Centrosome Reduplication Research Articles

The Crk4-Cyc4 complex regulates G2/M transition in Toxoplasma gondii

A versatile division of apicomplexan parasites and a dearth of conserved regulators have hindered the progress of apicomplexan cell cycle studies. While most apicomplexans divide in a multinuclear fashion, Toxoplasma gondii tachyzoites divide in the traditional binary mode. We previously identified five Toxoplasma CDK-related kinases (Crk). Here, we investigated TgCrk4 and its cyclin partner TgCyc4. We demonstrated that TgCrk4 regulates conventional G2 phase processes, such as repression of chromosome rereplication and centrosome reduplication, and acts upstream of the spindle assembly checkpoint. The spatial TgCyc4 dynamics supported the TgCrk4–TgCyc4 complex role in the coordination of chromosome and centrosome cycles. We also identified a dominant TgCrk4–TgCyc4 complex interactor, TgiRD1 protein, related to DNA replication licensing factor CDT1 but played no role in licensing DNA replication in the G1 phase. Our results showed that TgiRD1 also plays a role in controlling chromosome and centrosome reduplication. Global phosphoproteome analyses identified TgCrk4 substrates, including TgORC4, TgCdc20, TgGCP2, and TgPP2ACA. Importantly, the phylogenetic and structural studies suggest the Crk4–Cyc4 complex is limited to a minor group of the binary dividing apicomplexans.

Structural basis for the ORC1-Cyclin A association.

Progression of cell cycle is regulated by sequential expression of cyclins, which associate with distinct cyclin kinases to drive the transition between different cell cycle phases. The complex of Cyclin A with cyclin-dependent kinase 2 (CDK2) controls the DNA replication activity through phosphorylation of a set of chromatin factors, which critically influences the S phase transition. It has been shown that the direct interaction between the Cyclin A-CDK2 complex and origin recognition complex subunit 1 (ORC1) mediates the localization of ORC1 to centrosomes, where ORC1 inhibits cyclin E-mediated centrosome reduplication. However, the molecular basis underlying the specific recognition between ORC1 and cyclins remains elusive. Here we report the crystal structure of Cyclin A-CDK2 complex bound to a peptide derived from ORC1 at 2.54 å resolution. The structure revealed that the ORC1 peptide interacts with a hydrophobic groove, termed cyclin binding groove (CBG), of Cyclin A via a KXL motif. Distinct from other identified CBG-binding sequences, an arginine residue flanking the KXL motif of ORC1 inserts into a neighboring acidic pocket, contributing to the strong ORC1-Cyclin A association. Furthermore, structural and sequence analysis of cyclins reveals divergence on the ORC1-binding sites, which may underpin their differential ORC1-binding activities. This study provides a structural basis of the specific ORC1-cyclins recognition, with implication in development of novel inhibitors against the cyclin/CDK complexes.

LRWD1 Regulates Microtubule Nucleation and Proper Cell Cycle Progression in the Human Testicular Embryonic Carcinoma Cells.

Leucine-rich repeats and WD repeat domain containing protein 1 (LRWD1) is a testis-specific protein that mainly expressed in the sperm neck where centrosome is located. By using microarray analysis, LRWD1 is identified as a putative gene that involved in spermatogenesis. However, its role in human male germ cell development has not been extensively studied. When checking in the semen of patients with asthenozoospermia, teratozoospermia, and asthenoteratozoospermia, the level of LRWD1 in the sperm neck was significantly reduced with a defective neck or tail. When checking the sub-cellular localization of LRWD1 in the cells, we found that LRWD1 resided in the centrosome and its centrosomal residency was independent of microtubule transportation in NT2/D1, the human testicular embryonic carcinoma, cell line. Depletion of LRWD1 did not induce centrosome re-duplication but inhibited microtubule nucleation. In addition, the G1 arrest were observed in LRWD1 deficient NT2/D1 cells. Upon LRWD1 depletion, the levels of cyclin E, A, and phosphorylated CDK2, were reduced. Overexpression of LRWD1 promoted cell proliferation in NT2/D1, HeLa, and 239T cell lines. In addition, we also observed that autophagy was activated in LRWD1 deficient cells and inhibition of autophagy by chloroquine or bafilomycin A1 promoted cell death when LRWD1 was depleted. Thus, we found a novel function of LRWD1 in controlling microtubule nucleation and cell cycle progression in the human testicular embryonic carcinoma cells. J. Cell. Biochem. 119: 314-326, 2018. © 2017 Wiley Periodicals, Inc.

Investigating The Spatiotemporal Distribution Patterns of PCID2 between The Nucleus And The Centrosome

PCID2 is a subunit of the TREX‐2 mRNA export complex that is also involved in the process of CRM1‐mediated nuclear protein export. Nuclear export is driven by RanGTP, which triggers a structural modification of CRM1 resulting in increased affinity for the leucine‐rich NES. Subsequent GTP hydrolysis following translocation across the NPC causes the CRM1‐cargo complex to dissociate. Karyopherins can also serve functional roles at non‐nuclear locations in the cell. For example, both CRM1 and Ran are found at the centrosome in addition to the nucleus. Specific factors facilitate mitosis by restricting the centrosome to duplicate once during the cell cycle through alternating nuclear and centrosomal localization. Specifically, CRM1 has an integral role in localizing nucleophosmin to the centrosomes to facilitate this “licensing” process. Leptomycin B inhibition of CRM1 causes dissociation of nucleophosmin from the centrosomes. In doing so it leads to centrosome reduplication within the same cell cycle. In addition to its nuclear function, PCID2 is also localized to the centrosome in a subset of cells. The function of PCID2 at the centrosome is not yet elucidated, but we demonstrate here that PCID2 is in fact present in CRM1 complexes. This evidence gives us reason to believe that PCID2 may serve a role at the centrosome that is supplementary to that of CRM1 and Ran. We hypothesize that PCID2 is localized to the centrosome as a part of the CRM1‐RanGTP complex and may translocate from the nucleus to the centrosome. To investigate the relationship between PCID2 in the nucleus and at the centrosome, we performed immunofluorescence of PCID2 in HeLa cells treated with Leptomycin B, a CRM1‐dependent nuclear export inhibitor. Preliminary results shows that LMB treatment causes a decrease in nuclear intensity of CRM1 and a potential decrease in PCID2, while causing an increase in the incidence of cells exhibiting punctate staining of PCID2. Because we know that LMB treatment leads to unregulated duplication of centrosomes, these findings suggest a correlation between increased PCID2 punctate staining and multiple centrosome duplications. This is consistent with our previous work showing that PCID2 is most commonly localized to the centrosome in G2 when duplication is occurring. These findings could also imply that PCID2 has a role in the localization of centrosomal components or mitotic proteins to the centrosome from the nucleus. Future experiments will investigate whether increased PCID2 punctate staining is indicative of a role in excess centrosome duplication or whether LMB is instead altering the CRM1/PCID2 localization pattern, causing centrosomal accumulation of PCID2.

Cdc14 phosphatase directs centrosome re-duplication at the meiosis I to meiosis II transition in budding yeast.

Background Gametes are generated through a specialized cell division called meiosis, in which ploidy is reduced by half because two consecutive rounds of chromosome segregation, meiosis I and meiosis II, occur without intervening DNA replication. This contrasts with the mitotic cell cycle where DNA replication and chromosome segregation alternate to maintain the same ploidy. At the end of mitosis, CDKs are inactivated. This low CDK state in late mitosis/G1 allows for critical preparatory events for DNA replication and centrosome/spindle pole body (SPB) duplication. However, their execution is inhibited until S phase, where further preparatory events are also prevented. This "licensing" ensures that both the chromosomes and the centrosomes/SPBs replicate exactly once per cell cycle, thereby maintaining constant ploidy. Crucially, between meiosis I and meiosis II, centrosomes/SPBs must be re-licensed, but DNA re-replication must be avoided. In budding yeast, the Cdc14 protein phosphatase triggers CDK down regulation to promote exit from mitosis. Cdc14 also regulates the meiosis I to meiosis II transition, though its mode of action has remained unclear. Methods Fluorescence and electron microscopy was combined with proteomics to probe SPB duplication in cells with inactive or hyperactive Cdc14. Results We demonstrate that Cdc14 ensures two successive nuclear divisions by re-licensing SPBs at the meiosis I to meiosis II transition. We show that Cdc14 is asymmetrically enriched on a single SPB during anaphase I and provide evidence that this enrichment promotes SPB re-duplication. Cells with impaired Cdc14 activity fail to promote extension of the SPB half-bridge, the initial step in morphogenesis of a new SPB. Conversely, cells with hyper-active Cdc14 duplicate SPBs, but fail to induce their separation. Conclusion Our findings implicate reversal of key CDK-dependent phosphorylations in the differential licensing of cyclical events at the meiosis I to meiosis I transition.

Cdc14 phosphatase directs centrosome re-duplication at the meiosis I to meiosis II transition in budding yeast

Background: Gametes are generated through a specialized cell division called meiosis, in which ploidy is reduced by half because two consecutive rounds of chromosome segregation, meiosis I and meiosis II, occur without intervening DNA replication. This contrasts with the mitotic cell cycle where DNA replication and chromosome segregation alternate to maintain the same ploidy. At the end of mitosis, cyclin-dependent kinases (CDKs) are inactivated. This low CDK state in late mitosis/G1 allows for critical preparatory events for DNA replication and centrosome/spindle pole body (SPB) duplication. However, their execution is inhibited until S phase, where further preparatory events are also prevented. This “licensing” ensures that both the chromosomes and the centrosomes/SPBs replicate exactly once per cell cycle, thereby maintaining constant ploidy. Crucially, between meiosis I and meiosis II, centrosomes/SPBs must be re-licensed, but DNA re-replication must be avoided. In budding yeast, the Cdc14 protein phosphatase triggers CDK down regulation to promote exit from mitosis. Cdc14 also regulates the meiosis I to meiosis II transition, though its mode of action has remained unclear. Methods: Fluorescence and electron microscopy was combined with proteomics to probe SPB duplication in cells with inactive or hyperactive Cdc14. Results: We demonstrate that Cdc14 ensures two successive nuclear divisions by re-licensing SPBs at the meiosis I to meiosis II transition. We show that Cdc14 is asymmetrically enriched on a single SPB during anaphase I and provide evidence that this enrichment promotes SPB re-duplication. Cells with impaired Cdc14 activity fail to promote extension of the SPB half-bridge, the initial step in morphogenesis of a new SPB. Conversely, cells with hyper-active Cdc14 duplicate SPBs, but fail to induce their separation. Conclusion: Our findings implicate reversal of key CDK-dependent phosphorylations in the differential licensing of cyclical events at the meiosis I to meiosis II transition.

Cdc14 phosphatase directs centrosome re-duplication at the meiosis I to meiosis II transition in budding yeast

BackgroundGametes are generated through a specialized cell division called meiosis, in which ploidy is reduced by half because two consecutive rounds of chromosome segregation, meiosis I and meiosis II, occur without intervening DNA replication. This contrasts with the mitotic cell cycle where DNA replication and chromosome segregation alternate to maintain the same ploidy. At the end of mitosis, CDKs are inactivated. This low CDK state in late mitosis/G1 allows for critical preparatory events for DNA replication and centrosome/spindle pole body (SPB) duplication. However, their execution is inhibited until S phase, where further preparatory events are also prevented. This “licensing” ensures that both the chromosomes and the centrosomes/SPBs replicate exactly once per cell cycle, thereby maintaining constant ploidy. Crucially, between meiosis I and meiosis II, centrosomes/SPBs must be re-licensed, but DNA re-replication must be avoided. In budding yeast, the Cdc14 protein phosphatase triggers CDK down regulation to promote exit from mitosis. Cdc14 also regulates the meiosis I to meiosis II transition, though its mode of action has remained unclear.MethodsFluorescence and electron microscopy was combined with proteomics to probe SPB duplication in cells with inactive or hyperactive Cdc14.ResultsWe demonstrate that Cdc14 ensures two successive nuclear divisions by re-licensing SPBs at the meiosis I to meiosis II transition. We show that Cdc14 is asymmetrically enriched on a single SPB during anaphase I and provide evidence that this enrichment promotes SPB re-duplication. Cells with impaired Cdc14 activity fail to promote extension of the SPB half-bridge, the initial step in morphogenesis of a new SPB. Conversely, cells with hyper-active Cdc14 duplicate SPBs, but fail to induce their separation.ConclusionOur findings implicate reversal of key CDK-dependent phosphorylations in the differential licensing of cyclical events at the meiosis I to meiosis I transition.

Abstract 3786: Rad6 inhibition enhances paclitaxel sensitivity of triple negative breast cancer cells by inducing mitotic spindle defects

Abstract Triple negative breast cancers (TNBC) lack estrogen receptors, and progesterone receptors, and HER2/neu receptor amplifications, making targeted therapies unsuitable. The taxane based drug paclitaxel (PTX) is used as a first line chemotherapy. PTX is a microtubule stabilizer that induces G2/M arrest and mitotic catastrophe. Loss of BRCA1 often seen in TNBC patients is associated with PTX resistance. Rad6 is an E2 ubiquitin conjugating enzyme that has two human homologues, Rad6A (UBE2A) and Rad6B (UBE2B), and high Rad6B expression rather than Rad6A is associated with poor survival of TNBC patients. Rad6 is associated with centrosomes at all phases of the cell cycle, and constitutive overexpression of Rad6B in nontransformed MCF10A breast cells leads to centrosome amplification and aneuploidy. Inhibition of Rad6 enzymatic activity, with our novel Rad6-selective small molecule inhibitor (SMI#9) induces G2/M arrest, much like PTX. We hypothesize that the overexpression of Rad6B commonly found in BRCA1 wt and BRCA1 mut TNBCs contributes to taxane resistance by promoting centrosome amplification and enhancing microtubule dynamics. Thus, targeting Rad6 will confer taxane sensitivity by preventing centrosome reduplication and/or microtubule hypernucleation, strengthening G2/M arrest, and ensuring terminal mitotic catastrophe. Immunostaining shows Rad6 is overexpressed in BRCA1 wt and BRCA1 mut breast cancer tissues and TNBC cell lines. Immunostaining of the centrosomal protein pericentrin shows centrosome amplification in both BRCA1 wt and BRCA1 mut TNBC cells. MTT data show SMI#9 synergistically sensitizes both BRCA1 wt (MDA-MB-468) and BRCA1 mut (HCC1937) TNBC cell lines to PTX. Colony forming assays corroborate these data and show that addition of SMI#9 diminishes colony forming efficiency of PTX pretreated MDA-MB-468 cells. SMI#9 treatment of HCC1937 PTX resistant colonies results in increases in cells with enlarged and multiple nuclei (characteristic of mitotic catastrophe). Western blot analysis of Tau, a marker of PTX sensitivity, indicates PTX, SMI#9, and PTX+SMI#9 treatments decrease the steady state levels of the 1N3R and 0N4R, or 1N3R Tau isoforms in MDA-MB-468 and HCC1937 cells, respectively. Analysis of cyclin B1, a marker of G2/M arrest, shows increases in cyclin B1 levels in PTX, SMI#9, and PTX+SMI#9 treated MDA-MB-468 cells, whereas cyclin B1 is degraded in HCC1937 cells treated with these agents (indicative of mitotic catastrophe). Immunofluorescence staining shows that Tau localization to the mitotic spindles is unaffected by PTX, SMI#9, or PTX+SMI#9. However, treatments including SMI#9 resulted in mitotic cells with defective or monopolar mitotic spindles. These data implicate a role for Rad6 in centrosome duplication/separation and provide mechanistic support for inhibiting Rad6 to enhance PTX sensitivity. Supported by NCI R21 CA178117 and T32-CA009531. Citation Format: Brittany Haynes, Kristen Cunningham, Malathy Shekhar. Rad6 inhibition enhances paclitaxel sensitivity of triple negative breast cancer cells by inducing mitotic spindle defects. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3786.

The GTPase RAN regulates multiple steps of the centrosome life cycle.

Growing lines of evidence implicate the small GTPase RAN, its regulators and effectors--predominantly, nuclear transport receptors--in practically all aspects of centrosome biology in mammalian cells. These include duplication licensing, cohesion, positioning, and microtubule-nucleation capacity. RAN cooperates with the protein nuclear export vector exportin 1/CRM1 to recruit scaffolding proteins containing nuclear export sequences that play roles in the structural organization of centrosomes. Together, they also limit centrosome reduplication by regulating the localization of key "licensing" proteins during the centrosome duplication cycle. In parallel, RAN also regulates the capacity of centrosomes to nucleate and organize functional microtubules, and this predominanlty involves importin vectors: many factors regulating microtubule nucleation or function harbor nuclear localization sequences that interact with importin molecules and such interaction inhibits their activity. Active RANGTP binding to importin molecules removes the inhibition and releases microtubule regulatory factors in the free productive form. A dynamic scenario emerges, in which RAN is pivotal in linking spatiotemporal control of centrosome regulators to the cell cycle machinery.

Centrin 3 is an inhibitor of centrosomal Mps1 and antagonizes centrin 2 function.

Centrins are a family of small, calcium-binding proteins with diverse cellular functions that play an important role in centrosome biology. We previously identified centrin 2 and centrin 3 (Cetn2 and Cetn3) as substrates of the protein kinase Mps1. However, although Mps1 phosphorylation sites control the function of Cetn2 in centriole assembly and promote centriole overproduction, Cetn2 and Cetn3 are not functionally interchangeable, and we show here that Cetn3 is both a biochemical inhibitor of Mps1 catalytic activity and a biological inhibitor of centrosome duplication. In vitro, Cetn3 inhibits Mps1 autophosphorylation at Thr-676, a known site of T-loop autoactivation, and interferes with Mps1-dependent phosphorylation of Cetn2. The cellular overexpression of Cetn3 attenuates the incorporation of Cetn2 into centrioles and centrosome reduplication, whereas depletion of Cetn3 generates extra centrioles. Finally, overexpression of Cetn3 reduces Mps1 Thr-676 phosphorylation at centrosomes, and mimicking Mps1-dependent phosphorylation of Cetn2 bypasses the inhibitory effect of Cetn3, suggesting that the biological effects of Cetn3 are due to the inhibition of Mps1 function at centrosomes.

Centriolar satellite- and hMsd1/SSX2IP-dependent microtubule anchoring is critical for centriole assembly.

Centriolar satellites are numerous electron-dense granules dispersed around the centrosome. Mutations in their components are linked to various human diseases, but their molecular roles remain elusive. In particular, the significance of spatial communication between centriolar satellites and the centrosome is unknown. hMsd1/SSX2IP localizes to both the centrosome and centriolar satellites and is required for tethering microtubules to the centrosome. Here we show that hMsd1/SSX2IP-mediated microtubule anchoring is essential for proper centriole assembly and duplication. On hMsd1/SSX2IP knockdown, the centriolar satellites become stuck at the microtubule minus end near the centrosome. Intriguingly, these satellites contain many proteins that normally localize to the centrosome. Of importance, microtubule structures, albeit not being anchored properly, are still required for the emergence of abnormal satellites, as complete microtubule depolymerization results in the disappearance of these aggregates from the vicinity of the centrosome. We highlighted, using superresolution and electron microscopy, that under these conditions, centriole structures are faulty. Remarkably, these cells are insensitive to Plk4 overproduction-induced ectopic centriole formation, yet they accelerate centrosome reduplication upon hydroxyurea arrest. Finally, the appearance of satellite aggregates is cancer cell specific. Together our findings provide novel insights into the mechanism of centriole assembly and microtubule anchoring.

RABL6A, a Novel RAB-Like Protein, Controls Centrosome Amplification and Chromosome Instability in Primary Fibroblasts

RABL6A (RAB-like 6 isoform A) is a novel protein that was originally identified based on its association with the Alternative Reading Frame (ARF) tumor suppressor. ARF acts through multiple p53-dependent and p53-independent pathways to prevent cancer. How RABL6A functions, to what extent it depends on ARF and p53 activity, and its importance in normal cell biology are entirely unknown. We examined the biological consequences of RABL6A silencing in primary mouse embryo fibroblasts (MEFs) that express or lack ARF, p53 or both proteins. We found that RABL6A depletion caused centrosome amplification, aneuploidy and multinucleation in MEFs regardless of ARF and p53 status. The centrosome amplification in RABL6A depleted p53−/− MEFs resulted from centrosome reduplication via Cdk2-mediated hyperphosphorylation of nucleophosmin (NPM) at threonine-199. Thus, RABL6A prevents centrosome amplification through an ARF/p53-independent mechanism that restricts NPM-T199 phosphorylation. These findings demonstrate an essential role for RABL6A in centrosome regulation and maintenance of chromosome stability in non-transformed cells, key processes that ensure genomic integrity and prevent tumorigenesis.

Abnormal centrosomal structure and duplication in Cep135-deficient vertebrate cells

Centrosomes are key microtubule-organizing centers that contain a pair of centrioles, conserved cylindrical, microtubule-based structures. Centrosome duplication occurs once per cell cycle and relies on templated centriole assembly. In many animal cells this process starts with the formation of a radially symmetrical cartwheel structure. The centrosomal protein Cep135 localizes to this cartwheel, but its role in vertebrates is not well understood. Here we examine the involvement of Cep135 in centriole function by disrupting the Cep135 gene in the DT40 chicken B-cell line. DT40 cells that lack Cep135 are viable and show no major defects in centrosome composition or function, although we note a small decrease in centriole numbers and a concomitant increase in the frequency of monopolar spindles. Furthermore, electron microscopy reveals an atypical structure in the lumen of Cep135-deficient centrioles. Centrosome amplification after hydroxyurea treatment increases significantly in Cep135-deficient cells, suggesting an inhibitory role for the protein in centrosome reduplication during S-phase delay. We propose that Cep135 is required for the structural integrity of centrioles in proliferating vertebrate cells, a role that also limits centrosome amplification in S-phase-arrested cells.

Meier-Gorlin syndrome mutations disrupt an Orc1 CDK inhibitory domain and cause centrosome reduplication

Like DNA replication, centrosomes are licensed to duplicate once per cell division cycle to ensure genetic stability. In addition to regulating DNA replication, the Orc1 subunit of the human origin recognition complex controls centriole and centrosome copy number. Here we report that Orc1 harbors a PACT centrosome-targeting domain and a separate domain that differentially inhibits the protein kinase activities of Cyclin E-CDK2 and Cyclin A-CDK2. A cyclin-binding motif (Cy motif) is required for Orc1 to bind Cyclin A and inhibit Cyclin A-CDK2 kinase activity but has no effect on Cyclin E-CDK2 kinase activity. In contrast, Orc1 inhibition of Cyclin E-CDK2 kinase activity occurs by a different mechanism that is affected by Orc1 mutations identified in Meier-Gorlin syndrome patients. The cyclin/CDK2 kinase inhibitory domain of Orc1, when tethered to the PACT domain, localizes to centrosomes and blocks centrosome reduplication. Meier-Gorlin syndrome mutations that disrupt Cyclin E-CDK2 kinase inhibition also allow centrosome reduplication. Thus, Orc1 contains distinct domains that control centrosome copy number and DNA replication. We suggest that the Orc1 mutations present in some Meier-Gorlin syndrome patients contribute to the pronounced microcephaly and dwarfism observed in these individuals by altering centrosome duplication in addition to DNA replication defects.

A p27Kip1 mutant that does not inhibit CDK activity promotes centrosome amplification and micronucleation

Mitotic catastrophe occurs when cells enter mitosis with damaged DNA or excess centrosomes. Cells overexpressing the centrosome protein CP110 or depleted of cyclin F, which targets CP110 for destruction, have more than two centrosomes and undergo mitotic catastrophe. Our studies show centrosome reduplication and mitotic catastrophe in osteosarcoma cells inducibly expressing a p27Kip1 mutant (termed p27K) that binds cyclins but not cyclin-dependent kinases (CDKs). p27K inhibited cell proliferation but not CDK activity or cell cycle progression. It did not induce apoptosis; however, cells expressing p27K had more than two centrosomes and, indicative of mitotic catastrophe, irregularly shaped nuclei or multiple micronuclei. p27K interacted with cyclin F in vivo (as did endogenous p27Kip1) and displaced cyclin F from CP110. Depletion of CP110 rescued p27K-expressing cells from centrosome reduplication and mitotic catastrophe. Collectively, our data show that p27Kip1 can perturb mitosis and suggest that it does so by sequestering cyclin F, which prevents its interaction with and the subsequent degradation of CP110, ultimately resulting in centrosome reduplication, mitotic catastrophe and abrogation of cell proliferation.

An Integrated In Vitro and In Vivo High-Throughput Screen Identifies Treatment Leads for Ependymoma

Using a mouse model of ependymoma-a chemoresistant brain tumor-we combined multicell high-throughput screening (HTS), kinome-wide binding assays, and invivo efficacy studies, to identify potential treatments with predicted toxicity against neural stem cells (NSC). We identified kinases within the insulin signaling pathway and centrosome cycle as regulators of ependymoma cell proliferation, and their corresponding inhibitors as potential therapies. FDA approved drugs not currently used to treat ependymoma were also identified that posses selective toxicity against ependymoma cells relative to normal NSCs both invitro and invivo, e.g., 5-fluorouracil. Our comprehensive approach advances understanding of the biology and treatment of ependymoma including the discovery of several treatment leads for immediate clinical translation.

The SCF–FBXW5 E3-ubiquitin ligase is regulated by PLK4 and targets HsSAS-6 to control centrosome duplication

Deregulated centrosome duplication can result in genetic instability and contribute to tumorigenesis. Here, we show that centrosome duplication is regulated by the activity of an E3-ubiquitin ligase that employs the F-box protein FBXW5 (ref. 3) as its targeting subunit. Depletion of endogenous FBXW5 or overexpression of an F-box-deleted mutant version results in centrosome overduplication and formation of multipolar spindles. We identify the centriolar protein HsSAS-6 (refs 4,5) as a critical substrate of the SCF-FBXW5 complex. FBXW5 binds HsSAS-6 and promotes its ubiquitylation in vivo. The activity of SCF-FBXW5 is in turn negatively regulated by Polo-like kinase 4 (PLK4), which phosphorylates FBXW5 at Ser 151 to suppress its ability to ubiquitylate HsSAS-6. FBXW5 is a cell-cycle-regulated protein with expression levels peaking at the G1/S transition. We show that FBXW5 levels are controlled by the anaphase-promoting (APC/C) complex, which targets FBXW5 for degradation during mitosis and G1, thereby helping to reset the centrosome duplication machinery. In summary, we show that a cell-cycle-regulated SCF complex is regulated by the kinase PLK4, and that this in turn restricts centrosome re-duplication through degradation of the centriolar protein HsSAS-6.

The cyclin A centrosomal localization sequence recruits MCM5 and Orc1 to regulate centrosome reduplication

Centrosomes are the major microtubule-organizing centers in animal cells and regulate formation of a bipolar mitotic spindle. Aberrant centrosome number causes chromosome mis-segregation, and has been implicated in genomic instability and tumor development. Previous studies have demonstrated a role for the DNA replication factors MCM5 and Orc1 in preventing centrosome reduplication. Cyclin A-Cdk2 localizes on centrosomes by means of a modular centrosomal localization sequence (CLS) that is distinct from that of cyclin E. Here, we show that cyclin A interacts with both MCM5 and Orc1 in a CLS-dependent but Cdk-independent manner. Although the MRAIL hydrophobic patch is contained within the cyclin A CLS, binding of both MCM5 and Orc1 to cyclin A does not require a wild-type hydrophobic patch. The same domain in MCM5 that mediates interaction with cyclin E also binds cyclin A, resulting in centrosomal localization of MCM5. Finally, unlike its function in DNA synthesis, MCM5-mediated inhibition of centrosome reduplication in S-phase-arrested CHO cells does not require binding to other MCM family members. These results suggest that cyclins E and A sequentially prevent centrosome reduplication throughout interphase by recruitment of DNA replication factors such as MCM5 and Orc1.

Cdc2-mediated Phosphorylation of CLIP-170 Is Essential for Its Inhibition of Centrosome Reduplication

CLIP-170, the founding member of microtubule "plus ends tracking" proteins, is involved in many critical microtubule-related functions, including recruitment of dynactin to the microtubule plus ends and formation of kinetochore-microtubule attachments during metaphase. Although it has been reported that CLIP-170 is a phosphoprotein, neither have individual phosphorylation sites been identified nor have the associated kinases been extensively studied. Herein, we identify Cdc2 as a kinase that phosphorylates CLIP-170. We show that Cdc2 interacts with CLIP-170 mediating its phosphorylation on Thr(287) in vivo. Significantly, expression of CLIP-170 with a threonine 287 to alanine substitution (T287A) results in its mislocalization, accumulation of Plk1 and cyclin B, and block of the G2/M transition. Finally, we found that depletion of CLIP-170 leads to centrosome reduplication and that Cdc2 phosphorylation of CLIP-170 is required for the process. These results demonstrate that Cdc2-mediated phosphorylation of CLIP-170 is essential for the normal function of this protein during cell cycle progression.

BubR1 localizes to centrosomes and suppresses centrosome amplification via regulating Plk1 activity in interphase cells

BubR1 is a critical component of the mitotic checkpoint that delays the onset of anaphase until all chromosomes have established bipolar attachment to the microtubules. We previously reported that mutations of the BUB1B gene (encoding BubR1) caused premature chromatid separation (PCS) syndrome, a condition characterized by constitutional aneuploidy and a high risk of childhood cancer. We here report that the cells from PCS syndrome patients have loss of regulation of the centrosome duplication machinery, resulting in centrosome amplification and multipolar mitosis. PCS syndrome cells show increased activity of Polo-like kinase 1 (Plk1), whose knockdown suppresses centrosome amplification. BubR1 localizes to centrosomes, physically interacts with Plk1 and inhibits Plk1 phosphorylation and its kinase activity during interphase. These results unravel a crucial role of BubR1 in preventing centrosome reduplication through negative regulation of Plk1 in interphase cells.