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Single-instruction set architecture (ISA) heterogeneous multi-processor architecture is promising for developing multi-processor system-on-chips (MPSoCs). In this architecture, all processors execute the same instruction set, yet with various performance and power behavior, since processors may have various micro-architectures. Therefore, systems with this architecture have the advantages of easy to develop new functions as the homogeneous architecture, and easy to customize the resource allocation to achieve high energy efficiency as the heterogeneous architecture. However, for an MPSoC utilizing the target architecture, a key design issue is how to select the set of processors so that the target system can achieve good performance while the cost of the chip is constrained to the expected value. To solve this, in this paper, we propose a processor allocation method for MPSoCs with single-ISA heterogeneous multi-core architecture. The goal of the proposed method is to automatically synthesize the allocation of cores for the given workload so that the performance is optimized while the resource constraint is met. To the best of our knowledge, this is the first work that tackles the processor allocation problem for MPSoCs with the target architecture. To bring out the best performance of a hardware configuration, the proposed algorithm also synthesizes the software design of task mapping for a selected hardware configuration. The experimental results show that, compared with the homogeneous architecture with the least cost and lowest performance cores only, even if the number of core is set to the maximum parallelism degree of the target workload, the proposed method achieves up to 8.25% of performance improvement among all the cases we evaluated while the area constraint is met. Compared with the architecture with all high performance but large cores, when the number of cores is also set to the same as the maximum parallelism degree of the target workload, the proposed method has at most 11.5% of performance degradation, while the area cost is reduced by 60.7%.

Overexpression of Aurora kinases is largely observed in many cancers, including hematologic malignancies. In this study, we investigated the effects and molecular mechanisms of Aurora kinase inhibitors in acute lymphoblastic leukemia (ALL). Western blot analysis showed that both Aurora-A and Aurora-B are overexpressed in ALL cell lines and primary ALL cells. Both VE-465 and VX-680 effectively inhibited Aurora kinase activities in nine ALL cell lines, which exhibited different susceptibilities to the inhibitors. Cells sensitive to Aurora kinase inhibitors underwent apoptosis at an IC50 of ∼10-30 nM and displayed a phenotype of Aurora-A inhibition, whereas cells resistant to Aurora kinase inhibitors (with an IC50 more than 10 μM) accumulated polyploidy, which may have resulted from Aurora-B inhibition. Drug susceptibility of ALL cell lines was not correlated with the expression level or activation status of Aurora kinases. Interestingly, RS4;11 and MV4;11 cells, which contain the MLL-AF4 gene, were both sensitive to Aurora kinase-A inhibitors treatment. Complementary DNA (cDNA) microarray analysis suggested that CDKN1A might govern the drug responsiveness of ALL cell lines in a TP53-independent manner. Most importantly, primary ALL cells with MLL-AF4 and CDKN1A expression were sensitive to Aurora kinase inhibitors. Our study suggests CDKN1A could be a potential biomarker in determining the drug responsiveness of Aurora kinase inhibitors in ALL, particularly in MLL-AF4-positive patients.

This paper presents an analysis of the effect of the SiO"2/Si interfacial property on output (transfer) characteristics of organic thin film transistors (OTFTs). A SiO"2 layer was grown on the heavily doped p-type Si wafer using a dry oxidation process as a gate oxide layer. The electrical conduction investigations suggest that the leakage behavior is governed by the poor insulation of the oxygen deficient oxide between SiO"2 and Si. From the observed result, the relationship between gate leakage and output (transfer) characteristics of OTFTs was discussed.

Recent genetic studies in Drosophila identified Kibra as a novel regulator of the Hippo pathway, which controls tissue growth and tumorigenesis by inhibiting cell proliferation and promoting apoptosis. The cellular function and regulation of human KIBRA remain largely unclear. Here, we show that KIBRA is a phosphoprotein and that phosphorylation of KIBRA is regulated in a cell cycle-dependent manner with the highest level of phosphorylated KIBRA detected in mitosis. We further demonstrate that the mitotic kinases Aurora-A and -B phosphorylate KIBRA both in vitro and in vivo. We identified the highly conserved Ser(539) as the primary phosphorylation site for Aurora kinases. Moreover, we found that wild-type, but not catalytically inactive, protein phosphatase 1 (PP1) associates with KIBRA. PP1 dephosphorylated Aurora-phosphorylated KIBRA. KIBRA depletion impaired the interaction between Aurora-A and PP1. We also show that KIBRA associates with neurofibromatosis type 2/Merlin in a Ser(539) phosphorylation-dependent manner. Phosphorylation of KIBRA on Ser(539) plays a role in mitotic progression. Our results suggest that KIBRA is a physiological substrate of Aurora kinases and reveal a new avenue between KIBRA/Hippo signaling and the mitotic machinery.

Unperturbed mitosis is a prerequisite for the generation of two genetically identical daughter cells. Nucleolar-spindle associated protein (NuSAP) is an important mitotic regulator. The activity of NuSAP is essential for a variety of cellular events that occur during mitosis starting from spindle assembly to cytokinesis. In addition to playing crucial roles during mitosis, NuSAP has been in the spotlight recently due to different studies exhibiting its importance in embryogenesis and cancer. In this review, we have extensively mined the current literature and made connections between different studies involving NuSAP. Importantly, we have assembled data pertaining to NuSAP from several proteomic studies and analyzed it thoroughly. Our review focuses on the role of NuSAP in mitosis and cancer, and brings to light several unanswered questions regarding the regulation of NuSAP in mitosis and its role in carcinogenesis.

The small guanosine triphosphatase Ran loaded with GTP (RanGTP) can stimulate assembly of the type V intermediate filament protein lamin B into a membranous lamin B spindle matrix, which is required for proper microtubule organization during spindle assembly. Microtubules in turn enhance assembly of the matrix. Here we report that the isolated matrix contains known spindle assembly factors such as dynein and Nudel. Using spindle assembly assays in Xenopus egg extracts, we show that Nudel regulates microtubule organization during spindle assembly independently of its function at kinetochores. Importantly, Nudel interacts directly with lamin B to facilitate the accumulation and assembly of lamin-B-containing matrix on microtubules in a dynein-dependent manner. Perturbing either Nudel or dynein inhibited the assembly of lamin B matrix. However, depleting lamin B still allowed the formation of matrices containing dynein and Nudel. Therefore, dynein and Nudel regulate assembly of the lamin B matrix. Interestingly, we found that whereas depleting lamin B resulted in disorganized spindle and spindle poles, disrupting the function of Nudel or dynein caused a complete lack of spindle pole focusing. We suggest that Nudel regulates microtubule organization in part by facilitating assembly of the lamin B spindle matrix in a dynein-dependent manner.

Mitotic spindle morphogenesis is a series of highly coordinated movements that lead to chromosome segregation and cytokinesis. We report that the intermediate filament protein lamin B, a component of the interphase nuclear lamina, functions in spindle assembly. Lamin B assembled into a matrix-like network in mitosis through a process that depended on the presence of the guanosine triphosphate–bound form of the small guanosine triphosphatase Ran. Depletion of lamin B resulted in defects in spindle assembly. Dominant negative mutant lamin B proteins that disrupt lamin B assembly in interphase nuclei also disrupted spindle assembly in mitosis. Furthermore, lamin B was essential for the formation of the mitotic matrix that tethers a number of spindle assembly factors. We propose that lamin B is a structural component of the long-sought-after spindle matrix that promotes microtubule assembly and organization in mitosis.

The mitotic spindle apparatus has attracted the attention of cell biologists for decades. Whereas the main function of this microtubule-based system is to segregate chromosomes, spindle morphogenesis and chromosome segregation must also coordinate with the segregation of the whole cell. The finding that RanGTPase stimulates the assembly of a lamin B-containing membranous matrix in mitosis [1] may provide a connection between the segregation of mitotic chromosomes and the partitioning of membrane systems during cell division.

Overexpression of Aurora kinases is largely observed in many cancers, including hematologic malignancies. In this study, we investigated the effects and molecular mechanisms of Aurora kinase inhibitors in acute lymphoblastic leukemia (ALL). Western blot analysis showed that both Aurora-A and Aurora-B are overexpressed in ALL cell lines and primary ALL cells. Both VE-465 and VX-680 effectively inhibited Aurora kinase activities in nine ALL cell lines, which exhibited different susceptibilities to the inhibitors. Cells sensitive to Aurora kinase inhibitors underwent apoptosis at an IC50 of ∼10-30 nM and displayed a phenotype of Aurora-A inhibition, whereas cells resistant to Aurora kinase inhibitors (with an IC50 more than 10 μM) accumulated polyploidy, which may have resulted from Aurora-B inhibition. Drug susceptibility of ALL cell lines was not correlated with the expression level or activation status of Aurora kinases. Interestingly, RS4;11 and MV4;11 cells, which contain the MLL-AF4 gene, were both sensitive to Aurora kinase-A inhibitors treatment. Complementary DNA (cDNA) microarray analysis suggested that CDKN1A might govern the drug responsiveness of ALL cell lines in a TP53-independent manner. Most importantly, primary ALL cells with MLL-AF4 and CDKN1A expression were sensitive to Aurora kinase inhibitors. Our study suggests CDKN1A could be a potential biomarker in determining the drug responsiveness of Aurora kinase inhibitors in ALL, particularly in MLL-AF4-positive patients.

Nucleolar proteins have been in the limelight due to their involvement in multiple cellular processes other than their well-studied functions in maintaining the structural integrity of the nucleolus and ribosome synthesis. Many of these nucleolar proteins have now been shown to play roles in the processes of mitosis as well as tumorigenesis. In this book chapter, we have discussed the contribution of three such Aurora A-interacting nucleolar proteins: NuSAP, Arpc1b and Eg5, to mitosis and tumor progression. Importantly, we have also brought into light several unanswered questions pertaining to the function and regulation of these nucleolar proteins in both mitosis as well as cancer. Finally, we have attempted to reason why several nucleolar proteins have now been implicated as important players in mitosis.

Summary The cullin-RING family of ubiquitin ligases regulates diverse cellular functions, such as cell cycle control, via ubiquitylation of specific substrates. CUL3 targets its substrates through BTB proteins. Here we show that depletion of CUL3 and the BTB protein KLHL18 causes a delay in mitotic entry. Centrosomal activation of Aurora-A, a kinase whose activity is required for entry into mitosis, is also delayed in depleted cells. Moreover, we identify Aurora-A as a KLHL18-interacting partner. Overexpression of KLHL18 and CUL3 promotes Aurora-A ubiquitylation in vivo, and the CUL3-KLHL18-ROC1 ligase ubiquitylates Aurora-A in vitro. Our study reveals that the CUL3-KLHL18 ligase is required for timely entry into mitosis, as well as for the activation of Aurora-A at centrosomes. We propose that the CUL3-KLHL18 ligase regulates mitotic entry through an Aurora-A-dependent pathway.

The Hippo pathway controls organ size and tumorigenesis by inhibiting cell proliferation and promoting apoptosis. KIBRA was recently identified as a novel regulator of the Hippo pathway. Several of the components of the Hippo pathway are important regulators of mitosis-related cell cycle events. We recently reported that KIBRA is phosphorylated by the mitotic kinases Aurora-A and -B. However, the role KIBRA plays in mitosis has not been established. Here, we show that KIBRA activates the Aurora kinases and is required for full activation of Aurora kinases during mitosis. KIBRA also promotes the phosphorylation of large tumor suppressor 2 (Lats2) on Ser(83) by activating Aurora-A, which controls Lats2 centrosome localization. However, Aurora-A is not required for KIBRA to associate with Lats2. We also found that Lats2 inhibits the Aurora-mediated phosphorylation of KIBRA on Ser(539), probably via regulating protein phosphatase 1. Consistent with playing a role in mitosis, siRNA-mediated knockdown of KIBRA causes mitotic abnormalities, including defects of spindle and centrosome formation and chromosome misalignment. We propose that the KIBRA-Aurora-Lats2 protein complexes form a novel axis that regulates precise mitosis.

Aurora B kinase forms the enzymatic core of the Chromosomal Passenger Complex (CPC) and is a master regulator of mitosis. Understanding the regulation of Aurora B is critical to illuminate its role in mitosis. INCENP, Survivin and Borealin have all been known to promote Aurora B activation. In this study, we have identified the Aurora A activator protein TPX2 as a novel scaffold and co-activator protein of the CPC. Studies utilizing M-phase Xenopus egg extracts (XEE) revealed that the immunodepletion of endogenous TPX2 from XEE decreases Aurora B-Survivin and Aurora B-INCENP interactions, leading to a consequent reduction in Aurora B activity. Further, residues 138 to 328 of Xenopus TPX2 (TPX2 B) are sufficient to enhance Aurora B-Survivin association and Aurora B kinase activity in vitro. Importantly, experiments with pancreatic cancer cell lines suggest that this mechanism of Aurora B activation by TPX2 is likely to be conserved in human cells. Strikingly, the overexpression of human TPX2 B in HeLa cells causes defects in metaphase chromosome alignment and INCENP localization. Thus, in addition to its already established role as an Aurora A activator, our data support the role of TPX2 as a novel co-activator of Aurora kinase B.

A cDNA, Ch-GT2, encoding a glucose transporter was cloned by screening a chicken liver λgt11 cDNA library and by modified polymerase chain reaction (PCR) techniques. The encoded protein is highly homologous to rat and human GLUT 2. Within the predicted amino acid sequence, there are 12 putative transmembrane helices with a relatively large exofacial hydrophilic loop between transmembrane segments 1 and 2, which is characteristic of mammalian GLUT 2. Expression of Ch-GT2 gene in E. coli results in sixfold increase in the uptake of 2-deoxy-D-glucose uptake, and this increment can be reduced by incubation with phloretin. Thus, Ch-GT2 is a bona fide chicken liver glucose transporter. A deletion mutant generated by PCR was expressed in bacteria and shown to be functional, indicating that the N-terminal seven amino acid residues of Ch-GT2 is not essential for transport of glucose. From Northern blot analysis, Ch-GT2 is prominently expressed in chicken liver with a transcript of 4.0 kb, but not in chicken brain and heart, suggesting that distinct types of glucose transporters are present in various chicken tissues.

The recombinant 60-kDa fragment of rat hsc70 has been overexpressed in Escherichia coli. The recombinant protein is not capable of disassembling clathrin from coated vesicles. However, the affinity for peptides and the peptide-stimulated ATPase activity of the intact protein are retained in the 60-kDa fragment. The dissociation constants of peptide P3a (the recognition sequence of clathrin light chain LCa by hsc70) and S peptide of ribonuclease for 60-kDa protein are 13 and 7 microM, respectively. The maximal velocities of stimulated ATPase activity by peptides P3a and GT4 are 0.25 and 0.31 nmol/h/microgram of protein, respectively, and the EC50 values (the concentration of peptides that brought about half-maximum hydrolysis) for peptides P3a and GT4 are 0.56 and 0.30 mM, respectively. These results indicate that peptide-stimulated ATPase activity of hsc70 is not sufficient for clathrin uncoating. We suggest that other activities or cellular components as yet unidentified associated with the C-terminal 10-kDa fragment of hsc70 are required for clathrin uncoating.

Selective small-molecule kinase inhibitors have encouraging clinical efficacy in several malignancies. These agents are still limited to a subset of patients, indicating the need to develop therapeutic biomarkers that influence clinical benefit. In this study, we demonstrate that treatment with MK-8745, a novel Aurora-A specific inhibitor, leads to cell cycle arrest at the G2/M phase with accumulation of tetraploid nuclei followed by cell death in non-Hodgkin lymphoma (NHL) cell lines. The sensitivity of the cell lines to MK-8745 is correlated with the expression level of Aurora-A activator. The siRNA knockdown of Aurora-A activator TPX2 (targeting protein for Xenopus kinase-like protein 2) increased MK-8745 sensitivity in less-MK-8745-sensitive NHL cell lines, whereas overexpression of TPX2 in high-MK-8745-sensitive NHL cell lines increased drug resistance. Our results indicate that TPX2 may serve as a biomarker for identifying subpopulations of patients sensitive to Aurora-A inhibitor treatment.

Three mutants of rat hsc70 were constructed, overexpressed in Escherichia coli, purified, and characterized. First, site-directed mutation was utilized to substitute Asn for Asp-10. The recombinant protein, hsc70(D10N), loses not only its peptide-stimulated ATPase activity but also its basal ATPase activity. The measured dissociation constants of ATP (0.3 microM) and S-peptide (5 microM) for hsc70(D10N), however, are virtually identical to those of hsc70. The intrinsic fluorescence spectra of hsc70(D10N) also remain largely unchanged. Therefore, the overall structure of the hsc70 protein is most likely intact after mutation. Second, the entire C-terminal peptide-binding domain was deleted and the resultant mutant contains only the N-terminal ATPase domain of hsc70. This recombinant protein, Nt-hsc70, is a peptide-independent ATPase. The ATPase activity at 37 degrees C of the Nt-hsc70, 270 pmol/h/micrograms of protein, is comparable to that of maximally peptide-activated hsc70. Third, the Asp-10 of Nt-hsc70 was replaced by Asn. Despite that this mutant, Nt-hsc70(D10N), is capable of binding ATP and it loses the capability to hydrolyze ATP. Taken together, these results indicate that aspartyl residue 10 of hsc70 is essential for ATP hydrolysis. Purified hsc70 and its mutants autophosphorylate in vitro at a substoichiometric level. On average, less than 1% of the hsc70 and Nt-hsc70 proteins are phosphorylated. Although the amount of phosphate incorporated into hsc70(D10N) and Nt-hsc70(D10) is reduced, a significant level of phosphorylation can still be achieved in these two site-directed mutants. Hence, autophosphorylation of hsc70 and its mutants is not correlated with their ability to hydrolyze ATP.

In addition to its function as an Arp2/3 complex subunit, Arp1cb interacts with and stimulates Aurora A at centrosomes, functioning in cell cycle progression., Here we provide evidence in support of an inherent role for Arpc1b, a component of the Arp2/3 complex, in regulation of mitosis and demonstrate that its depletion inhibits Aurora A activation at the centrosome and impairs the ability of mammalian cells to enter mitosis. We discovered that Arpc1b colocalizes with γ-tubulin at centrosomes and stimulates Aurora A activity. Aurora A phosphorylates Arpc1b on threonine 21, and expression of Arpc1b but not a nonphosphorylatable Arpc1b mutant in mammalian cells leads to Aurora A kinase activation and abnormal centrosome amplification in a Pak1-independent manner. Together, these findings reveal a new function for Arpc1b in centrosomal homeostasis. Arpc1b is both a physiological activator and substrate of Aurora A kinase and these interactions help to maintain mitotic integrity in mammalian cells.

International audience; The activated form of Ran (Ran-GTP) stimulates spindle assembly in Xenopus laevis egg extracts, presumably by releasing spindle assembly factors, such as TPX2 (target protein for Xenopus kinesin-like protein 2) and NuMA (nuclear-mitotic apparatus protein) from the inhibitory binding of importin-alpha and -beta. We report here that Ran-GTP stimulates the interaction between TPX2 and the Xenopus Aurora A kinase, Eg2. This interaction causes TPX2 to stimulate both the phosphorylation and the kinase activity of Eg2 in a microtubule-dependent manner. We show that TPX2 and microtubules promote phosphorylation of Eg2 by preventing phosphatase I (PPI)-induced dephosphorylation. Activation of Eg2 by TPX2 and microtubules is inhibited by importin-alpha and -beta, although this inhibition is overcome by Ran-GTP both in the egg extracts and in vitro with purified proteins. As the phosphorylation of Eg2 stimulated by the Ran-GTP-TPX2 pathway is essential for spindle assembly, we hypothesize that the Ran-GTP gradient established by the condensed chromosomes is translated into the Aurora A kinase gradient on the microtubules to regulate spindle assembly and dynamics.

We present a definition of burstiness for delay sensitive traffic streams in terms of a function, i.e., the cell loss probability of a bufferless multiplexer at all non-negative link capacities. Some properties of the definition are proved. One property implies any definition of burstiness in terms of a real number may not be appropriate. The other property implies the commonly used moment matching method in approximating traffic sources may not be appropriate either. We also present a quantization algorithm for networks which provide only finitely many bit rates for describing traffic characteristics. Our proposed quantization algorithm guarantees the quantized version is always burstier than the original source and thus can be used to design call admission control scheme for an ATM network.

The nature of kinesin interactions with membrane-bound organelles and mechanisms for regulation of kinesin-based motility have both been surprisingly difficult to define. Most kinesin is recovered in supernatants with standard protocols for purification of motor proteins, but kinesin recovered on membrane-bound organelles is tightly bound. Partitioning of kinesin between vesicle and cytosolic fractions is highly sensitive to buffer composition. Addition of eitherN-ethylmaleimide or EDTA to homogenization buffers significantly increased the fraction of kinesin bound to organelles. Given that an antibody against kinesin light chain tandem repeats also releases kinesin from vesicles, these observations indicated that specific cytoplasmic factors may regulate kinesin release from membranes. Kinesin light tandem repeats contain DnaJ-like motifs, so the effects of hsp70 chaperones were evaluated. Hsc70 released kinesin from vesicles in an MgATP-dependent andN-ethylmaleimide-sensitive manner. Recombinant kinesin light chains inhibited kinesin release by hsc70 and stimulated the hsc70 ATPase. Hsc70 actions may provide a mechanism to regulate kinesin function by releasing kinesin from cargo in specific subcellular domains, thereby effecting delivery of axonally transported materials.

Abstract 4907 Introduction: Despite improved treatment outcome in acute lymphoblastic leukemia (ALL), drug resistance and disease recurrence remain major obstacles in specific subgroups. Thus, there is an urgent need to identify new targets for therapy. Several studies showed that Aurora kinases were therapeutic targets in cancer therapy, including solid tumors and hematological malignancies. Here we describe preclinical testing of Aurora kinase inhibitors in ALL and the molecular mechanism of different drug activity. Materials and methods: Quantitative RT-PCR and Western blot were used to assess the expressions of Aurora kinases and their activators in ALL. RT-PCR was used to detect the expression of MDR-1. To test activity against Aurora kinases, different ALL cell lines were treated with various concentrations of Aurora kinase inhibitors “VE-465 and VX-680”. The effects of Aurora kinase inhibitors on the cell cycle were evaluated by flow cytometry. Gene expression profiling was performed to identify the candidate targets which regulate the different drug sensitivity. Transient knockdown and overexpression of candidate genes in ALL cell lines were also employed in this study. Results: Nine ALL cell lines treated with Aurora kinase inhibitors (VE-465 and VX-680) exhibited different drug sensitivity. Five ALL cell lines were sensitive to drug treatment with IC50 around 10–40 nM, including MLL-AF4-positive and BCR-ABL-positive cell lines. However, RPMI-8402 was one of the three cell lines which were resistant to VE-465 and VX-680 with IC50 more than 10 μM. Among these sensitive ALL cell lines, treatment of Aurora kinase inhibitors resulted in an increased G2/M and sub-G1 populations. In contrast, drug-resistant ALL cell lines showed increased polyploidy status after exposure to Aurora kinase inhibitors. The different treatment efficacy was not related to the expression of Aurora kinases, their activators or MDR-1. In order to elucidate the molecular mechanism to regulate the different drug sensitivity, microarray study was performed. It showed that treatment of Aurora kinase inhibitors resulted in differential expressions of genes (75 up-regulated and 90 down-regulated genes) and CDKN1Awas one of the potential molecules which regulated the treatment diversity. RT-PCR and Western blot confirmed the cDNA microarray data: CDKN1A was up-regulated after treatment with Aurora kinase inhibitors in the drug-sensitive cell lines, but no change in the level of CDKN1A in the drug-resistance cell lines. Knockdown of CDKN1A in drug-sensitive cell lines impaired the treatment activity. Over-expression of CDKN1A in drug-resistant cell lines increased the anti-leukemia effect of Aurora kinase inhibitors. Conclusion: These data suggest that treatment with Aurora kinase inhibitors may be a novel and effective therapy in specific subgroups of ALL, including MLL-AF4-positive ALL. These data show that status of Aurora kinases, their activators or MDR-1 does not correlate with the drug susceptibility in ALL cell lines. The susceptibility to Aurora kinase inhibitors in ALL depends on the activation status of CDKN1A. Disclosures: No relevant conflicts of interest to declare.

Aurora B kinase is a master regulator of mitosis and is over-expressed in a variety of malignancies including astrocytomas, seminomas, ependymomas, prostate cancer and non small-cell lung cancer. Several Aurora B inhibitors are currently being evaluated for their efficacy in cancer therapy. Therefore, it is critical to understand the regulation of Aurora B activity by other proteins. Aurora B forms the enzymatic core of the Chromosomal Passenger Complex (CPC). The CPC consists of three other proteins besides Aurora B: INCENP, Survivin and Borealin. All these proteins have been known to promote Aurora B activation. The mitotic protein Targeting Protein for Xenopus kinesin-like protein 2 (TPX2) co-localizes with Aurora B at several stages of mitosis, hinting at the existence of a signaling cross-talk between Aurora B and TPX2. The main purpose of our study is to determine whether Aurora B and TPX2 can cross-talk with each other during mitosis, and if found so, to decode the mechanism by which this signaling cross-talk occurs. We hypothesize that TPX2 can transmit signals to regulate Aurora B activity during mitosis. To test our hypothesis, we have performed experiments utilizing M-phase Xenopus egg extracts (XEE) which possess an abundance of mitotic proteins, and human cell lines as model systems. By performing immunoprecipitation assays (IPs) in M-phase XEE, we detected an interaction between TPX2 and the CPC proteins - Aurora B, INCENP and Survivin. This validates the existence of a signaling cross-talk between TPX2 and the CPC proteins. Moreover, IPs and microtubule pelleting assays utilizing XEE revealed that, as is typical of scaffold proteins, either too much or too little TPX2 alters Aurora B activity. Significantly, immunodepletion of endogenous TPX2 from XEE decreases the association between Aurora B and its activators- Survivin and INCENP, leading to a consequent reduction in Aurora B activity. Additionally, in vitro binding assays demonstrate that, residues 138 to 328 of Xenopus TPX2 (TPX2 B) are sufficient to enhance the interaction between Aurora B and its activator, Survivin. Further, TPX2 B also increases Aurora B kinase activity in an in vitro kinase assay. These data suggest that under endogenous conditions, TPX2 functions as a co-activator of Aurora B by increasing Aurora B-Survivin and Aurora B-INCENP interactions. Importantly, IPs with Panc-1 and CFPAC-1 pancreatic cancer cell lines suggest that this mechanism of Aurora B activation by TPX2 is likely to be conserved in human cancer cell lines. Based upon our results, we conclude that TPX2 enhances Aurora B activity by serving as a scaffold protein for assembly of the CPC. 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 LB-287. doi:1538-7445.AM2012-LB-287

A plethora of proteins and processes regulate proper mitosis and are yet to be fully understood. Since mitotic cells are an emerging target for cancer therapy, it is of utmost importance to decipher the mechanisms that regulate mitosis. The purpose of our study is to identify the role CUL3 plays in regulating mitosis. Specifically, it is our aim to identify CUL3 interacting partners that play roles in spindle assembly, in order to understand the function of CUL3 in constructing the mitotic spindle. The CUL3 protein is an ubiquitin E3 ligase scaffold. By means of ubiquitination, CUL3 can regulate an array of proteins. We have performed CUL3 RNAi experiments, followed by cell cycle analysis using flow cytometry and immunostaining of DNA, α-tubulin and γ-tubulin, to identify the importance of CUL3 in regulating mitosis. Furthermore, we have performed CUL3 immunoprecipitation (IP) experiments in X.laevis mitotic egg extracts and in HeLa cells synchronized at mitosis, to identify the binding partners of CUL3 which orchestrate mitosis. Using treatment with either nocodazole (promotes microtubule depolymerization) or monastrol (causes microtubule stabilization), we performed interaction studies in HeLa cells to determine the effect of microtubule polymerization on the interaction of CUL3 with its mitotic binding partners. Similarly, in X.laevis egg extracts, we have used warm (for microtubule depolymerization) and cold (for microtubule stabilization) IP conditions to determine the effects of microtubule status on CUL3 interaction with its binding partners. Our results have shown that CUL3-depleted cells display a dramatic mitotic phenotype which includes: defective spindles, multiple centrosomes, enlarged cells and nuclei, G2/M accumulation and delayed cytokinesis. To understand how CUL3 may participate in the formation of the mitotic spindle, we determined the binding partners of CUL3 that are involved in spindle assembly. CUL3 IP results from both X.laevis egg extracts and HeLa cells indicate that CUL3 interacts with the kinesin motor protein, Eg5. Eg5 is an important spindle assembly factor, which is essential in mitosis for proper formation of a bipolar spindle. Interestingly, targeting Eg5 is emerging as an attractive means to inhibit the proliferation of cancer cells. Thus, better characterizing the interaction of CUL3 and Eg5 is critical to understanding the function of CUL3 as well as Eg5 in mitosis. Further characterization of the interaction between CUL3 and Eg5 demonstrated that this interaction is present only in the presence of depolymerized microtubules. Our results culminate to the following conclusions: CUL3 is required for cells to properly undergo mitosis, especially the correct formation of spindle apparatus. CUL3 interacts with the spindle assembly factor Eg5 in the presence of depolymerized microtubules, specifically during mitosis. We suspect that CUL3-Eg5 interaction is important for mitosis and in particular for bipolar spindle formation. 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 LB-288. doi:1538-7445.AM2012-LB-288

Summary The roles of the kinase Aurora A (AurA) in centrosome function and spindle assembly [1–3] have been established in Drosophila [4, 5], C. elegans [6], and Xenopus egg extracts [7–9]. Recently, we have shown that AurA acts downstream of the RanGTPase signaling pathway to stimulate spindle assembly in mitosis [10]. However, it is still not clear whether AurA can stimulate the formation of microtubule organizing centers (MTOC) on its own. Moreover, whether AurA is essential for spindle assembly in the absence of centrosomes has remained unclear [10, 11]. Here, we report the development of functional assays that allow us to show that activation of AurA by TPX2 is essential for Ran-stimulated spindle assembly in the presence or absence of centrosomes. Furthermore, AurA-coated magnetic beads function as MTOCs in the presence of RanGTP in Xenopus egg extracts and RanGTP stimulates AurA to recruit activities responsible for both MT nucleation and organization to the beads. The MTOC function of AurA-coated beads require both MT nucleators and motors. Compared to XMAP215-coated beads [12], AurA-coated beads increase the rate of bipolar spindle assembly in the presence of RanGTP, and the kinase activity of AurA is essential for the beads to function as MTOCs.