variable N-terminal domain, followed by WD repeats and a C-terminal coiled-coil dimerization domain. Transfection of coronin-1-GFP into RAW 264.7 cells revealed that coronin rapidly and transiently associates with the phagosome. To determine if coronin is involved in mammalian phagocytosis we used a dominant-negative approach by expressing only the central WD domains. However, this caused cell rounding and dissociation from the substratum, hampering analysis of their phenotype. We therefore developed TAT-fusion constructs of coronin-1 WD domains to acutely introduce the recombinant protein fragment into live cells. We show that although TAT-WD has no effect on binding of opsonized RBCs to RAW 264.7 cells, receptor clustering or several downstream signaling events, lamellipodial extensions, and actin accumulation at the base of the bound particle were diminished. Furthermore, Arp3 accumulation at the phagosome was impaired after TAT-WD treatment. Interestingly, whereas coronin-1 also accumulates at the sites of actin remodeling associated with Salmonella invasion, TAT-WD had no effect on this process. Together, our data demonstrates that coronin-1 is required for an early step in phagosome formation, consistent with a role in actin polymerization.     

INCENP is required for proper targeting of Survivin to the centromeres and the anaphase spindle during mitosis

Three lines of investigation have suggested that interactions between Survivin and the chromosomal passenger proteins INCENP and Aurora-B kinase may be important for mitotic progression. First, interference with the function of Survivin/BIR1, INCENP, or Aurora-B kinase leads to similar defects in mitosis and cytokinesis [1-7] (see [8] for review). Second, INCENP and Aurora-B exist in a complex in Xenopus eggs [9] and in mammalian cultured cells [7]. Third, interference with Survivin or INCENP function causes Aurora-B kinase to be mislocalized in mitosis in both C. elegans and vertebrates [5, 7, 9]. Here, we provide evidence that Survivin, Aurora-B, and INCENP interact physically and functionally. Direct visualization of Survivin-GFP in mitotic cells reveals that it localizes identically to INCENP and Aurora-B. Survivin binds directly to both Aurora-B and INCENP in yeast two-hybrid and in vitro pull-down assays. The in vitro interaction between Survivin and Aurora-B is extraordinarily stable in that it resists 3 M NaCl. Finally, Survivin and INCENP interact functionally in vivo; in cells in which INCENP localization is disrupted, Survivin adheres to the chromosomes and no longer concentrates at the centromeres or transfers to the anaphase spindle midzone. Our data provide the first biochemical evidence that Survivin can interact directly with members of the chromosomal passenger complex.     

Self-regulated Plk1 recruitment to kinetochores by the Plk1-PBIP1 interaction is critical for proper chromosome segregation

The polo-box domain (PBD) of mammalian polo-like kinase 1 (Plk1) is essential in targeting its catalytic activity to specific subcellular structures critical for mitosis. The mechanism underlying Plk1 recruitment to the kinetochores and the role of Plk1 at this site remain elusive. Here, we demonstrate that a PBD-binding protein, PBIP1, is crucial for recruiting Plk1 to the interphase and mitotic kinetochores. Unprecedentedly, Plk1 phosphorylated PBIP1 at T78, creating a self-tethering site that specifically interacted with the PBD of Plk1, but not Plk2 or Plk3. Later in mitosis, Plk1 also induced PBIP1 degradation in a T78-dependent manner, thereby enabling itself to interact with other components critical for proper kinetochore functions. Absence of the p-T78-dependent Plk1 localization induced a chromosome congression defect and compromised the spindle checkpoint, ultimately leading to aneuploidy. Thus, Plk1 self-regulates the Plk1-PBIP1 interaction to timely localize to the kinetochores and promote proper chromosome segregation.     

The centrosomal kinase Plk1 localizes to the transition zone of primary cilia and induces phosphorylation of nephrocystin-1

Polo-like kinase (Plk1) plays a central role in regulating the cell cycle. Plk1-mediated phosphorylation is essential for centrosome maturation, and for numerous mitotic events. Although Plk1 localizes to multiple subcellular sites, a major site of action is the centrosomes, which supports mitotic functions in control of bipolar spindle formation. In G0 or G1 untransformed cells, the centriolar core of the centrosome differentiates into the basal body of the primary cilium. Primary cilia are antenna-like sensory organelles dynamically regulated during the cell cycle. Whether Plk1 has a role in ciliary biology has never been studied. Nephrocystin-1 (NPHP1) is a ciliary protein; loss of NPHP1 in humans causes nephronophthisis (NPH), an autosomal-recessive cystic kidney disease. We here demonstrate that Plk1 colocalizes with nephrocystin-1 to the transition zone of primary cilia in epithelial cells. Plk1 co-immunoprecipitates with NPHP1, suggesting it is part of the nephrocystin protein complex. We identified a candidate Plk1 phosphorylation motif (D/E-X-S/T-φ-X-D/E) in nephrocystin-1, and demonstrated in vitro that Plk1 phosphorylates the nephrocystin N-terminus, which includes the specific PLK1 phosphorylation motif. Further, induced disassembly of primary cilia rapidly evoked Plk1 kinase activity, while small molecule inhibition of Plk1 activity or RNAi-mediated downregulation of Plk1 limited the first and second phase of ciliary disassembly. These data identify Plk1 as a novel transition zone signaling protein, suggest a function of Plk1 in cilia dynamics, and link Plk1 to the pathogenesis of NPH and potentially other cystic kidney diseases.     

From Plk1 to Plk5: Functional evolution of polo-like kinases

Mammalian polo-like kinases (Plks) are characterized by the presence of an N-terminal protein kinase domain and a C-terminal polo-box domain (PBD) involved in substrate binding and regulation of kinase activity. Plk1-4 have traditionally been linked to cell cycle progression, genotoxic stress and, more recently, neuron biology. Recently, a fifth mammalian Plk family member, Plk5, has been characterized in murine and human cells. Plk5 is expressed mainly in differentiated tissues such as the cerebellum. Despite apparent loss of catalytic activity and a stop codon in the middle of the human gene, Plk5 proteins retain important functions in neuron biology. Notably, its expression is silenced by epigenetic alterations in brain tumors, such as glioblastomas, and its re-expression prevents cell proliferation of these tumor cells. In this review, we will focus on the non-cell cycle roles of Plks, the biology of the new member of the family and the possible kinase- and PBD-independent functions of polo-like kinases.Key words: cell cycle, kinase evolution, neuron differentiation, polo-box domain, polo-like kinases, tumor suppression     

PICH, a centromere-associated SNF2 family ATPase, is regulated by Plk1 and required for the spindle checkpoint

We identify PICH (Plk1-interacting checkpoint "helicase"), a member of the SNF2 ATPase family, as an interaction partner and substrate of Plk1. Following phosphorylation of PICH on the Cdk1 site T1063, Plk1 is recruited to PICH and controls its localization. Starting in prometaphase, PICH accumulates at kinetochores and inner centromeres. Moreover, it decorates threads that form during metaphase before increasing in length and progressively diminishing during anaphase. PICH-positive threads connect sister kinetochores and are dependent on tension, sensitive to DNase, and exacerbated in response to premature loss of cohesins or inhibition of topoisomerase II, suggesting that they represent stretched centromeric chromatin. Depletion of PICH causes the selective loss of Mad2 from kinetochores and completely abrogates the spindle checkpoint, resulting in massive chromosome missegregation. These data identify PICH as a novel essential component of checkpoint signaling. We propose that PICH binds to catenated centromere-related DNA to monitor tension developing between sister kinetochores.     

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.     

Tex14, a Plk1-regulated protein, is required for kinetochore-microtubule attachment and regulation of the spindle assembly checkpoint

Proper assembly of kinetochores (KTs) during mitosis is required for bipolar attachment of spindle microtubules (MTs) and the accumulation of spindle assembly checkpoint (SAC) components. Here we show that testis-expressed protein 14 (Tex14), which has been implicated in midbody function, is recruited to KTs by Plk1 in a Cdk1-dependent manner during early mitosis. Exclusion of Tex14 from kinetochores results in an inability to efficiently localize outer KT components, impaired KT-MT attachment, chromosome congression defects, and whole-chromosome instability. In addition, we demonstrate that phosphorylation of Tex14 by Plk1 during metaphase promotes APC(Cdc20)-mediated Tex14 degradation. Inhibition of this phosphorylation event causes retention of Tex14 at KTs and results in delayed metaphase-to-anaphase transition and chromosome segregation defects. Our findings identify Tex14 as an important mediator of KT structure and function and the fidelity of chromosome separation.     

Mad2-independent spindle assembly checkpoint activation and controlled metaphase-anaphase transition in Drosophila S2 cells

The spindle assembly checkpoint is essential to maintain genomic stability during cell division. We analyzed the role of the putative Drosophila Mad2 homologue in the spindle assembly checkpoint and mitotic progression. Depletion of Mad2 by RNAi from S2 cells shows that it is essential to prevent mitotic exit after spindle damage, demonstrating its conserved role. Mad2-depleted cells also show accelerated transit through prometaphase and premature sister chromatid separation, fail to form metaphases, and exit mitosis soon after nuclear envelope breakdown with extensive chromatin bridges that result in severe aneuploidy. Interestingly, preventing Mad2-depleted cells from exiting mitosis by a checkpoint-independent arrest allows congression of normally condensed chromosomes. More importantly, a transient mitotic arrest is sufficient for Mad2-depleted cells to exit mitosis with normal patterns of chromosome segregation, suggesting that all the associated phenotypes result from a highly accelerated exit from mitosis. Surprisingly, if Mad2-depleted cells are blocked transiently in mitosis and then released into a media containing a microtubule poison, they arrest with high levels of kinetochore-associated BubR1, properly localized cohesin complex and fail to exit mitosis revealing normal spindle assembly checkpoint activity. This behavior is specific for Mad2 because BubR1-depleted cells fail to arrest in mitosis under these experimental conditions. Taken together our results strongly suggest that Mad2 is exclusively required to delay progression through early stages of prometaphase so that cells have time to fully engage the spindle assembly checkpoint, allowing a controlled metaphase-anaphase transition and normal patterns of chromosome segregation.