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.     

Distinct Dynamics of Aurora B and Survivin During Mitosis

We have studied the dynamics of Aurora B and Survivin during mitosis in living cells, using C-terminal GFP chimeras of the two proteins. These chimeras showed identical localization and behave as bona fide wild type proteins. The mobility of Aurora B-GFP and Survivin-GFP was analyzed by FRAP. The data show that Survivin-GFP, in contrast to Aurora B-GFP, is highly mobile at prometaphase and metaphase. At telophase and cell cleavage, both chimeras are found to be fully immobile. The ablation of Aurora B by siRNA results in a dramatic decrease of the Survivin-GFP mobility. These results demonstrate that Survivin, but not Aurora B, is weakly associated with the centromeric chromatin at prometaphase and metaphase. The weak association of Survivin with centromeric chromatin is dependent on the presence of Aurora B and is not affected by treatment with either nocodazole or taxol. The rapid and conditional interchange between passenger proteins that we show by live imaging indicates that the high affinity interactions demonstrated with in vitro analysis of passenger protein binding are, in fact, static “snapshots” of highly dynamic and regulated in vivo interactions in mitotic cells.     

The Cdc48/p97-Ufd1-Npl4 Complex: Its Potential Role in Coordinating Cellular Morphogenesis During the M-G1 Transition

The AAA ATPase Cdc48/p97 together with its adaptors, Ufd1-Npl4, regulate membrane-related functions and mitotic spindle disassembly by directly binding to membrane-associated proteins or spindle assembly factors, modulating their interactions with membranes or spindles, respectively. Here, we discuss the possibility that the Cdc48/p97-Ufd1-Npl4 complex has a more general role in mediating morphological transitions as the cell exits mitosis and enters G1.     

Localization of Aurora A and Aurora B kinases during interphase: Role of the N-Terminal domain

Aurora kinases possess a conserved catalytic domain (CD) and a N-terminal domain (ND) that varies in size and sequence. We have previously reported that the N-terminal domain of AuroraA (AurA) participates in the localization of the kinase to the centrosome in interphase. AuroraB (AurB) is a chromosome passenger protein and its N-terminal domain is not necessary for its localization or function during mitosis. Using various combinations of GFP-AurA and AurB protein domains we show that AurB N-terminal domain is required for nuclear localization in Xenopus XL2 cells in interphase. In human cells, however, we found both AurA and AurB kinases in the nucleus, AurA being mainly cytoplasmic and AurB mainly nuclear. Both proteins are actively excluded from the nucleus by a CRM1 dependent pathway. Interestingly, at a functional level, in interphase, every combination of Aurora kinase domains (ND-CD) rescues histone H3 Serine10 phosphorylation defect induced by AurB knockdown. This clearly indicates the presence of a functional AurA in the nucleus. However, the chimera ND-AurA/CD-AurB was much more efficient than the ND-AurB/CD-AurA to rescue multinucleation also induced by AurB knockdown. This indicates that the catalytic domain of AurB is required to fulfill specific functions during mitosis that cannot be fulfilled by the catalytic domain of AurA, probably for localization reasons during mitosis.     

The survivin/Aurora B complex: its role in coordinating tension and attachment

Proper chromosome segregation relies on the action of the spindle checkpoint. Recent data have shown that the chromosomal passenger proteins survivin and Aurora B play an important auxiliary role in spindle checkpoint surveillance. Knock-down experiments in human cells indicate that the function of the survivin/Aurora B complex is required to correct improper microtubule-kinetochore interactions. Combined data of four different groups show that the survivin/Aurora B complex is not an integral component of the spindle checkpoint, but it enables the cell to communicate lack of tension back to the attached microtubules. Moreover, they show that the affinity of BubR1 for kinetochores is directly influenced by the absence or presence of the survivin/Aurora B complex. These functions of the survivin/Aurora B complex are essential for chromosome biorientation, a prerequisite for proper chromosome segregation. As such, this complex plays an important role in the maintenance of a stable genome.     

Indomethacin promotes apoptosis in gastric cancer cells through concomitant degradation of Survivin and Aurora B kinase proteins

Regular usage of nonsteroidal anti-inflammatory drugs (NSAIDs) is associated with reduced incidence of a variety of cancers. The molecular mechanisms underlying these chemopreventive effects remain poorly understood. This current investigation showed that in gastric cancer cells: (1) Indomethacin treatment enhanced the degradation of chromosomal passenger proteins, Survivin and Aurora B kinase; (2) Indomethacin treatment down-regulated Aurora B kinase activity in a cell cycle-independent fashion; (3) siRNA knockdown of Survivin level promoted Aurora B kinase protein degradation, and vice versa; (4) ectopic overexpression of Survivin blocked reduction of Aurora B kinase level and activity by indomethacin treatment, and vice versa; (5) siRNA knockdown of Aurora B kinase level and AZD1152 inhibition of its activity induced apoptosis, and overexpression of Aurora B kinase inhibited indomethacin-induced apoptosis; (6) indomethacin treatment reduced Aurora B kinase level, coinciding with reduction of Survivin level and induction of apoptosis, in KATO III and HT-29 cells, and in mouse gastric mucosa. A role for Aurora B kinase function in NSAID-induced apoptosis was not previously explored. Thus this report provides better understanding of the molecular mechanisms underlying the anti-cancer effect of NSAIDs by elucidating a significant role for Aurora B kinase in indomethacin-induced apoptosis.     

Survivin mutant (Surv-DD70, 71AA) disrupts the interaction of Survivin with Aurora B and causes multinucleation in HeLa cells

Survivin is associated with Aurora B, inner centromere protein (INCENP), and borealin to form a chromosomal passenger complex that plays multiple roles during cell division. We used mutational analysis to study interaction of Survivin with Aurora B and the effect of this interaction on cell division. A Survivin mutant with the terminal domain deleted (Survivin 1-107) bound Aurora B as efficiently as Survivin wild type. This indicated that the proximal BIR domain of Survivin was responsible for Survivin binding to Aurora B. Survivin mutants (Surv-R18A, Surv-D53A, and Sur-KK78, and 79AA) all bound to Aurora B efficiently, but mutation in the conserved amino acid residues of the acidic patch on Survivin (Surv-DD70, 71AA) abolished the direct interaction of Survivin and Aurora B. The Survivin mutant (Surv-DD70, 71AA) localized diffusely in metaphase and failed to successfully accumulate in the midbody during cytokinesis. Furthermore, over-expression of the Survivin mutant (Surv-DD70, 71AA) severely disturbed cytokinesis, resulting in multinucleation in HeLa cell. This indicated that the direct interaction of Survivin and Aurora B was critical for the correct location of Survivin and the function of the Survivin complex in cell division.     

嗅鞘细胞治疗脊髓损伤的研究进展

本文通过广泛查阅近几年嗅鞘细胞治疗脊髓损伤的国内外相关文献,发现嗅鞘细胞可以分泌众多的神经营养因子并表达相应受体,且能够调节星型胶质细胞的反应性,降低其神经胶质酸性蛋白和硫酸软骨素糖蛋白的表达水平,且能与其更好的融合在一起.在临床应用方面,嗅鞘细胞供应来源、应用时机及纯度正引起关注及研究.嗅鞘细胞对于治疗脊髓损伤有巨大的应用前景,对嗅鞘细胞进行基因改造、联合应用其他有促进作用的治疗方法将是未来的研究方向.     

The tumor suppressor Lats2 is pivotal in Aurora A and Aurora B signaling during mitosis

Accurate coordination between chromosome segregation and cytokinesis by various mitotic kinases, such as Aurora, prevent tetraploidization and subsequent tumorigensis. The tumor suppressors Lats1 and Lats2 are serine/threonine kinases that localize to the centrosome and regulate cell cycle progression and apoptosis. In the present study, Aurora A was demonstrated to phosphorylate Lats2 on serine 380 (S380) during mitosis. Immunocytochemical observations revealed that the subcellular localization of Lats2 was distinct during the cell cycle and depended on which site was phosphorylated. Interestingly, the S380-phosphorylated Lats2 protein (pS380) colocalized at the central spindle with Aurora B. Physical interactions were observed between Aurora A, Lats2, Lats1 and Aurora B. The Lats1 kinase was shown to phosphorylate Aurora B. Cells expressing a nonphosphorylated mutant (S380A) of Lats2 caused chromosome missegregation and cytokinesis failure, similar to cells with aberrantly expressed Aurora B. Together, the results suggest that the Aurora A-Lats1/2-Aurora B axis might be a novel pathway that regulates accurate mitotic progression by ensuring the proper mitotic localization of Lats2.     

VX-680 Inhibits Aurora A and Aurora B Kinase Activity in Human Cells

VX-680, also known as MK-0457, is a member of a diverse group of small molecules that inhibit the Aurora kinases, and has shown significant potential as an anti-cancer agent. In keeping with many protein kinase inhibitors, this compound is not a monospecific agent, and its cellular specificity remains largely unknown. In cells, VX-680 blocks mitotic Histone H3 phosphorylation and induces polyploidy and apoptosis, consistent with inhibition of the mitotic protein kinase Aurora B. In this study, we have investigated the effects of VX-680 in proliferating human cancer cells, and demonstrate that it blocks the phosphorylation and activation of both Aurora A and B. Additionally, VX-680 suppresses the phosphorylation of specific substrates of each enzyme, including the Aurora A target TACC3 on Ser558. Exposure to VX-680 induces a monopolar spindle phenotype, delays mitotic progression and rapidly overrides the spindle assembly checkpoint in the presence of spindle poisons. VX-680 also exhibits potent cytotoxicity when compared to the well documented Aurora B inhibitor ZM447439. Taken together, these data identify Aurora A and Aurora B as dual intracellular targets of VX-680.     

The Survivin Isoform Survivin-3B is Cytoprotective and can Function as a Chromosomal Passenger Complex Protein

Survivin is described as a bifunctional protein inhibiting apoptosis and regulating mitosis. However, the biological functions and contributions to cancer progression of survivin splicevariants are controversially discussed. We here show that the intracellular localization of 5 these splice variants depends on a Crm1-dependent nuclear export signal (NES) present in survivin, survivin-2B and survivin-3B, but absent in survivin-ΔEx3 and survivin-2α. Survivin isoforms lack an active nuclear import signal and are able to enter the nucleus by passive diffusion. Only survivin-3B but none of the other splice variants is cytoprotective and able to efficiently interact with chromosomal passenger complex (CPC) proteins. The NES together 10 with efficient CPC formation is required for the cytoprotective activity of survivin isoforms, aswell as for their correct localization and function during cell division. In the tumours from breast, colorectal, head and neck cancer, lymphoma and leukemia patients, survivin and survivin-2B were found overexpressed. However, survivin was the predominant form detected, and the other survivin isoforms were only expressed at low levels in tumours. Our data 15 provide a molecular rationale for the localization and activity of survivin variants, and conclude that survivin isoforms are unlikely to modulate survivin in trans in cancer patients.     

The Aurora A and Aurora B Protein Kinases: A Single Amino Acid Difference Controls Intrinsic Activity and Activation by TPX2

The Aurora A and B protein kinases are key players in mitotic control and the etiology of human cancer. Despite the near identity of amino acid sequence in the catalytic domain, monomeric Aurora B is 50 fold lower in activity than monomeric Aurora A, and previous studies have shown that TPX2 binding to the catalytic domain activates Aurora A but not Aurora B. Here we identify G205 in Aurora A as a key determinant of both intrinsic activity and regulation by TPX2. Mutation of G205 in Aurora A to N, the equivalent residue in Aurora B, had no effect on autophosphorylation of the T-loop but led to a 20-fold loss of specific activity, whereas mutation of N158 in Aurora B to G caused a 350-fold increase in specific activity. G205 N Aurora A was still activated by TPX2, but protection of pT295 from dephosphorylation by protein phosphatase 1 was abolished. Structural analysis of these effects suggests that the G198 forms a pivot point in the enzyme that results in movement of the N-terminal domain glycine-rich loop closer to the ATP binding site of the enzyme and also moves the C-helix slightly closer to the activation loop. Changes in these positions are comparable to those reported for other protein kinases and demonstrate that phosphorylation of the activation loop alone is not sufficient for enzyme activation. The generation of an activated mutant of Aurora B will be important for studying its role in cell cycle control and tumorigenesis.     

The Mre11/Rad50/Nbs1 Complex and Its Role as a DNA Double-Strand Break Sensor for ATM

Abstract not yet available.     

Tension and Force-Resistant Attachment Are Essential for Myofibrillogenesis in Drosophila Flight Muscle

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Aurora蛋白激酶及其抑制剂的研究进展

Aurora激酶是一类在细胞周期调控中具有重要作用的丝/苏氨酸蛋白激酶,包括三个成员:Aurora A、B、C.Aurora激酶在大多数肿瘤中均高表达,其异常调控与肿瘤的发生发展密切相关.近年来,Aurora激酶抑制剂作为抗肿瘤候选药物受到广泛的关注,已有一些进入临床试验,且表现出良好的抗肿瘤活性.本文围绕Aurora家族的三个成员,讨论了其在细胞有丝分裂过程中的生物学功能、与肿瘤发生发展的关系及其抑制荆的研究进展,指出其作为抗肿瘤药物靶点的潜在价值.     

Abscission checkpoint control: stuck in the middle with Aurora B

At the end of cell division, the cytoplasmic bridge joining the daughter cells is severed through a process that involves scission of the plasma membrane. The presence of chromatin bridges 'stuck' in the division plane is sensed by the chromosomal passenger complex (CPC) component Aurora B kinase, triggering a checkpoint that delays abscission until the chromatin bridges have been resolved. Recent work has started to shed some light on the molecular mechanism by which the CPC controls the timing of abscission.     

Sensing centromere tension: Aurora B and the regulation of kinetochore function

Maintaining genome integrity during cell division requires regulated interactions between chromosomes and spindle microtubules. To ensure that daughter cells inherit the correct chromosomes, the sister kinetochores must attach to opposite spindle poles. Tension across the centromere stabilizes correct attachments, whereas phosphorylation of kinetochore substrates by the conserved Ipl1/Aurora B kinase selectively eliminates incorrect attachments. Here, we review our current understanding of how mechanical forces acting on the kinetochore are linked to biochemical changes to control chromosome segregation. We discuss models for tension sensing and regulation of kinetochore function downstream of Aurora B, and mechanisms that specify Aurora B localization to the inner centromere and determine its interactions with substrates at distinct locations.     

The Role of Membrane Lateral Tension in Calcium-Induced Membrane Fusion

Calcium-induced fusion of liposomes was studied with a view to understand the role of membrane tension in this process. Lipid mixing due to fusion was monitored by following fluorescence of rhodamine-phosphatidyl-ethanolamine incorporated into liposomal membrane at a self-quenching concentration. The extent of lipid mixing was found to depend on the rate of calcium addition: at slow rates it was significantly lower than when calcium was injected instantly. The vesicle inner volume was then made accessible to external calcium by adding calcium ionophore A23187. No effect on fusion was observed at high rates of calcium addition while at slow rates lipid mixing was eliminated. Fusion of labeled vesicles with a planar phospholipid membrane (BLM) was studied using fluorescence microscopy. Above a threshold concentration specific for each ion, Ca²⁺, Mg²⁺, Cd²⁺ and La³⁺ induce fusion of both charged and neutral membranes. The threshold calcium concentration required for fusion was found to be dependent on the vesicle charge, but not on the BLM charge. Pretreatment of vesicles with ionophore and calcium inhibited vesicle fusion with BLM. This effect was reversible: chelation of calcium prior to the application of vesicle to BLM completely restored their ability to fuse. These results support the hypothesis that tension in the outer monolayer of lipid vesicle is a primary reason for membrane destabilization promoting membrane fusion. How this may be a common mechanism for both purely lipidic and protein-mediated membrane fusion is discussed. Received: 27 September 1999/Revised: 22 March 2000     

The Regulative Role of Neurite Mechanical Tension in Network Development

A bewildering series of dynamical processes take part in the development of the nervous system. Neuron branching dynamics, the continuous formation and elimination of neural interconnections, are instrumental in constructing distinct neuronal networks, which are the functional building blocks of the nervous system. In this study, we investigate and validate the important regulative role of mechanical tension in determining the final morphology of neuronal networks. To single out the mechanical effect, we cultured relatively large invertebrate neurons on clean quartz surfaces. Applied to these surfaces were isolated anchoring sites consisting of carbon nanotube islands to which the cells and the neurites could mechanically attach. Inspection of branching dynamics and network wiring upon development revealed an innate selection mechanism in which one axon branch wins over another. The apparent mechanism entails the build-up of mechanical tension in developing axons. The tension is maintained by the attachment of the growth cone to the substrate or, alternatively, to the neurites of a target neuron. The induced tension promotes the stabilization of one set of axon branches while causing retraction or elimination of axon collaterals. We suggest that these findings represent a crucial, early step that precedes the formation of synapses and regulates neuronal interconnections. Mechanical tension serves as a signal for survival of the axonal branch and perhaps for the subsequent formation of synapses.