Key Mitotic Regulator Research Articles

Abstract 3600: Preclinical pharmacokinetic and biomarker analysis of AMG 900, an orally available small molecule inhibitor of aurora kinases, in human xenograft tumor and surrogate tissues

Abstract Background: The aurora family of serine-threonine kinases (Aurora-A, -B, -C) regulate cell-cycle progression in mammalian cells. Aurora kinases A and B are essential for proper chromosome congression, segregation, and cytokinesis during mitosis, whereas aurora kinase C function appears restricted to male meiosis. Aurora kinase B is responsible for the direct phosphorylation of histone H3 on serine-10 (p-histone H3). Aurora kinase A and B expression is elevated in a variety of human cancers and is associated with advanced clinical staging and poor prognosis. With the emergence of aurora kinases as key mitotic regulators and their potential role in tumorigenesis, we developed AMG 900, a novel ATP competitive small molecule inhibitor, that is highly potent and selective for aurora kinases A, B, and C. In vivo, AMG 900 inhibits the growth of multiple human tumor xenografts, including multidrug-resistant models. Objective: The aim of this study was to establish a pharmacokinetic-pharmacodynamic (PK-PD) relationship for AMG 900 in human tumor xenografts and surrogate tissues. Results: In nude mice, oral administration of AMG 900 inhibited p-histone H3 in tumors and bone marrow in a concentration- and dose-dependent manner. To further refine the AMG 900 PK-PD relationship, p-histone H3 IC50 values for bone marrow and dissociated COLO 205 tumors from mice were associated with plasma concentrations of 294 and 273 ng/mL, respectively. The duration of target coverage required for anti-tumor efficacy was determined to be > 6 hours per day above the in vivo p-histone H3 IC50 using different dosing schedules. To explore whether skin could be a suitable surrogate tissue for measuring p-histone H3 inhibition, tumor-bearing mice were administered AMG 900 at 3.75, 7.5, and 15 mg/kg for 3 hours. Significant suppression of p-histone H3 in skin was only observed at 15 mg/kg (p < 0.0001), whereas in tumor, a dose-dependent inhibition was achieved at all doses (p < 0.0015). To determine the feasibility of measuring p-histone H3 using fine-needle aspirate (FNA) tumor biopsies, tumor-bearing mice were administered AMG 900 at 15 mg/kg for 3 hours. Treatment with AMG 900 significantly inhibited p-histone H3 (> 99%, p < 0.0001) in COLO 205 tumors, suggesting that FNA biopsies may be a viable approach for assessing AMG 900 PD effects in the clinic. Conclusion: These studies demonstrate that AMG 900 inhibits p-histone H3 in tumors and surrogate tissues, although tissues such as skin may be less sensitive to assess PD effects. The use of p-histone H3 as a biomarker to determine plasma levels of AMG 900 required to inhibit aurora B activity may be useful in clinical studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3600.

Abstract 1082: BI 811283, a potent inhibitor of the mitotic kinase Aurora B, shows dose- and schedule-dependent efficacy in human cancer xenograft models

Abstract Background. The serine/threonine kinase Aurora B is involved in the regulation of several mitotic processes, including chromosome condensation, congression and segregation as well as cytokinesis. The essential functions of Aurora B and its overexpression in many cancer types render this protein kinase an attractive target for therapeutic intervention. BI 811283, a potent inhibitor of this key mitotic regulator, inhibits proliferation of a wide range of cultured human cancer cells at low nanomolar concentrations by inducing polyploidy, senescence and apoptosis. Methods. BomTac:NMRI-Foxn1nu mice were grafted subcutaneously with NCI-H460 non-small cell lung carcinoma (mutant KRAS, wild-type p53), HCT 116 colon carcinoma (mutant KRAS, wild-type p53) or BxPC-3 pancreas adenocarcinoma cells (wild-type KRAS, mutant p53). Treatment was initiated when the tumors had reached a volume of ∼50 mm3. BI 811283 was injected intravenously once or twice weekly as a single bolus or b.i.d. Alternatively, the compound was administered once-weekly by a continuous 24 h infusion via subcutaneously implanted osmotic mini-pumps. Multiple dose levels and dosing schedules were evaluated. Results. In models of human non-small cell lung cancer, colon carcinoma and pancreas carcinoma, multiple cycles of treatment with BI 811283 at total weekly doses of 20 to 75 mg/kg resulted in dose-dependent inhibition of tumor growth or tumor regression. Continuous s.c. infusion at 20 mg/kg over 24 h once-weekly was clearly superior to all bolus injection schedules delivering weekly doses up to 75 mg/kg. Furthermore, regression of large tumors (350 mm3) was induced in the HCT 116 colon carcinoma model. Biomarker analyses of HCT 116 tumors revealed that therapeutic doses of BI 811283 inhibited phosphorylation of histone H3, a direct substrate of Aurora B. Histological examination showed an accumulation of enlarged, multinucleated cells in accordance with the expected mechanism of action. Conclusions. BI 811283 has demonstrated potent antitumor activity in multiple cancer models at well-tolerated doses; treated tumors show hallmarks of Aurora B inhibition. Continuous infusion over 24 h provides a superior therapeutic index compared with bolus administration. The compound is currently under investigation in Phase I clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1082.

Abstract 1642: Preclinical studies of MK-8745: Correlation between Aurora-A inhibitor sensitivity and Aurora-A activator

Abstract Selective small-molecule kinase inhibitors have emerged as an important class of anti-cancer agents, and have demonstrated encouraging clinical efficacy in several different malignancies. However, it has become clear that the clinical activity of these agents is typically limited to a subset of patients, indicating the need to develop a clear understanding of the various factors that influence clinical benefit. To date, most studies have identified cancer patients whose tumors have genetic alterations of the targeted kinase and have demonstrated their greatest clinical efficacy for the kinase inhibitors. However, recent drug discovery efforts are also being aimed at targeting kinases within oncogene-associated signaling pathways and the pathway-specific determinants of sensitivity. Aurora-kinase family members (Aurora kinases -A, B, C) have been implicated as key mitotic regulators essential for genomic stability. Aurora kinase A (AURKA) is highly expressed in multiple human cancers and has been shown to be a promising therapeutic target. In this study, we examined the effects of small molecule AURKA specific inhibitor MK-8745 on non-Hodgkin lymphoma (NHL) cells. Our results indicate that MK-8745 treatment leads to cell cycle arrest at G2/M phase with accumulation of tetraploid DNA followed by mitotic catastrophe and cell death in five cell lines (Granta 519, Jeko1, JVM2, Z138C and Akata). More importantly, we found that the sensitivity of the cell to the AURKA inhibitor is strongly correlated to the expression level of AURKA activators. We found that siRNA knockdown of one of the AURKA activators TPX2 increased MK-8745 sensitivity in drug resistant cell lines (Granta 519), whereas overexpression of TPX2 in MK-8745 sensitive cell (Z138C) lines increased drug resistance. This differential drug-sensitivity is specific to MK-8745 since treatment with pan-Aurora inhibitor (VE465, inhibitor of Aurora-A, B and C) and another mitotic drug, Taxol, did not show a differential sensitivity in these cell lines. The AURKA phosphorylation was decreased by MK-8745 treatment with simultaneous reduction in endogenous AURKA substrate TPX2, Eg5 and TACC3 protein levels. Our results suggest, beside p53, AURKA activators should be potential biomarkers for selecting subpopulations of patients for AURKA inhibitor treatment, while AURKA substrates can be the potential biomarkers for the effect of Aurora-A inhibitor treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1642.

Overexpression of the E2 ubiquitin–conjugating enzyme UbcH10 causes chromosome missegregation and tumor formation

The anaphase-promoting complex/cyclosome (APC/C) E3 ubiquitin ligase functions with the E2 ubiquitin-conjugating enzyme UbcH10 in the orderly progression through mitosis by marking key mitotic regulators for destruction by the 26-S proteasome. UbcH10 is overexpressed in many human cancer types and is associated with tumor progression. However, whether UbcH10 overexpression causes tumor formation is unknown. To address this central question and to define the molecular and cellular consequences of UbcH10 overexpression, we generated a series of transgenic mice in which UbcH10 was overexpressed in graded fashion. In this study, we show that UbcH10 overexpression leads to precocious degradation of cyclin B by the APC/C, supernumerary centrioles, lagging chromosomes, and aneuploidy. Importantly, we find that UbcH10 transgenic mice are prone to carcinogen-induced lung tumors and a broad spectrum of spontaneous tumors. Our results identify UbcH10 as a prominent protooncogene that causes whole chromosome instability and tumor formation over a wide gradient of overexpression levels.

Cdc20 proteolysis requires p38 MAPK signaling and Cdh1‐independent APC/C ubiquitination during spindle assembly checkpoint activation by cadmium

Cdc20, an activator of the anaphase promoting complex/cyclosome (APC/C) ubiquitin ligase, initiates the destruction of key mitotic regulators to facilitate mitosis, while it is negatively regulated by the spindle assembly checkpoint (SAC) to prevent premature anaphase entry. Activation of the p38 mitogen-activated protein kinase could contribute to mitotic arrest, but the underlying mechanism is unknown. Here we report a novel pathway in which the p38 signaling triggers Cdc20 destruction under SAC elicited by cadmium, a human carcinogen. We found that the cadmium-induced prometaphase arrest was linked to decreased Cdc20 and accumulated cyclin A protein levels in human cells, whereas the activity of cyclin B1-Cdk1 was unaffected. The Cdc20 half-life was markedly shortened along with its ubiquitination and degradation via 26S proteasome in cadmium-treated asynchronous or G(2)-enriched cells. Depletion of APC3 markedly suppressed the cadmium-induced Cdc20 ubiquitination and proteolysis, while depletion of Cdh1, another activator of APC/C, did not. Intriguingly, blockage of p38 activity restored the Cdc20 levels for continuing mitosis under cadmium, while inhibition of JNK activity had no effect. The cadmium-induced Cdc20 proteolysis was also suppressed during transient depletion of p38alpha or stable expression a dominant negative form of p38. Inhibition of p38 abolished the induction of Mad2-Cdc20-APC3 complex by cadmium. Moreover, forced expression of MKK6-p38 signaling could promote Cdc20 degradation in a Cdh1-independent APC/C pathway. In summary, accelerated ubiquitination and proteolysis of Cdc20 is essential for prometaphase arrest that is mediated via the p38 signaling during SAC activation.

Abstract C194: In vivo characterization of AMG 900, an orally active small molecule inhibitor of aurora kinases in phase 1 clinical trials

Abstract Background: The aurora family of serine-threonine kinases (Aurora-A, -B, -C) regulate cell-cycle progression in mammalian cells. Aurora kinases A and B are essential for proper chromosome congression, segregation, and cytokinesis during mitosis, whereas aurora kinase C function appears restricted to male meiosis. Aurora kinase B is responsible for the direct phosphorylation of histone H3 on serine-10 (p-histone H3). Aurora kinase A and B expression is elevated in a variety of human cancers and is associated with advanced clinical staging and poor prognosis. The emergence of aurora kinases as key mitotic regulators and their potential role in tumorigenesis has focused substantial interest in developing selective small molecule inhibitors for the treatment of human cancers. AMG 900 is a novel ATP competitive small molecule inhibitor that is highly potent and selective for aurora kinases A, B, and C. Objective: The aim of this study was to characterize the in vivo effects of AMG 900 by establishing a pharmacokinetic-pharmacodynamic relationship and profiling its antitumor activity across multiple human xenografts models using a variety of dosing schedules. Results: In nude mice, oral administration of AMG 900 inhibited phosphorylation of histone H3 in tumors and bone marrow in a time- and dose-dependent manner. The degree of p-histone H3 suppression in cells correlated with plasma drug concentrations. The effect of AMG 900 was tested in a panel of human tumor xenograft models. Oral administration of AMG 900 at 15 mg/kg BID for two consecutive days per week or 3 mg/kg BID every day inhibited tumor growth (50–97% inhibition; p ≤ 0.0044) in all nine of the xenograft models tested compared with the vehicle control group. Importantly, three of these xenograft models are resistant to paclitaxel, docetaxel, and doxorubicin. Mice treated with efficacious doses of AMG 900 showed a transient loss of body weight (0–10%) and bone marrow cellularity, consistent with its on-mechanism effects on normal proliferating cells. Since aurora kinase inhibitors are known to cause neutropenia, studies were performed to investigate the effects of prophylactic treatment of PEGylated-G-CSF (SD/02). In mice, administration of SD/02 shortened the duration of AMG 900-induced neutropenia. Conclusion: Based on these preclinical activities, AMG 900 has the potential to treat advanced cancers, including tumors resistant to standard-of-care chemotherapeutic agents such as paclitaxel and docetaxel. AMG 900 is currently undergoing phase 1 clinical evaluation in patients with advanced solid tumors. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C194.

Abstract A261: In vitro characterization of AMG 900, an orally active small molecule inhibitor of aurora kinases in phase 1 clinical trials

Abstract Background: The aurora family of serine-threonine kinases (Aurora-A, -B, -C) regulate cell-cycle progression in mammalian cells. Aurora kinases A and B are essential for proper chromosome congression, segregation, and cytokinesis during mitosis, whereas aurora kinase C function appears restricted to male meiosis. Aurora kinase B is responsible for the direct phosphorylation of histone H3 on serine-10 (p-histone H3). Aurora kinase A and B expression is elevated in a variety of human cancers and is associated with advanced clinical staging and poor prognosis. The emergence of aurora kinases as key mitotic regulators and their potential role in tumorigenesis has focused substantial interest in developing selective small molecule inhibitors for the treatment of human cancers. Objective: The aim of this study was to characterize the in vitro effects of AMG 900 in tumor cell lines, including cell lines resistant to paclitaxel and three well-characterized aurora kinase inhibitors. Results: AMG 900 is a novel ATP competitive small molecule inhibitor that is highly potent and selective for aurora kinases A, B, and C. In cells, AMG 900 inhibits autophosphorylation of aurora kinases A and B as well as the phosphorylation of histone H3. The predominant cellular response of tumor cells to AMG 900 treatment is aborted cell division without a mitotic arrest, which leads to endoreduplication and cell death. The effect of AMG 900 was tested in a panel of 26 tumor cell lines to evaluate its potential to inhibit cell proliferation across multiple tumor types. AMG 900 inhibited the proliferation of tumor cell lines at low nanomolar concentrations (EC50 values 1–6 nM). Importantly, AMG 900 was equally potent against multidrugresistant (MDR) cell lines expressing ATP-binding cassette transporters (P-gp and BCRP1), whereas paclitaxel and three aurora kinase inhibitors (AZD1152, MK-0457, and PHA-739358) showed a loss of potency in these MDR-positive cell lines compared with matched parental cell lines. To further investigate potential mechanisms of resistance, an HCT116 cell line was adapted to grow in the presence of AZD1152, a selective inhibitor of aurora kinase B. The resulting AZD1152-resistant HCT116 subline harbors a mutation in one allele of the aurora kinase B gene (W221L substitution) and was negative for P-gp and BCRP1 expression. AMG 900 induced an accumulation of cells with ≥4N DNA content (EC50 value 5 nM) and inhibited colony formation in this AZD1152-resistant HCT116 subline. Conclusion: To our knowledge, AMG 900 is the first described inhibitor of aurora kinases A, B, and C that has uniform in vitro potency in MDR-positive tumor cell lines. AMG 900 is currently undergoing phase 1 clinical evaluation in patients with advanced solid tumors. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A261.

Plk1-dependent phosphorylation of FoxM1 regulates a transcriptional programme required for mitotic progression

Proper control of entry into and progression through mitosis is essential for normal cell proliferation and the maintenance of genome stability. The mammalian mitotic kinase Polo-like kinase 1 (Plk1) is involved in multiple stages of mitosis5. Here we report that Forkhead Box M1 (FoxM1), a substrate of Plk1, controls a transcriptional programme that mediates Plk1-dependent regulation of cell-cycle progression. The carboxy-terminal domain of FoxM1 binds Plk1, and phosphorylation of two key residues in this domain by Cdk1 is essential for Plk1-FoxM1 interaction. Formation of the Plk1-FoxM1 complex allows for direct phosphorylation of FoxM1 by Plk1 at G2/M and the subsequent activation of FoxM1 activity, which is required for expression of key mitotic regulators, including Plk1 itself. Thus, Plk1-dependent regulation of FoxM1 activity provides a positive-feedback loop ensuring tight regulation of transcriptional networks essential for orderly mitotic progression.

Midzone activation of aurora B in anaphase produces an intracellular phosphorylation gradient

Proper partitioning of the contents of a cell between two daughters requires integration of spatial and temporal cues. The anaphase array of microtubules that self-organize at the spindle midzone contributes to positioning the cell-division plane midway between the segregating chromosomes. How this signalling occurs over length scales of micrometres, from the midzone to the cell cortex, is not known. Here we examine the anaphase dynamics of protein phosphorylation by aurora B kinase, a key mitotic regulator, using fluorescence resonance energy transfer (FRET)-based sensors in living HeLa cells and immunofluorescence of native aurora B substrates. Quantitative analysis of phosphorylation dynamics, using chromosome- and centromere-targeted sensors, reveals that changes are due primarily to position along the division axis rather than time. These dynamics result in the formation of a spatial phosphorylation gradient early in anaphase that is centred at the spindle midzone. This gradient depends on aurora B targeting to a subpopulation of microtubules that activate it. Aurora kinase activity organizes the targeted microtubules to generate a structure-based feedback loop. We propose that feedback between aurora B kinase activation and midzone microtubules generates a gradient of post-translational marks that provides spatial information for events in anaphase and cytokinesis.

Role of Survivin Phosphorylation by Aurora B in Mitosis

The chromosomal protein passenger complex, a key mitotic regulator, consists of at least four proteins, INCENP, Aurora B, Survivin and Borealin. Survivin, in contrast to the other members of the chromosomal protein passenger complex (CPC), is mobile at metaphase. This protein is also phosphorylated by Aurora B at Threonine 117. In this work we have studied the role of the phosphorylation of Survivin in mitotosis by using non phosphorylable T117A and phosphomimic T117E silent resistant Survivin mutants, inducible cell lines expressing these mutants and a combination of siRNA, time-lapse microscopy and FRAP analysis. Time lapse microscopy and FRAP analysis show that Survivin T117A mutant is very stably associated with centromeres and its expression induces a prometaphasic arrest in endogenous survivin depleted cells. In addition, Survivin T117A was unable to rescue the phenotypes of the endogenous survivin depleted cells. Expressed in these cells, the phosphomimic Survivin T117E mutant exhibits a very weak interaction with the centromeres and behaves as a dominant negative mutant inducing severe mitotic defects. Our data suggest that the Aurora B generated phosphorylation/dephosphorylation cycle of Survivin is required for proper proceeding of mitosis.

Evaluation of ON 01910.Na, a novel modulator of polo-like kinase 1 (Plk1) pathway, and development of a cyclin-B1-based predictive assay in pancreatic cancer

3569 Background: Plk1 is a key mitotic regulator of the transition through the G2/M checkpoint in the cell cycle. This work aimed to evaluate the activity of ON 01910.Na, a Plk1 pathway modulator, in in vitro and in vivo models of pancreatic cancer (PaCa) and to discover biomarkers predictive of efficacy. Methods: ON 01910.Na was tested in 12 PaCa cell lines. Studies assessing Plk1 related markers were conducted to identify biomarkers. For validation a live collection of PaCa xenografts from fresh tumor samples obtained at the time of surgical resection was used (PancXenoBank). The ex vivo assay was based on fine-needle aspirate (FNA) biopsies. Results: ON 01910.Na showed equal activity to gemcitabine against PaCa cell lines. The activity of ON 01910.Na correlated with suppression of two downstream mediators of PLK1, CDC25C and cyclin B1 (by mRNA and protein). ON 01910.Na was tested in xenografts from representative pancreatic cell lines. The selected markers were evaluated in an ex vivo assay, using intra-tumor pharmacokinetics to select the dose of the assay. Cyclin B1 mRNA evaluation yielded the most optimal combination of accuracy and reproducibility. Knockdown of cyclin B1 by siRNA had no effect per se or in the response of the resistant MiaPaca2 to either of the drugs. We next used the ex vivo assay to profile ten patient-derived cases from the PancXenoBank. Two cases were catalogued as potential responders. From each of these ten cases, a group of mice bearing at least 20 tumors received vehicle or ON 01910.Na for 28 days. There was a correlation between the ex vivo cyclin B1 assay and the sensitivity to the tested agent, as the 2 cases prospectively identified as sensitive met pre-specified criteria for response. Of the 8 tumors predicted to be resistant, only one was sensitive. In IHC testing cases showing ex vivo cyclin B1 down-regulation had also decreases in cyclin B1 protein, and there was a correlation between activity and IHC changes in cyclin B1. Conclusions: ON 01910.Na demonstrated significant activity in a preclinical model of PaCa. A rationally designed ex vivo cyclin B1-based assay not only identified cases sensitive to ON 01910.Na, but also replicated the pharmacodynamic events occurring after in vivo exposure. No significant financial relationships to disclose.

Separase, securin and Rad21 in neural cell growth

The key mitotic regulator securin is expressed at low levels in fetal brain compared with adult, and modulates the proliferation of human embryonic neuronal N-Tera2 (NT2) cells. We now examine the function and expression of securin's interacting partner separase, along with Rad21, the functional component of cohesin, which is cleaved by separase following interaction with securin. In contrast to securin, the cleaved forms of separase and Rad21 were highly expressed in human fetal cerebral cortex compared with adult. In a murine model of absent securin expression - the PTTG knock-out mouse - separase and Rad21 were over-expressed in multiple brain regions. In addition, cDNA array analysis of other key mitotic regulators additionally identified cyclin C and sestrin 2 to be induced in the brains of securin-null mice compared with wild type. Further, Rad21 mRNA expression was highly correlated with that of securin, separase, cyclin C and sestrin 2 in fetal brains. In embryonic neuronal NT2 cells, siRNA repression of separase failed to significantly alter cell turnover, whereas repression of securin expression resulted in increased levels of the activated forms of Rad21 and separase, and promoted cell proliferation. Our data suggest that the co-ordinated expression of separase, securin and Rad21 is fundamental for the developing brain.

Mitotic regulation of the anaphase-promoting complex.

Orderly progression through mitosis is regulated by the anaphase-promoting complex/cyclosome (APC/C), a large multiprotein E3 ubiquitin ligase that targets key mitotic regulators for destruction by the proteasome. APC/C has two activating subunits, Cdc20 and Cdh1. The well-established view is that Cdc20 activates APC/C from the onset of mitosis through the metaphase-anaphase transition, and that Cdh1 does so from anaphase through G1. Recent work, however, indicates that Cdh1 also activates APC/C in early mitosis and that this APC/C pool targets the anaphase inhibitor securin. To prevent premature degradation of securin, the nuclear transport factors Nup98 and Rae1 associate with APC/C(Cdh1)-securin complexes. In late metaphase, when all kinetochores are attached to spindle microtubules and the spindle assembly checkpoint is satisfied, Nup98 and Rae1 are released from these complexes, thereby allowing for prompt ubiquitination of securin by APC/C(Cdh1). This, and other mechanisms by which the catalytic activity of APC/C is tightly regulated to ensure proper timing of degradation of each of its mitotic substrates, are highlighted.

Polo kinase controls cell-cycle-dependent transcription by targeting a coactivator protein

Polo kinases have crucial conserved functions in controlling the eukaryotic cell cycle through orchestrating several events during mitosis. An essential element of cell cycle control is exerted by altering the expression of key regulators. Here we show an important function for the polo kinase Cdc5p in controlling cell-cycle-dependent gene expression that is crucial for the execution of mitosis in the model eukaryote Saccharomyces cerevisiae. In particular, we find that Cdc5p is temporally recruited to promoters of the cell-cycle-regulated CLB2 gene cluster, where it targets the Mcm1p-Fkh2p-Ndd1p transcription factor complex, through direct phosphorylation of the coactivator protein Ndd1p. This phosphorylation event is required for the normal temporal expression of cell-cycle-regulated genes such as CLB2 and SWI5 in G2/M phases. Furthermore, severe defects in cell division occur in the absence of Cdc5p-mediated phosphorylation of Ndd1p. Thus, polo kinase is required for the production of key mitotic regulators, in addition to previously defined roles in controlling other mitotic events.

Destruction Box–Dependent Degradation of Aurora B Is Mediated by the Anaphase-Promoting Complex/Cyclosome and Cdh1

Aurora B kinase, a subunit of the chromosomal passenger protein complex, plays essential roles in spindle assembly, chromosome bi-orientation, and cytokinesis. The kinase activity of Aurora B, which peaks in mitosis, is tightly controlled in the cell cycle. Modulation of Aurora B protein levels could partly account for the regulation of its kinase activity in the cell cycle. However, little is known on the molecular mechanism of regulation of Aurora B levels. Here, we examined Aurora B protein levels and confirmed that they fluctuate during the cell cycle, peaking in mitosis and dropping drastically in G1. This profile for Aurora B in the cell cycle is reminiscent of those for substrates of the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase essential for mitotic progression. Indeed, Aurora B is a substrate of APC/C both in vitro and in vivo. Aurora B is efficiently ubiquitinated in an in vitro reconstituted system by APC/C that had been activated by Cdh1. The recognition of Aurora B by APC/C-Cdh1 is specific as it requires the presence of a conserved D-box at the COOH terminus of Aurora B. Furthermore, endogenous Aurora B and Cdh1 form a complex exclusively in mitotic cells. Degradation of Aurora B at the end of mitosis requires Cdh1 in vivo as a reduction of the Cdh1 level by RNA interference stabilizes the Aurora B protein. We conclude that, as a key mitotic regulator, Aurora B is regulated both by its activation during early mitosis and by its destruction by APC/C-Cdh1 in late mitosis and in G1.

NIPA Targets Nuclear Cyclin B1 in a Cell-Cycle Dependent Manner.

Ubiquitin-mediated destruction of regulatory proteins marks the vital means of controlling cell cycle progresssion. The E3 ubiquitin-ligases are prominent in this process, as they allow the transfer of ubiquitin to the target protein and mediate substrate binding specificity. Recently, a new class of E3 ligases referred to as SCF complexes has been identified that consists of four subunits:SKP1, Cul1, Roc1 and an F-box protein, the latter of which determines substrate specifity.We previously reported the cloning of NIPA (nuclear interaction partner of ALK) in complex with constitutively-active oncogenic fusions of ALK, which contribute to the development of certain lymphomas and sarcomas. Subsequently we characterized NIPA as a human F-box protein that determines a novel SCF complex (SCFNIPA) whose cell cycle regulated activity is restricted to interphase to allow for substrate expression at G2/M and mitosis. Phosphorylation of NIPA in late S-phase was found to be the underlying mechanism of SCFNIPA inactivation.We have now identified the key mitotic regulator cyclin B1 to serve as the relevant substrate of the SCFNIPA complex. This targeting process is restricted to interphase and directed towards the nuclear pool of cyclin B1. Inactivation of NIPA by siRNAs results in nuclear accumulation of cyclin B1 in interphase and an elevation of cells in S-phase and mitosis. In contrast, expression of a phosphorylation deficient NIPA mutant that retains constitutive SCFNIPA activity throughout the cell cycle arrests cells at early prophase thus delaying mitotic entry. Both effects are likely attributable to either cyclin B1 accumulation in the case of NIPA inactivation by siRNA or untimely cyclin B degradation at G2/M upon expression of the constitutively active SCFNIPA complex. Cyclin B1 is physiologically kept cytoplasmic during interphase and premature nuclear accumulation has been associated with untimely mitotic entry, loss of checkpoint control and genomic instability. Our data provides a mechanism to inhibit premature nuclear accumulation of cyclin B1 in the mammalian cell cycle. NIPAs association with NPM-ALK of ALCL has been shown to be associated with NIPA phosphorylation and thus to the inactivation of the SCFNIPA complex. The mechanism described above may therefore provide a framework for understanding how this oncogene interferes with the physiologic regulation of cyclin B - a potential mechanism by which NPM-ALK transforms hematopoietic cells.

The identification of Pcl1-interacting proteins that genetically interact with Cla4 may indicate a link between G1 progression and mitotic exit.

In budding yeast, Cla4 and Ste20, two p21-activated kinases, contribute to numerous morphogenetic processes. Loss of Ste20 or Cla4 individually confers distinct phenotypes, implying that they regulate different processes. However, loss of both proteins is lethal, suggesting some functional overlap. To explore the role(s) of Cla4, we and others have sought mutations that are lethal in a cla4 Delta strain. These mutations define >60 genes. Recently, both Ste20 and Cla4 have been implicated in mitotic exit. Here, we identify a genetic interaction between PHO85, which encodes a cyclin-dependent kinase, and CLA4. We further show that the Pho85-coupled G(1) cyclins Pcl1 and Pcl2 contribute to this Pho85 role. We performed a two-hybrid screen with Pcl1. Three Pcl1-interacting proteins were identified: Ncp1, Hms1, and a novel ATPase dubbed Epa1. Each of these proteins interacts with Pcl1 in GST pull-down experiments and is specifically phosphorylated by Pcl1.Pho85 complexes. NCP1, HMS1, and EPA1 also genetically interact with CLA4. Like Cla4, the proteins Hms1, Ncp1, and Pho85 appear to affect mitotic exit, a conclusion that follows from the mislocalization of Cdc14, a key mitotic regulator, in strains lacking these proteins. We propose a model in which the G(1) Pcl1.Pho85 complex regulates mitotic exit machinery.

Ordered proteolysis in anaphase inactivates Plk1 to contribute to proper mitotic exit in human cells.

We have found that key mitotic regulators show distinct patterns of degradation during exit from mitosis in human cells. Using a live-cell assay for proteolysis, we show that two of these regulators, polo-like kinase 1 (Plk1) and Aurora A, are degraded at different times after the anaphase-promoting complex/cyclosome (APC/C) switches from binding Cdc20 to Cdh1. Therefore, events in addition to the switch from Cdc20 to Cdh1 control the proteolysis of APC/CCdh1 substrates in vivo. We have identified a putative destruction box in Plk1 that is required for degradation of Plk1 in anaphase, and have examined the effect of nondegradable Plk1 on mitotic exit. Our results show that Plk1 proteolysis contributes to the inactivation of Plk1 in anaphase, and that this is required for the proper control of mitotic exit and cytokinesis. Our experiments reveal a role for APC/C-mediated proteolysis in exit from mitosis in human cells.

Orchestrating the cell cycle in yeast: sequential localization of key mitotic regulators at the spindle pole and the bud neck.

Orchestrating the cell cycle in yeast: sequential localization of key mitotic regulators at the spindle pole and the bud neck.

Cell cycle trials in Salamanca: workshop on G2/M progression and associated checkpoints.

This EMBO workshop was organised by Angel Nebreda, Tim Hunt, Sergio Moreno and Paul Nurse and was held in Salamanaca, Spain, September 6–9, 2001. ![][1] The time‐trial stage of the annual ‘La vuelta’ cycle race was held in Salamanca in early September 2001. By coincidence, the cell cycle went on trial in Salamanca at the same time, at an EMBO workshop on G2/M progression and associated checkpoints organized by Angel Nebreda and Sergio Moreno—and by Tim Hunt and Paul Nurse in their pre‐Nobel‐laureate stage! This was a highly enjoyable and stimulating meeting that covered a wide range of topics concerning the less fashionable part of the cell cycle: p53 was hardly mentioned and Rb got very short shrift. In this report, I will try to highlight some of the novel trends and concepts that emerged during the meeting rather than review all the talks. My apologies to those speakers whose talks I have been unable to include. ### Polo‐like kinases: wresting mitosis from the cyclin–CDK family The plenary lecture was given by Stephen Elledge (Houston, TX), who introduced some of the themes that would recur during the meeting. He showed that, in budding yeast, the Bfa1/Byr4 protein, which makes up a two‐component GAP (GTPase activating protein) with Bub2, is regulated by phosphorylation. The Bfa1–Bub2 pathway regulates exit from mitosis in response to the structure and position of the mitotic spindle and in response to DNA damage. Elledge presented evidence that Cdc5, the polo‐like kinase in budding yeast, phosphorylates Bfa1 in anaphase to facilitate exit from mitosis. This phosphorylation was blocked by the spindle assembly and orientation checkpoints. In addition, DNA damage could block mitosis by two pathways: phosphorylation of the anaphase inhibitor securin to prevent its destruction; and hyperphosphorylation of Bfa1, which may help to prevent mitotic exit. During the meeting, it became apparent that the polo family … [1]: /embed/graphic-1.gif