Roles of polo-like kinase 1 in the assembly of functional mitotic spindles

BACKGROUND: The stable association of chromosomes with both poles of the mitotic spindle (biorientation) depends on spindle pulling forces. These forces create tension across sister kinetochores and are thought to stabilize microtubule-kinetochore interactions and to silence the spindle checkpoint. Polo-like kinase 1 (Plk1) has been implicated in regulating centrosome maturation, mitotic entry, sister chromatid cohesion, the anaphase-promoting complex/cyclosome (APC/C), and cytokinesis, but it is unknown if Plk1 controls chromosome biorientation. RESULTS: We have analyzed Plk1 functions in synchronized mammalian cells by RNA interference (RNAi). Plk1-depleted cells enter mitosis after a short delay, accumulate in a preanaphase state, and subsequently often die by apoptosis. Spindles in Plk1-depleted cells lack focused poles and are not associated with centrosomes. Chromosomes attach to these spindles, but the checkpoint proteins Mad2, BubR1, and CENP-E are enriched at many kinetochores. When Plk1-depleted cells are treated with the Aurora B inhibitor Hesperadin, which silences the spindle checkpoint by stabilizing microtubule-kinetochore interactions, cells degrade APC/C substrates and exit mitosis without chromosome segregation and cytokinesis. Experiments with monopolar spindles that are induced by the kinesin inhibitor Monastrol indicate that Plk1 is required for the assembly of spindles that are able to generate poleward pulling forces. CONCLUSIONS: Our results imply that Plk1 is not essential for mitotic entry and APC/C activation but is required for proper spindle assembly and function. In Plk1-depleted cells spindles may not be able to create enough tension across sister kinetochores to stabilize microtubule-kinetochore interactions and to silence the spindle checkpoint.     

Plk1 Regulates Both ASAP Localization and Its Role in Spindle Pole Integrity

Bipolar spindle formation is essential for faithful chromosome segregation at mitosis. Because centrosomes define spindle poles, abnormal number and structural organization of centrosomes can lead to loss of spindle bipolarity and genetic integrity. ASAP (aster-associated protein or MAP9) is a centrosome- and spindle-associated protein, the deregulation of which induces severe mitotic defects. Its phosphorylation by Aurora A is required for spindle assembly and mitosis progression. Here, we show that ASAP is localized to the spindle poles by Polo-like kinase 1 (Plk1) (a mitotic kinase that plays an essential role in centrosome regulation and mitotic spindle assembly) through the γ-TuRC-dependent pathway. We also demonstrate that ASAP is a novel substrate of Plk1 phosphorylation and have identified serine 289 as the major phosphorylation site by Plk1 in vivo. ASAP phosphorylated on serine 289 is localized to centrosomes during mitosis, but this phosphorylation is not required for its Plk1-dependent localization at the spindle poles. We show that phosphorylated ASAP on serine 289 contributes to spindle pole stability in a microtubule-dependent manner. These data reveal a novel function of ASAP in centrosome integrity. Our results highlight dual ASAP regulation by Plk1 and further confirm the importance of ASAP for spindle pole organization, bipolar spindle assembly, and mitosis.     

Polo-like kinase 1 triggers the initiation of cytokinesis in human cells by promoting recruitment of the RhoGEF Ect2 to the central spindle

Cytokinesis of animal cells requires ingression of the actomyosin-based contractile ring between segregated sister genomes. Localization of the RhoGEF Ect2 to the central spindle at anaphase promotes local activation of the RhoA GTPase, which induces assembly and ingression of the contractile ring. Here we have used BI 2536, an inhibitor of the mitotic kinase Plk1, to analyze the functions of this enzyme during late mitosis in human cells. We show that Plk1 acts after Cdk1 inactivation and independently from Aurora B to promote RhoA accumulation at the equator, contractile ring formation, and cleavage furrow ingression. Inhibition of Plk1 abolishes the interaction of Ect2 with its activator and midzone anchor, HsCyk-4, thereby preventing localization of Ect2 to the central spindle. We propose that late mitotic Plk1 activity promotes recruitment of Ect2 to the central spindle, triggering the initiation of cytokinesis and contributing to cleavage plane specification in human cells.     

Furry Protein Promotes Aurora A-mediated Polo-like Kinase 1 Activation

Bipolar mitotic spindle organization is fundamental to faithful chromosome segregation. Furry (Fry) is an evolutionarily conserved protein implicated in cell division and morphology. In human cells, Fry localizes to centrosomes and spindle microtubules in early mitosis, and depletion of Fry causes multipolar spindle formation. However, it remains unknown how Fry controls bipolar spindle organization. This study demonstrates that Fry binds to polo-like kinase 1 (Plk1) through the polo-box domain of Plk1 in a manner dependent on the cyclin-dependent kinase 1-mediated Fry phosphorylation at Thr-2516. Fry also binds to Aurora A and promotes Plk1 activity by binding to the polo-box domain of Plk1 and by facilitating Aurora A-mediated Plk1 phosphorylation at Thr-210. Depletion of Fry causes centrosome and centriole splitting in mitotic spindles and reduces the kinase activity of Plk1 in mitotic cells and the accumulation of Thr-210-phosphorylated Plk1 at the spindle poles. Our results suggest that Fry plays a crucial role in the structural integrity of mitotic centrosomes and in the maintenance of spindle bipolarity by promoting Plk1 activity at the spindle poles in early mitosis.     

Plk1 Inhibition Causes Post-Mitotic DNA Damage and Senescence in a Range of Human Tumor Cell Lines

Plk1 is a checkpoint protein whose role spans all of mitosis and includes DNA repair, and is highly conserved in eukaryotes from yeast to man. Consistent with this wide array of functions for Plk1, the cellular consequences of Plk1 disruption are diverse, spanning delays in mitotic entry, mitotic spindle abnormalities, and transient mitotic arrest leading to mitotic slippage and failures in cytokinesis. In this work, we present the in vitro and in vivo consequences of Plk1 inhibition in cancer cells using potent, selective small-molecule Plk1 inhibitors and Plk1 genetic knock-down approaches. We demonstrate for the first time that cellular senescence is the predominant outcome of Plk1 inhibition in some cancer cell lines, whereas in other cancer cell lines the dominant outcome appears to be apoptosis, as has been reported in the literature. We also demonstrate strong induction of DNA double-strand breaks in all six lines examined (as assayed by γH2AX), which occurs either during mitotic arrest or mitotic-exit, and may be linked to the downstream induction of senescence. Taken together, our findings expand the view of Plk1 inhibition, demonstrating the occurrence of a non-apoptotic outcome in some settings. Our findings are also consistent with the possibility that mitotic arrest observed as a result of Plk1 inhibition is at least partially due to the presence of unrepaired double-strand breaks in mitosis. These novel findings may lead to alternative strategies for the development of novel therapeutic agents targeting Plk1, in the selection of biomarkers, patient populations, combination partners and dosing regimens.     

Mio depletion links mTOR regulation to Aurora A and Plk1 activation at mitotic centrosomes

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.     

sSgo1, a major splice variant of Sgo1, functions in centriole cohesion where it is regulated by Plk1

Shugoshin 1 (Sgo1) functions as a protector of centromeric cohesion of sister chromatids in higher eukaryotes. Here, we provide evidence for a previously unrecognized role for Sgo1 in centriole cohesion. Sgo1 depletion via RNA interference induces the formation of multiple centrosome-like structures in mitotic cells that result from the separation of paired centrioles. Sgo1+/- mitotic murine embryonic fibroblasts display split centrosomes. Localization study of two major endogenous splice variants of Sgo1 indicates that the smaller variant, sSgo1, is found at the centrosome in interphase and at spindle poles in mitosis. sSgo1 interacts with Plk1 and its spindle pole localization is Plk1 dependent. Centriole splitting induced by Sgo1 depletion or expression of a dominant negative mutant is suppressed by ectopic expression of sSgo1 or by Plk1 knockdown. Our studies strongly suggest that sSgo1 plays an essential role in protecting centriole cohesion, which is partly regulated by Plk1.     

Regulation of sister chromatid cohesion between chromosome arms

Sister chromatid separation in anaphase depends on the removal of cohesin complexes from chromosomes. In vertebrates, the bulk of cohesin is already removed from chromosome arms during prophase and prometaphase, whereas cohesin remains at centromeres until metaphase, when cohesin is cleaved by the protease separase. In unperturbed mitoses, arm cohesion nevertheless persists throughout metaphase and is principally sufficient to maintain sister chromatid cohesion. How arm cohesion is maintained until metaphase is unknown. Here we show that small amounts of cohesin can be detected in the interchromatid region of metaphase chromosome arms. If prometaphase is prolonged by treatment of cells with microtubule poisons, these cohesin complexes dissociate from chromosome arms, and arm cohesion is dissolved. If cohesin dissociation in prometaphase-arrested cells is prevented by depletion of Plk1 or inhibition of Aurora B, arm cohesion is maintained. These observations imply that, in unperturbed mitoses, small amounts of cohesin maintain arm cohesion until metaphase. When cells lacking Plk1 and Aurora B activity enter anaphase, chromatids lose cohesin. This loss is prevented by proteasome inhibitors, implying that it depends on separase activation. Separase may therefore be able to cleave cohesin at centromeres and on chromosome arms.     

Purification and proteomic identification of putative upstream regulators of polo-like kinase-1 from mitotic cell extracts

Polo-like kinase-1 (Plk1) is phosphorylated on Thr210 for activation during mitosis. Here, we investigated the question of which kinase(s) is the specific upstream kinase of mitotic Plk1. Upstream kinases of Plk1 were purified from mitotic cell extracts through column chromatography procedures, and identified by mass spectrometry. Candidates for Plk1 kinase included p21-activated kinase, aurora A, and mammalian Ste20-like kinases. Immunoprecipitates of these proteins from mitotic cell extracts phosphorylated Plk1 on Thr210. Even if the activity of Aurora A was blocked with a specific inhibitor, Plk1 phosphorylation still occurred, suggesting that function of Plk1 could be controlled by these kinases for proper mitotic progression, as well as by Aurora A in very late G2 phase for the beginning of mitosis.

Structured abstract

MINT-7996332: PAK1 (uniprotkb:Q13153) physically interacts (MI:0915) with PLK1 (uniprotkb:P53350) by pull down (MI:0096)MINT-7996345: PAK3 (uniprotkb:O75914) physically interacts (MI:0915) with PLK1 (uniprotkb:P53350) by pull down (MI:0096)     

Inhibition of Plk1 induces mitotic infidelity and embryonic growth defects in developing zebrafish embryos

Polo-like kinase 1 (Plk1) is central to cell division. Here, we report that Plk1 is critical for mitosis in the embryonic development of zebrafish. Using a combination of several cell biology tools, including single-cell live imaging applied to whole embryos, we show that Plk1 is essential for progression into mitosis during embryonic development. Plk1 morphant cells displayed mitotic infidelity, such as abnormal centrosomes, irregular spindle assembly, hypercondensed chromosomes, and a failure of chromosome arm separation. Consequently, depletion of Plk1 resulted in mitotic arrest and finally death by 6 days post-fertilization. In comparison, Plk2 or Plk3 morphant embryos did not display any significant abnormalities. Treatment of embryos with the Plk1 inhibitor, BI 2536, caused a block in mitosis, which was more severe when used to treat plk1 morphants. Finally, using an assay to rescue the Plk1 morphant phenotype, we found that the kinase domain and PBD domains are both necessary for Plk1 function in zebrafish development. Our studies demonstrate that Plk1 is required for embryonic proliferation because its activity is crucial for mitotic integrity. Furthermore, our study suggests that zebrafish will be an efficient and economical in vivo system for the validation of anti-mitotic drugs.     

Shugoshin 1 Plays a Central Role in Kinetochore Assembly and is Required for Kinetochore Targeting of Plk1

Physical connection between the sister chromatids is mediated by the cohesin protein complex. During prophase, cohesin is removed from the chromosome arms while the centromeres remain united. Shugoshin1 (Sgo1) is required for maintenance of centromeric cohesion from prophase to the metaphase-anaphase transition. Furthermore, Sgo1 has been proposed to regulate kinetochore microtubule stability and sense interkinetochore tension, two tasks which are tightly coupled with the function of the Chromosomal Passenger Complex (CPC) and Polo-like kinase 1 (Plk1). Here we show that depletion or chemical inhibition of Aurora B kinase (AurB), the catalytic subunit of the CPC, disrupts accumulation of Sgo1 on the kinetochores in HeLa cells and causes Sgo1 to localize on the chromosome arms. RNAi assays show that depletion of Sgo1 did not affect AurB localization but diminished Plk1 kinetochore binding. Furthermore, we demonstrate that vertebrate Sgo1 is phosphorylated by both AurB and Plk1 in vitro. The data presented here includes an extensive analysis of kinetochore targeting interdependencies of mitotic proteins that propose a novel branch in kinetochore assembly where Sgo1 and Plk1 have central roles. Furthermore our studies implicate Sgo1 in the tension sensing mechanism of the spindle checkpoint by regulating Plk1 kinetochore affinity.     

Stathmin and microtubules regulate mitotic entry in HeLa cells by controlling activation of both Aurora kinase A and Plk1

Depletion of stathmin, a microtubule (MT) destabilizer, delays mitotic entry by ∼4 h in HeLa cells. Stathmin depletion reduced the activity of CDC25 and its upstream activators, Aurora A and Plk1. Chemical inhibition of both Aurora A and Plk1 was sufficient to delay mitotic entry by 4 h, while inhibiting either kinase alone did not cause a delay. Aurora A and Plk1 are likely regulated downstream of stathmin, because the combination of stathmin knockdown and inhibition of Aurora A and Plk1 was not additive and again delayed mitotic entry by 4 h. Aurora A localization to the centrosome required MTs, while stathmin depletion spread its localization beyond that of γ-tubulin, indicating an MT-dependent regulation of Aurora A activation. Plk1 was inhibited by excess stathmin, detected in in vitro assays and cells overexpressing stathmin–cyan fluorescent protein. Recruitment of Plk1 to the centrosome was delayed in stathmin-depleted cells, independent of MTs. It has been shown that depolymerizing MTs with nocodazole abrogates the stathmin-depletion induced cell cycle delay; in this study, depolymerization with nocodazole restored Plk1 activity to near normal levels, demonstrating that MTs also contribute to Plk1 activation. These data demonstrate that stathmin regulates mitotic entry, partially via MTs, to control localization and activation of both Aurora A and Plk1.     

The Plk1-dependent Phosphoproteome of the Early Mitotic Spindle

Polo-like kinases regulate many aspects of mitotic and meiotic progression from yeast to man. In early mitosis, mammalian Polo-like kinase 1 (Plk1) controls centrosome maturation, spindle assembly, and microtubule attachment to kinetochores. However, despite the essential and diverse functions of Plk1, the full range of Plk1 substrates remains to be explored. To investigate the Plk1-dependent phosphoproteome of the human mitotic spindle, we combined stable isotope labeling by amino acids in cell culture with Plk1 inactivation or depletion followed by spindle isolation and mass spectrometry. Our study identified 358 unique Plk1-dependent phosphorylation sites on spindle proteins, including novel substrates, illustrating the complexity of the Plk1-dependent signaling network. Over 100 sites were validated by in vitro phosphorylation of peptide arrays, resulting in a broadening of the Plk1 consensus motif. Collectively, our data provide a rich source of information on Plk1-dependent phosphorylation, Plk1 docking to substrates, the influence of phosphorylation on protein localization, and the functional interaction between Plk1 and Aurora A on the early mitotic spindle.During mitosis, multiple processes, such as mitotic entry, spindle assembly, chromosome segregation, and cytokinesis, must be carefully coordinated to ensure the error-free distribution of chromosomes into the newly forming daughter cells. The physical separation of the chromosomes to opposite poles of the cell is driven by the mitotic spindle, a proteinaceous and highly dynamic microtubule (MT)1-based macromolecular machine. Spindle assembly begins early in mitosis and is completed when the bipolar attachment of microtubules to kinetochore (KT) pairs is achieved (1, 2). Polo-like kinase 1 (Plk1), a serine/threonine-specific kinase first identified in Drosophila (3), is one of the key regulators of this essential mitotic process and has therefore attracted much attention (4–6). In agreement with its diverse functions, the localization of Plk1 during mitosis is dynamic. Plk1 first associates with centrosomes in prophase before it localizes to spindle poles and KTs in prometaphase and metaphase. During anaphase, Plk1 is recruited to the central spindle and finally accumulates at the midbody during telophase. Proteomics studies using oriented peptide libraries have shown that two so-called polo boxes at the C-terminal end of Plk1, the polo box domain (PBD), are crucial for the localization of this kinase to cellular structures (7, 8). This domain binds to specific phosphorylated sequence motifs that are created by other priming kinases or are self-primed by Plk1 itself, thus providing an efficient mechanism to regulate localization and substrate selectivity in time and space (9–11).Despite the pleiotropic and critical functions of Plk1 during mitosis, only a limited number of target proteins and phosphorylation sites on substrates have so far been identified or studied in detail (4–6, 12). The difficulties in identification of bona fide Plk1 substrates stem from the low abundance of some substrates, technical limitations for determining in vivo phosphorylation sites, the requirement for Plk1 localization for recognition of some substrates, and the possibility that Plk1 may phosphorylate a broader consensus motif than determined previously (13). Recent developments in mass spectrometry (MS)-based proteomics have allowed the identification of a large number of in vivo phosphorylation sites from complex samples (14). However, the nature of the kinase(s) responsible for most of these phosphorylation events is still unclear, and the assignment of phosphorylation sites to individual kinases remains a challenging task. Previously, we explored the human mitotic spindle by MS and successfully identified a large number of novel spindle proteins and phosphorylation sites (15, 16). Now, the development of quantitative methods to monitor in vivo phosphorylation changes in complex samples (17–19) represents a unique opportunity to address the role of individual kinases in spindle function.To study Plk1 function at the mitotic spindle, we combined quantitative proteomics using stable isotope labeling by amino acids in cell culture (SILAC) (20) with the isolation of human mitotic spindles and phosphopeptide enrichment. To expand the experimental coverage of Plk1 substrates and gain further insight into direct and indirect functions of Plk1, we compared the phosphoproteomes of mitotic spindles isolated from cells lacking Plk1 activity with spindles from cells with fully active kinase. Two independent approaches were used to interfere with Plk1 activity: protein depletion using an inducible small hairpin (shRNA) cell line and selective inhibition of the kinase by the small molecule inhibitor ZK-thiazolidinone (TAL) (21). Phosphorylation sites found to be down-regulated after Plk1 inhibition/depletion were subsequently validated using in vitro phosphorylation of synthetic peptide arrays. This approach identified many candidate Plk1 substrates, allowed confirmation of direct phosphorylation by Plk1 of more than 100 sites identified in vivo, and suggested a broader phosphorylation consensus motif for this kinase. Collectively, our data set provides a rich resource for in-depth studies on the spindle-associated Plk1-dependent phosphoproteome. This is illustrated by selective follow-up studies in which we validated the Plk1-dependent localization of substrates to centrosomes and kinetochores. In particular, using a phosphospecific antibody, we confirmed Plk1-dependent CENP-F phosphorylation in vivo and demonstrated that CENP-F localization to kinetochores depends on Plk1 kinase activity. Furthermore, we identified several Aurora A-dependent phosphorylation events that are regulated by Plk1, supporting the emerging view of an intimate functional relationship between Plk1 and Aurora A kinase (22, 23).     

有丝分裂激酶Aurora B的研究进展

真核生物细胞通过有丝分裂将遗传物质均等地分配到两个子细胞中,从而维持基因组的稳定性。有丝分裂的每一环节都需要精准而细致的调控,这依赖于一系列调节机制,尤其需要多个相关激酶的共同协调。Aurora B是一个关键的有丝分裂调控激酶,伴随有丝分裂的进行,其先后在染色体臂、内着丝粒、中央纺锤体、中体上动态分布。与其高度时空动态性相一致的是,Aurora B在有丝分裂的多个环节,如姐妹染色体粘连、动粒微管连接、纺锤体检验点和胞质分裂过程中都发挥着一系列重要功能。本文将概述近年来Aurora B激酶功能与调控方面的研究进展。     

Bub3p Facilitates Spindle Checkpoint Silencing in Fission Yeast

Although critical for spindle checkpoint signaling, the role kinetochores play in anaphase promoting complex (APC) inhibition remains unclear. Here we show that spindle checkpoint proteins are severely depleted from unattached kinetochores in fission yeast cells lacking Bub3p. Surprisingly, a robust mitotic arrest is maintained in the majority of bub3Δ cells, yet they die, suggesting that Bub3p is essential for successful checkpoint recovery. During recovery, two defects are observed: (1) cells mis-segregate chromosomes and (2) anaphase onset is significantly delayed. We show that Bub3p is required to activate the APC upon inhibition of Aurora kinase activity in checkpoint-arrested cells, suggesting that Bub3p is required for efficient checkpoint silencing downstream of Aurora kinase. Together, these results suggest that spindle checkpoint signals can be amplified in the nucleoplasm, yet kinetochore localization of spindle checkpoint components is required for proper recovery from a spindle checkpoint-dependent arrest.     

A High-Content Small Molecule Screen Identifies Sensitivity of Glioblastoma Stem Cells to Inhibition of Polo-Like Kinase 1

Glioblastoma multiforme (GBM) is the most common primary brain cancer in adults and there are few effective treatments. GBMs contain cells with molecular and cellular characteristics of neural stem cells that drive tumour growth. Here we compare responses of human glioblastoma-derived neural stem (GNS) cells and genetically normal neural stem (NS) cells to a panel of 160 small molecule kinase inhibitors. We used live-cell imaging and high content image analysis tools and identified JNJ-10198409 (J101) as an agent that induces mitotic arrest at prometaphase in GNS cells but not NS cells. Antibody microarrays and kinase profiling suggested that J101 responses are triggered by suppression of the active phosphorylated form of polo-like kinase 1 (Plk1) (phospho T210), with resultant spindle defects and arrest at prometaphase. We found that potent and specific Plk1 inhibitors already in clinical development (BI 2536, BI 6727 and GSK 461364) phenocopied J101 and were selective against GNS cells. Using a porcine brain endothelial cell blood-brain barrier model we also observed that these compounds exhibited greater blood-brain barrier permeability in vitro than J101. Our analysis of mouse mutant NS cells (INK4a/ARF^−/−, or p53^−/−), as well as the acute genetic deletion of p53 from a conditional p53 floxed NS cell line, suggests that the sensitivity of GNS cells to BI 2536 or J101 may be explained by the lack of a p53-mediated compensatory pathway. Together these data indicate that GBM stem cells are acutely susceptible to proliferative disruption by Plk1 inhibitors and that such agents may have immediate therapeutic value.     

A MEK-independent role for CRAF in mitosis and tumor progression

RAF kinases regulate cell proliferation and survival and can be dysregulated in tumors. The role of RAF in cell proliferation has been linked to its ability to activate mitogen-activated protein kinase kinase 1 (MEK) and mitogen-activated protein kinase 1 (ERK). Here we identify a MEK-independent role for RAF in tumor growth. Specifically, in mitotic cells, CRAF becomes phosphorylated on Ser338 and localizes to the mitotic spindle of proliferating tumor cells in vitro as well as in murine tumor models and in biopsies from individuals with cancer. Treatment of tumors with allosteric inhibitors, but not ATP-competitive RAF inhibitors, prevents CRAF phosphorylation on Ser338 and localization to the mitotic spindle and causes cell-cycle arrest at prometaphase. Furthermore, we identify phospho-Ser338 CRAF as a potential biomarker for tumor progression and a surrogate marker for allosteric RAF blockade. Mechanistically, CRAF, but not BRAF, associates with Aurora kinase A (Aurora-A) and Polo-like kinase 1 (Plk1) at the centrosomes and spindle poles during G2/M. Indeed, allosteric or genetic inhibition of phospho-Ser338 CRAF impairs Plk1 activation and accumulation at the kinetochores, causing prometaphase arrest, whereas a phospho-mimetic Ser338D CRAF mutant potentiates Plk1 activation, mitosis and tumor progression in mice. These findings show a previously undefined role for RAF in tumor progression beyond the RAF-MEK-ERK paradigm, opening new avenues for targeting RAF in cancer.     

The small-molecule inhibitor BI 2536 reveals novel insights into mitotic roles of polo-like kinase 1

BACKGROUND: The mitotic kinases, Cdk1, Aurora A/B, and Polo-like kinase 1 (Plk1) have been characterized extensively to further understanding of mitotic mechanisms and as potential targets for cancer therapy. Cdk1 and Aurora kinase studies have been facilitated by small-molecule inhibitors, but few if any potent Plk1 inhibitors have been identified. RESULTS: We describe the cellular effects of a novel compound, BI 2536, a potent and selective inhibitor of Plk1. The fact that BI 2536 blocks Plk1 activity fully and instantaneously enabled us to study controversial and unknown functions of Plk1. Cells treated with BI 2536 are delayed in prophase but eventually import Cdk1-cyclin B into the nucleus, enter prometaphase, and degrade cyclin A, although BI 2536 prevents degradation of the APC/C inhibitor Emi1. BI 2536-treated cells lack prophase microtubule asters and thus polymerize mitotic microtubules only after nuclear-envelope breakdown and form monopolar spindles that do not stably attach to kinetochores. Mad2 accumulates at kinetochores, and cells arrest with an activated spindle-assembly checkpoint. BI 2536 prevents Plk1's enrichment at kinetochores and centrosomes, and when added to metaphase cells, it induces detachment of microtubules from kinetochores and leads to spindle collapse. CONCLUSIONS: Our results suggest that Plk1's accumulation at centrosomes and kinetochores depends on its own activity and that this activity is required for maintaining centrosome and kinetochore function. Our data also show that Plk1 is not required for prophase entry, but delays transition to prometaphase, and that Emi1 destruction in prometaphase is not essential for APC/C-mediated cyclin A degradation.     

Kicking off the polo game

Polo-like kinase 1 (Plk1) is essential for checkpoint recovery and the activation of key mitotic enzymes; however, its own activation mechanism has remained elusive. Recent findings show that Bora, a G(2)-M expressed protein, facilitates Plk1 activation by the oncogenic kinase Aurora A in G(2). During mitosis, Plk1-dependent Bora degradation promotes Aurora A localization to the centrosome and/or spindle. Bora-dependent regulation provides important new insights into interactions between key mitotic kinases.     

A unique insertion in STARD9's motor domain regulates its stability

STARD9 is a largely uncharacterized mitotic kinesin and putative cancer target that is critical for regulating pericentriolar material cohesion during bipolar spindle assembly. To begin to understand the mechanisms regulating STARD9 function and their importance to cell division, we took a multidisciplinary approach to define the cis and trans factors that regulate the stability of the STARD9 motor domain. We show that, unlike the other ∼50 mammalian kinesins, STARD9 contains an insertion in loop 12 of its motor domain (MD). Working with the STARD9-MD, we show that it is phosphorylated in mitosis by mitotic kinases that include Plk1. These phosphorylation events are important for targeting a pool of STARD9-MD for ubiquitination by the SCFβ-TrCP ubiquitin ligase and proteasome-dependent degradation. Of interest, overexpression of nonphosphorylatable/nondegradable STARD9-MD mutants leads to spindle assembly defects. Our results with STARD9-MD imply that in vivo the protein levels of full-length STARD9 could be regulated by Plk1 and SCFβ-TrCP to promote proper mitotic spindle assembly.