Role In Centrosome Duplication Research Articles

Aurora Kinase A Is an Independent Predictor of Invasive Recurrence in Breast Ductal Carcinoma in situ

Introduction: Aurora Kinase A (AURKA/STK15) has a role in centrosome duplication and is a regulator of mitotic cell proliferation. It is over-expressed in breast cancer and other cancers, however; its role in ductal carcinoma in situ (DCIS) remains to be defined. This study aims to characterize AURKA protein expression in DCIS and evaluate its prognostic significance. Methods: AURKA was assessed immunohistochemically in a large well-characterized cohort of DCIS (n = 776 pure DCIS and 239 DCIS associated with invasive breast cancer [DCIS-mixed]) with long-term follow-up data (median = 105 months) and basic molecular characterization. Results: High AURKA expression was observed in 15% of DCIS cases and was associated with features of aggressiveness including larger tumour size, high nuclear grade, hormone receptor negativity, HER2 positivity, and high Ki67 proliferation index. AURKA expression was higher in DCIS associated with invasive breast cancer than in pure DCIS (p < 0.0001). In the DCIS-mixed cohort, the invasive component showed higher AURKA expression than the DCIS component (p < 0.0001). Outcome analysis revealed that AURKA was a predictor of invasive recurrence (p = 0.002). Conclusion: High AURKA expression is associated with poor prognosis in DCIS and might be a potential marker to predict DCIS progression to invasive disease.

Role for polo-like kinase 4 in mediation of cytokinesis

The mitotic protein polo-like kinase 4 (PLK4) plays a critical role in centrosome duplication for cell division. By using immunofluorescence, we confirm that PLK4 is localized to centrosomes. In addition, we find that phospho-PLK4 (pPLK4) is cleaved and distributed to kinetochores (metaphase and anaphase), spindle midzone/cleavage furrow (anaphase and telophase), and midbody (cytokinesis) during cell division in immortalized epithelial cells as well as breast, ovarian, and colorectal cancer cells. The distribution of pPLK4 midzone/cleavage furrow and midbody positions pPLK4 to play a functional role in cytokinesis. Indeed, we found that inhibition of PLK4 kinase activity with a small-molecule inhibitor, CFI-400945, prevents translocation to the spindle midzone/cleavage furrow and prevents cellular abscission, leading to the generation of cells with polyploidy, increased numbers of duplicated centrosomes, and vulnerability to anaphase or mitotic catastrophe. The regulatory role of PLK4 in cytokinesis makes it a potential target for therapeutic intervention in appropriately selected cancers.

Impacts of AURKA Genetic Polymorphism on Urothelial Cell Carcinoma Development.

Urothelial cell carcinoma (UCC) is the most common primary malignancy of the urinary system and the second-most common type of renal cell carcinoma. Aurora kinase A (AURKA), a serine/threonine kinase, has a critical role in centrosome duplication, spindle assembly checkpoint, and cytokinesis. Here, we determined the correlation between UCC susceptibility and AURKA polymorphisms. We used real-time polymerase chain reaction to compare the genotype distributions and allelic frequencies of four single-nucleotide polymorphisms (SNPs) of AURKA, namely rs1047972, rs2273535, rs2064863, and rs6024836, between 431 UCC cases and 862 healthy controls. Logistic regression models demonstrated that the G allele of rs2064863, a genetic polymorphism of AURKA, exhibited a significant protective effect against UCC among the 862 nonsmokers. Moreover, patients with rs2064863 G allele exhibited a slightly lower risk of lymph node metastasis and those with rs6024836 G allele exhibited a lower risk of distant metastases. Our study suggested that several variants of AURKA SNPs rs2064863 and rs6024836 may serve as critical predictors for the clinical status of UCC.

CEP131 indicates poor prognosis and promotes cell proliferation and migration in hepatocellular carcinoma

Centrosomal proteins have been implicated in the progression of human diseases. CEP131 plays important roles in centrosome duplication and genome stability, but its role in cancers remains largely unknown. Here, we showed that CEP131 expression was increased in hepatocellular carcinoma (HCC), compared to the paracarcinoma tissues, at both mRNA and protein levels. High CEP131 expression was closely associated with tumor size (P=0.020), tumor capsule (P=0.043), TNM stage (P=0.007) and tumor differentiation (P=0.019). Furthermore, patients with high expression of CEP131 were accompanied with worse overall and disease-free survivals in our and TCGA cohorts consisting of a total of 802 cases. The prognostic value of CEP131 was further confirmed by stratified survival analysis. Multivariate cox regression model indicated that CEP131 was an independent factor for overall survival (hazard ratio=1.762, 95% confident interval: 1.443–2.151, P<0.001). In vitro data demonstrated that nucleophosmin (NPM) physically bound to CEP131 and maintained its protein stability. Overexpression of CEP131 in HCC cell lines enhanced cell proliferation and migration, whereas the knockdown of CEP131 led to the opposite phenotypes. Further studies demonstrated that CEP131 exhibited oncogenic activity via activation of PI3K/AKT signaling pathway. Taken together, our findings suggest CEP131 serves as a potential prognostic biomarker in HCC, and functions as an oncogene in this deadly disease.

Abstract 2736: Regulation of cytokinesis by polo-like kinase 4

Abstract Introduction. The serine/threonine mitotic kinase, polo-like kinase 4 (PLK4), is known to play a critical role in centrosome duplication in preparation for cell division. Based on our preliminary observations with a PLK4 kinase inhibitor, we investigated the possibility that PLK4 may also play a role in regulation of cytokinesis. Experimental Procedures. Immunofluorescence was used to localize PLK4 and phospho-PLK4 in cultured human breast, ovarian, and colorectal cancer cell lines throughout the cell cycle without and with CFI-400945 PLK4 inhibitor and MG-115 protease inhibitor. Flow cytometry and videomicroscopy were used to analyze the consequences of PLK4 inhibition on cytokinesis. Results. Using immunofluorescence, PLK4 was localized to centrosomes; however, we also found that phospho-PLK4 was cleaved and distributed to kinetochores (metaphase and anaphase), cleavage furrow (telophase), and middle body (cytokinesis) during cell division in colorectal, ovarian, and breast cancer cells. Distribution of phospho-PLK4 to the cleavage furrow and middle body raised the possibility that this kinase plays a functional role in cytokinesis. Using either CFI-400945 PLK4 kinase inhibitor or the MG-115 protease inhibitor, we found that PLK4 accumulated in centrosomes with inhibition of translocation of PLK4 to the middle body. This change in subcellular distribution of PLK4 was associated with generation of large, multi-nucleated tumor cells or tumor cells with polyploidy. Videomicroscopy confirmed that treatment with CFI-400945 PLK4 inhibitor was associated with prevention of cellular abscission in treated cells. Conclusions. These observations demonstrate a role for phospho-PLK4 in facilitation of cytokinesis. A regulatory role for PLK4 in cytokinesis makes it a potential target for therapeutic intervention in appropriately selected cancers. Citation Format: Michael F. Press, Bin Xie, Simon Davenport, Yu Zhou, Neil O’Brien, Michael Palazzolo, Tak Mak, Joan Brugge, Dennis J. Slamon. Regulation of cytokinesis by polo-like kinase 4. [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 2736.

Frameshift Mutations of AKAP9 Gene in Gastric and Colorectal Cancers with High Microsatellite Instability.

A-kinase-anchoring protein 9 (AKAP9) coordinates the cellular location and function of protein kinase A. AKAP9 plays an important role in centrosome duplication, cell cycle progression and maintenance of cell membrane integrity, alterations of which contribute to tumorigenesis. Somatic mutations of AKAP9 gene have been detected in many cancers including gastric (GC) and colorectal cancers (CRC), but the mutation status with respect to microsatellite instability (MSI) has not been reported. In a public database, we found that AKAP9 gene had mononucleotide repeats in the coding sequences that might be mutation targets in the cancers with MSI. We analyzed the mutations in 79 GCs and 124 CRCs including high MSI (MSI-H) and microsatellite stable/low MSI (MSS/MSI-L) cases by single-strand conformation polymorphism analysis and DNA sequencing. Overall, we found AKAP9 frameshift mutations in 4 (11.7%) GCs and 20 (17.7%) CRCs with MSI-H (24/113), but not in MSS/MSI-L cancers (0/90) (p<0.001). In addition, we analyzed intratumoral heterogeneity (ITH) of AKAP9 frameshift mutations in 16 CRCs and found that five CRCs (31.3%) harbored regional ITH of the AKAP9 frameshift mutations. Our data indicate that AKAP9 gene harbors not only somatic frameshift mutations but also mutational ITH, which together may be features of GC and CRC with MSI-H. Our results also suggest that regional mutation analysis is needed for a better evaluation of mutation status in these tumors to overcome ITH.

Aurora A Kinase Inhibition Selectively Synergizes with Histone Deacetylase Inhibitor through Cytokinesis Failure in T-cell Lymphoma.

Aurora A kinase (AAK) is expressed exclusively during mitosis, and plays a critical role in centrosome duplication and spindle formation. Alisertib is a highly selective AAK inhibitor that has demonstrated marked clinical activity of alisertib across a spectrum of lymphomas, though particularly in patients with T-cell lymphoma (TCL). We sought to compare and contrast the activity of alisertib in preclinical models of B-cell lymphoma (BCL) and TCL, and identify combinations worthy of clinical study. High-throughput screening of pralatrexate, the proteasome inhibitor (ixazomib), and the histone deacetylase (HDAC) inhibitor (romidepsin) revealed that only romidepsin synergized with alisertib, and only in models of TCL. We discovered that the mechanism of synergy between AAK inhibitors and HDAC inhibitors appears to be mediated through cytokinesis failure. A high-throughput screening approach was used to identify drugs that were potentially synergistic in combination with alisertib. Live-cell imaging was used to explore the mechanistic basis for the drug: drug interaction between alisertib and romidepsin. An in vivo xenograft TCL model was used to confirm in vitro results. In vitro, alisertib exhibited concentration-dependent cytotoxicity in BCL and TCL cell lines. Alisertib was synergistic with romidepsin in a T-cell-specific fashion that was confirmed in vivo. Live-cell imaging demonstrated that the combination treatment resulted in profound cytokinesis failure. These data strongly suggest that the combination of alisertib and romidepsin is highly synergistic in TCL through modulation of cytokinesis and merits clinical development.

Association of genetic polymorphisms in AURKA, BRCA1, CCNE1 and CDK2 with the risk of endometrial carcinoma and clinicopathological parameters among Chinese Han women

ObjectiveCentrosome aberrations and cell-cycle deregulations have important implications for the development of endometrial carcinoma. AURKA, BRCA1, CCNE1 and CDK2 genes play pivotal roles in centrosome duplication and cell-cycle regulation. This study aimed to investigate whether genetic polymorphisms in these four genes may contribute to endometrial carcinoma susceptibility and progression in Chinese Han women. Study design: A total of twenty-two single nucleotide polymorphisms (SNPs) were selected according to the public HapMap database (HapMap Data Release #27; Chinese Beijing population), and genotyped using TaqMan Realtime PCR method in 530 endometrial adenocarcinoma cases and 825 age-matched controls from Chinese Han women. Then, haplotype blocks were reconstructed according to our genotyping data. ResultsFor AURKA, the rs911162 was associated with increased risk of endometrial carcinoma [in co-dominant model: adjusted odds ratio (aOR)=4.92, 95% CI=1.24–19.55, P=0.0235]. The haplotype GA (rs6064391+rs911162) in block 1 of AURKA was also associated with increased risk of endometrial carcinoma (aOR=1.25, 95% CI=1.07–2.06, P=0.0189). For BRCA1, the minor allele homozygotes of rs8067269 could decrease the risk of endometrial carcinoma (in co-dominant models: aOR=0.52, 95% CI=0.34–0.80, P=0.0032), so was the haplotype CTCAG (rs8176323+rs8176199+rs3737559+rs8067269+rs2070833) (aOR=0.69, 95% CI=0.50–0.95, P=0.0234). Moreover, we found several associations between genetic variations in CCNE1, BRCA1 and AURKA and clinicopathological parameters. ConclusionsThis study indicates that genetic polymorphisms of AURKA, BRCA1 and CCNE1 may contribute to endometrial carcinoma susceptibility or progression in Chinese Han women.

Centrin: Another target of monastrol, an inhibitor of mitotic spindle

Monastrol, a cell-permeable inhibitor, considered to specifically inhibit kinesin Eg5, can cause mitotic arrest and monopolar spindle formation, thus exhibiting antitumor properties. Centrin, a ubiquitous protein associated with centrosome, plays a critical role in centrosome duplication. Moreover, a correlation between centrosome amplification and cancer has been reported. In this study, it is proposed for the first time that centrin may be another target of the anticancer drug monastrol since monastrol can effectively inhibit not only the growth of the transformed Escherichia coli cells in vivo, but also the Lu3+-dependent self-assembly of EoCen in vitro. The two closely related compounds (Compounds 1 and 2) could not take the same effect. Fluorescence titration experiments suggest that four monastrols per protein is the optimum binding pattern, and the binding constants at different temperatures were obtained. Detailed thermodynamic analysis indicates that hydrophobic force is the main acting force between monastrol and centrin, and the extent of monastrol inhibition of centrin self-assembly is highly dependent upon the hydrophobic region of the protein, which is largely exposed by the binding of metal ions.

Constitutively active NDR1-PIF kinase functions independent of MST1 and hMOB1 signalling

The human MST1/hMOB1/NDR1 tumour suppressor cascade regulates important cellular processes, such as centrosome duplication. hMOB1/NDR1 complex formation appears to be essential for NDR1 activation by autophosphorylation on Ser281 and hydrophobic motif (HM) phosphorylation at Thr444 by MST1. To dissect these mechanistic relationships in MST1/hMOB1/NDR signalling, we designed NDR1 variants carrying modifications that mimic HM phosphorylation and/or abolish hMOB1/NDR1 interactions. Significantly, the analyses of these variants revealed that NDR1-PIF, an NDR1 variant containing the PRK2 hydrophobic motif, remains hyperactive independent of hMOB1/NDR1-PIF complex formation. In contrast, as reported for the T444A phospho-acceptor mutant, NDR1 versions carrying single phospho-mimicking mutations at the HM phosphorylation site, namely T444D or T444E, do not display increased kinase activities. Collectively, these observations suggest that in cells Thr444 phosphorylation by MST1 depends on the hMOB1/NDR1 association, while Ser281 autophosphorylation of NDR1 can occur independently. By testing centrosome-targeted NDR1 variants in NDR1- or MST1-depleted cells, we further observed that centrosome-enriched NDR1-PIF requires neither hMOB1 binding nor MST1 signalling to function in centrosome overduplication. Taken together, our biochemical and cell biological characterisation of NDR1 versions provides novel unexpected insights into the regulatory mechanisms of NDR1 and NDR1's role in centrosome duplication.

G protein–coupled receptor kinase 2 (GRK2) is localized to centrosomes and mediates epidermal growth factor–promoted centrosomal separation

G protein-coupled receptor kinases (GRKs) play a central role in regulating receptor signaling, but recent studies suggest a broader role in modulating normal cellular functions. For example, GRK5 has been shown to localize to centrosomes and regulate microtubule nucleation and cell cycle progression. Here we demonstrate that GRK2 is also localized to centrosomes, although it has no role in centrosome duplication or microtubule nucleation. Of interest, knockdown of GRK2 inhibits epidermal growth factor receptor (EGFR)-mediated separation of duplicated centrosomes. This EGFR/GRK2-mediated process depends on the protein kinases mammalian STE20-like kinase 2 (Mst2) and Nek2A but does not involve polo-like kinase 1. In vitro analysis and dominant-negative approaches reveal that GRK2 directly phosphorylates and activates Mst2. Collectively these findings demonstrate that GRK2 is localized to centrosomes and plays a central role in mitogen-promoted centrosome separation most likely via its ability to phosphorylate Mst2.

Genetic polymorphisms in AURKA, BRCA1, CCNE1 and CDK2 are associated with ovarian cancer susceptibility among Chinese Han women

IntroductionCentrosome aberrations and cell-cycle deregulation have important implications for ovarian cancer development. The AURKA, BRCA1, CCNE1 and CDK2 genes play pivotal roles in centrosome duplication and cell-cycle regulation. MethodsUsing a haplotype-based analysis, this study aimed to investigate whether genetic polymorphisms in these four genes may contribute to ovarian cancer susceptibility. A total of 22 single nucleotide polymorphisms (SNPs) in these four genes were genotyped in 287 cases of ovarian serous cystadenocarcinomas and 618 age-matched cancer-free controls from the Chinese Han population, and then haplotype blocks were reconstructed according to our genotyping data and linkage disequilibrium (LD) status of these SNPs. ResultsFor AURKA, we found that haplotype GA [rs6064391 (T→G)+rs911162 (G→A)] was strongly associated with decreased ovarian cancer risk (adjusted OR=0.31, 95% CI=0.15–0.63, P=0.0012). For BRCA1, we found that haplotype CGTAG was associated with decreased ovarian cancer risk (adjusted OR=0.64, 95% CI=0.41–0.98, P=0.0417). Moreover, women harboring homozygous GA/CGTAG haplotypes showed the lowest risk (OR=0.12, 95% CI=0.02–0.94, P=0.0438). In CCNE1, the SNPs rs3218035 and rs3218042 were significantly associated with increased ovarian cancer risk (rs3218035: adjusted OR=5.20, 95% CI=1.85–14.52, P=0.0017; rs3218042: adjusted OR=4.98, 95% CI=1.75–14.19, P=0.0027). For CDK2, no significant association was found. ConclusionsThis study indicates that genetic polymorphisms of AURKA, BRCA1 and CCNE1 may affect ovarian cancer susceptibility in Chinese Han women.

Abstract 1751: The E2F activators signal centrosome amplification in breast cancer cells through centrosome regulatory molecules.

Abstract Based on the elevated frequencies of centrosome amplification in premalignant breast lesions and in breast tumors, centrosome amplification is proposed to play a role in breast tumorigenesis. The coordination between cell and centrosome cycles is of paramount importance to maintain genomic stability and to the prevention of mammary tumorigenesis. Thus, both cell cycle regulators and molecules that regulate centrosome duplication work together to prevent abnormal centrosome duplication. E2Fs are transcription factors that regulate various cellular processes, such as DNA replication, DNA repair, cell proliferation and apoptosis; their role on centrosomal and mitotic regulation is understood poorly. We hypothesize that the E2F activators (E2F1, E2F2, E2F3a) play key roles in regulating centrosome amplification in HER2 positive breast cancer cells. We found that the E2Fs activators are deregulated or overexpressed in HER2 positive breast cancer cells displaying centrosome amplification, since serum withdrawal does not downregulate the E2F activators (which occurs in control MCF10A cells upon serum-withdrawal). Upregulated E2Fs correlated with the transcriptional overexpression of cell cycle and centrosome regulatory targets, including cylin D, cyclin E, Nek2 and Plk4. Knockdown of E2Fs in HER2 positive cells decreased centrosome amplification without affecting the cell cycle, or entry into S phase. The chromatin immunoprecipitation (ChIP) assay showed binding of E2F1, E2F2 and E2F3 into the promoter of Nek2 and between exons 1 and 2 of Plk4. In two out of three of the Her2+ breast cancer cell lines displaying centrosome amplification, extinguishing E2F expression leads to downregulation of Nek2 and cyclin D1 -potent inducers of centrosome amplification-. In conclusion, centrosome amplification in Her2+ breast cancer cells is dependent on the E2Fs activators and is mediated by molecules that play critical roles in centrosome duplication. We have identified a novel mechanism where E2F-generated centrosome amplification may contribute to breast carcinogenesis. Citation Format: Mi-Young Lee, Harold Ivan Saavedra. The E2F activators signal centrosome amplification in breast cancer cells through centrosome regulatory molecules. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1751. doi:10.1158/1538-7445.AM2013-1751

Abstract 113: Effect of targeting Aurora kinase A in human oral squamous cell carcinoma cells

Abstract Oncogene addiction has provided therapeutic opportunities in many human malignancies, but molecular targeted therapy for oral squamous cell carcinoma (OSCC) is not available. In this study, we identified an appropriate molecule for treatment of patients with OSCC. We determined the gene expression profiles in 10 human OSCC cell lines and a non-neoplastic keratinocyte cell line by microarray analysis, and then identified Aurora kinase A (AURKA) as a cancer-related gene which was commonly overexpressed in OSCC cells. We also confirmed the overexpression of AURKA mRNA in OSCC tissues and compared to normal oral mucosa tissues by quantitative RT-PCR. AURKA plays an essential role in centrosome duplication, mitoic entry, and spindle assembly. Overexpression of AURKA in human malignancies is associated with gene amplification, genetic instability, poor histological differentiation, and poor prognosis. To determine if the AURKA is a plausible therapeutic target, we investigated the effect of small interfering RNAs specific for AURKA(siAURKA) and an AURKA selective inhibitor MLN8237 on the growth of human OSCC cells in vitro and in vivo. Knockdown of AURKA by RNA interference (RNAi) markedly inhibited the growth of OSCC cells and observed the change of colony formation and cell size looked like cell cycle arrest. We examined the RNAi effect of siAURKA in OSCC cells by Western blotting. Compared with non-target siRNA, the protein expression level of AURKA was almost completely suppressed. MLN8237 also reduced the cell growth, but showed relatively resistant to cells expressing AURKA at high levels. Transfection of siAURKA inhibited the growth of OSCC cells more potent than treatment with MLN8237 in vitro. Furthermore, we confirmed the effect of siAURKA and MLN8237 on the growth of human OSCC tumors which had been subcutaneously xenografted in athymic nude mice. Atelocollagen-siAURKA (8 nmol) complexes were injected into the tail vein every 3 days and MLN8237 (30 mg/kg) were received orally for 14 consecutive days. We found that siAURKA and MLN8237 significantly reduced the size of subcutaneously xenografted OSSC tumors. Interestingly, the combination of siAURKA and MLN8237 induced remarkable anti-tumor activity compared with single agent groups. These results suggest that AURKA functions as a critical gene for supporting the growth of human OSCC cells and targeting AURKA may be a useful therapeutic strategy for OSCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 113. doi:1538-7445.AM2012-113

Geminin is partially localized to the centrosome and plays a role in proper centrosome duplication

Background information. Centrosome duplication normally parallels with DNA replication and is responsible for correct segregation of replicated DNA into the daughter cells. Although geminin interacts with Cdt1 to prevent loading of MCMs (minichromosome maintenance proteins) on to the replication origins, inactivation of geminin nevertheless causes centrosome over-duplication in addition to the re-replication of the genome, suggesting that geminin may play a role in centrosome duplication. However, the exact mechanism by which loss of geminin affects centrosomal duplication remains unclear and the possible direct interaction of geminin with centrosomal-localized proteins is still unidentified.Results. We report in the present study that geminin is physically localized to the centrosome. This unexpected geminin localization is cell-cycle dependent and mediated by the actin-related protein, Arp1, one subunit of the dynein–dynactin complex. Disruption of the integrity of the dynein–dynactin complex by overexpression of dynamitin/p50, a well-characterized inhibitor of dynactin, reduces the centrosomal localization of both geminin and Arp1. Enrichment of geminin on centrosomes was enhanced when cellular ATP production was suppressed in the ATP-inhibitor assay, whereas the accumulation of geminin on the centrosome was disrupted by depolymerization of the microtubules using nocodazole. We further demonstrate that the coiled-coil motif of geminin is required for its centrosomal localization and the interaction of geminin with Arp1. Depletion of geminin by siRNA (small interfering RNA) in MDA-MB-231 cells led to centrosome over-duplication. Conversely, overexpression of geminin inhibits centrosome over-duplication induced by HU in S-phase-arrested cells, and the coiled-coil-motif-mediated centrosomal localization of geminin is required for its inhibition of centrosome over-duplication. Centrosomal localization of geminin is conserved among mammalian cells and geminin might perform as an inhibitor of centrosome duplication.Conclusions. The results of the present study demonstrate that a fraction of geminin is localized on the centrosome, and the centrosomal localization of geminin is Arp1-mediated and dynein–dynactin-dependent. The coiled-coil motif of geminin is required for its targeting to the centrosome and inhibition of centrosome duplication. Thus the centrosomal localization of geminin might perform an important role in regulation of proper centrosome duplication.

Differential phosphorylation patterns between the Cyclin-A2/CDK2 complex and their monomers

The Cyclin-A2/CDK2 is a protein heterodimer with kinase activity that plays a key role in centrosome duplication and meiotic cell division. To check the suitability of the insect cells for the production and characterization of phosphorylated mammalian proteins, both proteins were expressed individually or as a complex using the baculovirus expression system. In this study, we used a linear ion trap mass spectrometer to identify the phosphorylated residues in mouse Cyclin-A2 and CDK2 recombinant proteins, after individual expression and after formation of the heterodimer complex, both in baculovirus. By using multi-protease digestion and data dependent neutral loss analysis, we identified a differential phosphorylation pattern before and after formation of the protein complex. The analysis of the monomeric proteins showed that Cyclin-A2 was phosphorylated on two Ser residues (Ser 14 and Ser 421) and CDK2 on a single residue (Thr 160). After heterodimer formation, Cyclin-A2 was phosphorylated only on Ser 14, whereas CDK2 contained two phosphorylated residues (Thr 39 and Thr 160). These findings may clarify relevant aspects of the functionality of the Cyclin-A2/CDK2 protein complex and its role in cell cycle and support the efficiency of the baculovirus system for the production of phosphorylated proteins mimicking the mammalian situation.

Centrosome overduplication and mitotic instability in PKD2 transgenic lines

Polycystin-2 (PC-2), a protein encoded by PKD2 and involved in autosomal dominant polycystic kidney disease (ADPKD), is a non-selective cationic channel recently implicated in the function of primary cilia. We recently constructed a new animal model in the form of a transgenic mouse with a BAC-containing human PKD2 inserted in its genome. Two transgenic mouse lines overexpressing human PKD2 showed mitotic instability. Fibroblasts from these transgenic mouse lines have abnormal chromosomal numbers. These lines also have supernumerary centrosomes. PC-2 overexpression is associated with mitotic instability and centrosome overduplication. PC-2 therefore seems to play a role in centrosome duplication, and this hypothesis is being evaluated in other models.

Cep97 and CP110 Suppress a Cilia Assembly Program

Mammalian centrioles play a dynamic role in centrosome function, but they also have the capacity to nucleate the assembly of cilia. Although controls must exist to specify these different fates, the key regulators remain largely undefined. We have purified complexes associated with CP110, a protein that plays an essential role in centrosome duplication and cytokinesis, and have identified a previously uncharacterized protein, Cep97, that recruits CP110 to centrosomes. Depletion of Cep97 or expression of dominant-negative mutants results in CP110 disappearance from centrosomes, spindle defects, and polyploidy. Remarkably, loss of Cep97 or CP110 promotes primary cilia formation in growing cells, and enforced expression of CP110 in quiescent cells suppresses their ability to assemble cilia, suggesting that Cep97 and CP110 collaborate to inhibit a ciliogenesis program. Identification of Cep97 and other genes involved in regulation of cilia assembly may accelerate our understanding of human ciliary diseases, including renal disease and retinal degeneration.

312 POSTER Preclinical evaluation of a rationally designed novel class of non-hydroxamate Histone Deacetylase inhibitors (HDACi)

Monastrol, a cell-permeable inhibitor, considered to specifically inhibit kinesin Eg5, can cause mitotic arrest and monopolar spindle formation, thus exhibiting antitumor properties. Centrin, a ubiquitous protein associated with centrosome, plays a critical role in centrosome duplication. Moreover, a correlation between centrosome amplification and cancer has been reported. In this study, it is proposed for the first time that centrin may be another target of the anticancer drug monastrol since monastrol can effectively inhibit not only the growth of the transformed Escherichia coli cells in vivo, but also the Lu3+-dependent self-assembly of EoCen in vitro. The two closely related compounds (Compounds 1 and 2) could not take the same effect. Fluorescence titration experiments suggest that four monastrols per protein is the optimum binding pattern, and the binding constants at different temperatures were obtained. Detailed thermodynamic analysis indicates that hydrophobic force is the main acting force between monastrol and centrin, and the extent of monastrol inhibition of centrin self-assembly is highly dependent upon the hydrophobic region of the protein, which is largely exposed by the binding of metal ions.

Crystallization and preliminary X-ray studies of mouse centrin1

Centrins belong to a family of Ca2+-binding EF-hand proteins that play a fundamental role in centrosome duplication and the function of cilia. To shed light on the structure-function relationship of these proteins, mouse centrin1 has been crystallized. The mouse centrin1 has been expressed in Escherichia coli as a GST-centrin fusion protein containing a thrombin protease cleavage site between the fusion partners. Two constructs with different linking-sequence lengths were expressed and purified. Thrombin cleavage yielded functional centrin1 and N-terminally extended centrin1 containing 25 additional residues upstream of its N-terminus. Only N-terminally extended centrin1 (MW approximately 22 240 Da) could be crystallized at room temperature, using 20-25%(w/v) PEG 1500, 5-10%(v/v) ethylene glycol and 1-2%(v/v) dioxane. Crystals were suitable for X-ray analysis, diffracting to 2.9 A at 295 K using a rotating-anode X-ray source. They belong to space group C2, with unit-cell parameters a = 60.7, b = 59.6, c = 58.3 A, beta = 109.4 degrees. Assuming the asymmetric cell to be occupied by one centrin1 molecule of 22.2 kDa, the unit cell contains 45% solvent with a crystal volume per protein weight, VM, of 2.2 A3 Da(-1).