XMog1, a nuclear ran-binding protein in Xenopus, is a functional homologue of Schizosaccharomyces pombe mog1p that co-operates with RanBP1 to control generation of Ran-GTP.

Ran is a multifunctional small GTPase of the Ras superfamily that plays roles in nucleocytoplasmic transport, mitotic spindle assembly and nuclear envelope formation. By screening a Xenopus oocyte cDNA library for Ran-GTP-binding proteins using the two-hybrid system of co-expression in yeast, we identified XMog1, a 20.4 kDa polypeptide related to Mog1p in Saccharomyces cerevisiae and similar gene products in Schizosaccharomyces pombe, Arabidopsis and mammals. We show that cDNAs encoding XMog1 and S. cerevisiae Mog1p rescue the growth defect of S. pombe cells lacking mog1, demonstrating conservation of their functions. In Xenopus somatic cells and transfected mammalian cells, XMogl is localised to the nucleus. XMog1 alone does not stimulate Ran GTPase activity or nucleotide exchange, but causes nucleotide release from Ran-GTP and forms a complex with nucleotide-free Ran. However, in combination with Ran-binding protein 1 (RanBP1), XMog1 promotes the release of GDP and the selective binding of GTP to Ran. XMog1 and RanBP1 also promote selective GTP loading onto Ran catalysed by the nuclear guanine nucleotide exchange factor, RCC1. We propose that Mog1-related proteins, together with RanBP1, facilitate the generation of Ran-GTP from Ran-GDP in the nucleus.

Ran GTPase: a master regulator of nuclear structure and function during the eukaryotic cell division cycle?

Ran is an abundant GTPase that is highly conserved in eukaryotic cells and has been implicated in many aspects of nuclear structure and function, especially determining the directionality of nucleocytoplasmic transport during interphase. However, cell-free systems have recently shown that Ran plays distinct roles in mitotic spindle assembly and nuclear envelope (NE) formation in vitro. During spindle assembly, Ran controls the formation of complexes with importins, the same effectors that control nucleocytoplasmic transport. Here, we review these advances and discuss a general model for Ran in the coordination of nuclear processes throughout the cell division cycle via common biochemical mechanisms.

Roles of Ran-GTP and Ran-GDP in precursor vesicle recruitment and fusion during nuclear envelope assembly in a human cell-free system.

The molecular mechanism of nuclear envelope (NE) assembly is poorly understood, but in a cell-free system made from Xenopus eggs NE assembly is controlled by the small GTPase Ran [1,2]. In this system, Sepharose beads coated with Ran induce the formation of functional NEs in the absence of chromatin [1]. Both generation of Ran-GTP by the guanine nucleotide exchange factor RCC1 and GTP hydrolysis by Ran are required for NE assembly, although the roles of the GDP- and GTP-bound forms of Ran in the recruitment of precursor vesicles and their fusion have been unclear. We now show that beads coated with either Ran-GDP or Ran-GTP assemble functional nuclear envelopes in a cell-free system derived from mitotic human cells, forming pseudo-nuclei that actively transport proteins across the NE. Both RCC1 and the GTPase-activating protein RanGAP1 are recruited to the beads, allowing interconversion between Ran-GDP and Ran-GTP. However, addition of antibodies to RCC1 and RanGAP1 shows that Ran-GDP must be converted to Ran-GTP by RCC1 before precursor vesicles are recruited, whereas GTP hydrolysis by Ran stimulated by RanGAP1 promotes vesicle recruitment and is necessary for vesicle fusion to form an intact envelope. Thus, the GTP-GDP cycle of Ran controls both the recruitment of vesicles and their fusion to form NEs.

Inhibition of the G2 DNA damage checkpoint and of protein kinases Chk1 and Chk2 by the marine sponge alkaloid debromohymenialdisine.

Cells can respond to DNA damage by activating checkpoints that delay cell cycle progression and allow time for DNA repair. Chemical inhibitors of the G(2) phase DNA damage checkpoint may be used as tools to understand better how the checkpoint is regulated and may be used to sensitize cancer cells to DNA-damaging therapies. However, few inhibitors are known. We used a cell-based assay to screen natural extracts for G(2) checkpoint inhibitors and identified debromohymenialdisine (DBH) from a marine sponge. DBH is distinct structurally from previously known G(2) checkpoint inhibitors. It inhibited the G(2) checkpoint with an IC(50) of 8 micrometer and showed moderate cytotoxicity (IC(50) = 25 micrometer) toward MCF-7 cells. DBH inhibited the checkpoint kinases Chk1 (IC(50) = 3 micrometer) and Chk2 (IC(50) = 3.5 micrometer) but not ataxia-telangiectasia mutated (ATM), ATM-Rad3-related protein, or DNA-dependent protein kinase in vitro, indicating that it blocks two major branches of the checkpoint pathway downstream of ATM. It did not cause the activation or inhibition of different signal transduction proteins, as determined by mobility shift analysis in Western blots, suggesting that it inhibits a narrow range of protein kinases in vivo.

Ran alters nuclear pore complex conformation.

Transport across the nuclear membranes occurs through the nuclear pore complex (NPC), and is mediated by soluble transport factors including Ran, a small GTPase that is generally GDP-bound during import and GTP-bound for export. The dynamic nature of the NPC structure suggests a possible active role for it in driving translocation. Here we show that RanGTP but not RanGDP causes alterations of NPC structure when injected into the cytoplasm of Xenopus oocytes, including compaction of the NPC and extension of the cytoplasmic filaments. RanGTP caused accumulation of nucleoplasmin-gold along the length of extended cytoplasmic filaments, whereas RanGDP caused accumulation around the cytoplasmic rim of the NPC. This suggests a possible role for Ran in altering the conformation of the cytoplasmic filaments during transport.

Chromatin-independent nuclear envelope assembly induced by Ran GTPase in Xenopus egg extracts.

The nuclear envelope (NE) forms a controlled boundary between the cytoplasm and the nucleus of eukaryotic cells. To facilitate investigation of mechanisms controlling NE assembly, we developed a cell-free system made from Xenopus laevis eggs to study the process in the absence of chromatin. NEs incorporating nuclear pores were assembled around beads coated with the guanosine triphosphatase Ran, forming pseudo-nuclei that actively imported nuclear proteins. NE assembly required the cycling of guanine nucleotides on Ran and was promoted by RCC1, a nucleotide exchange factor recruited to beads by Ran-guanosine diphosphate (Ran-GDP). Thus, concentration of Ran-GDP followed by generation of Ran-GTP is sufficient to induce NE assembly.

Substrate specificity determinants of the checkpoint protein kinase Chk1.

The Chk1 protein kinase plays a critical role in a DNA damage checkpoint pathway conserved between fission yeast and animals. We have developed a quantitative assay for Chk1 activity, using a peptide derived from a region of Xenopus Cdc25C containing Ser-287, a known target of Chk1. Variants of this peptide were used to determine the residues involved in substrate recognition by Chk1, revealing the phosphorylation motif Phi-X-beta-X-X-(S/T)*, where * indicates the phosphorylated residue, Phi is a hydrophobic residue (M>I>L>V), beta is a basic residue (R>K) and X is any amino acid. This motif suggests that Chk1 is a member of a group of stress-response protein kinases which phosphorylate target proteins with related specificities.

Ran-GTP stabilises microtubule asters and inhibits nuclear assembly in Xenopus egg extracts.

Ran is an abundant GTPase of the Ras superfamily that is highly conserved in eukaryotes. In interphase cells, Ran is mainly nuclear and thought to be predominantly GTP-bound, but it is also present in the cytoplasm, probably GDP-bound. This asymmetric distribution plays an important role in directing nucleocytoplasmic transport. Ran has also been implicated in cell cycle control, including the transition from mitosis to interphase when the compartmentalisation of the nucleus is established. Here, we have examined the role of Ran in this transition using a cell-free system of Xenopus egg extracts supplemented with sperm heads that provides a model for microtubule aster formation and post-M phase nuclear assembly. Ran-GTP, added as wild-type protein, a mutant defective in GTPase activity (Q69L), or generated by addition of the specific nucleotide exchange factor RCC1, stabilises large microtubule asters nucleated at the sperm centrosome, prevents the redistribution of NuMA from the aster to the nucleus and blocks chromatin decondensation. In contrast, Ran GDP does not stabilise microtubules or inhibit nuclear assembly. RanT24N and RanBP1, which oppose the generation of Ran-GTP by RCC1, arrest nuclear growth after disappearance of the aster. Ran associates with microtubule asters in egg extracts and with mitotic spindles in somatic Xenopus cells, suggesting that it may affect microtubule stability directly. These results show that Ran has a novel function in the control of microtubule stability that is clearly distinct from nucleocytoplasmic transport. The Ran GDP/GTP switch may play a role in co-ordinating changes in the structure of microtubules and the assembly of the nucleus associated with the transition from mitosis to interphase.

Bcl-2 regulates amplification of caspase activation by cytochrome c.

Caspases, a family of specific proteases, have central roles in apoptosis [1]. Caspase activation in response to diverse apoptotic stimuli involves the relocalisation of cytochrome c from mitochondria to the cytoplasm where it stimulates the proteolytic processing of caspase precursors. Cytochrome c release is controlled by members of the Bcl-2 family of apoptosis regulators [2] [3]. The anti-apoptotic members Bcl-2 and Bcl-xL may also control caspase activation independently of cytochrome c relocalisation or may inhibit a positive feedback mechanism [4] [5] [6] [7]. Here, we investigate the role of Bcl-2 family proteins in the regulation of caspase activation using a model cell-free system. We found that Bcl-2 and Bcl-xL set a threshold in the amount of cytochrome c required to activate caspases, even in soluble extracts lacking mitochondria. Addition of dATP (which stimulates the procaspase-processing factor Apaf-1 [8] [9]) overcame inhibition of caspase activation by Bcl-2, but did not prevent the control of cytochrome c release from mitochondria by Bcl-2. Cytochrome c release was accelerated by active caspase-3 and this positive feedback was negatively regulated by Bcl-2. These results provide evidence for a mechanism to amplify caspase activation that is suppressed at several distinct steps by Bcl-2, even after cytochrome c is released from mitochondria.

The role of the ran GTPase in nuclear assembly and DNA replication: characterisation of the effects of Ran mutants.

The Ran GTPase plays a critical role in nucleocytoplasmic transport and has been implicated in the maintenance of nuclear structure and cell cycle control. Here, we have investigated its role in nuclear assembly and DNA replication using recombinant wild-type and mutant Ran proteins added to a cell-free system of Xenopus egg extracts. RanQ69L and RanT24N prevent lamina assembly, PCNA accumulation and DNA replication. These effects may be due to the disruption of nucleocytoplasmic transport, since both mutants inhibit nuclear import of a protein carrying a nuclear localisation signal (NLS). RanQ69L, which is deficient in GTPase activity, sequesters importins in stable complexes that are unable to support the docking of NLS-proteins at the nuclear pore complex (NPC). RanT24N, in contrast to wild-type Ran-GDP, interacts only weakly with importin alpha and nucleoporins, and not at all with the import factor p10, consistent with its poor activity in nuclear import. However, RanT24N does interact stably with importin beta, Ran binding protein 1 and RCC1, an exchange factor for Ran. We show that Ran-GDP is essential for proper nuclear assembly and DNA replication, the requirement being primarily before the initiation of DNA replication. Ran-GDP therefore mediates the active transport of necessary factors or otherwise controls the onset of S-phase in this system.

Restriction fragment length polymorphism analysis of Mycobacterium bovis isolates from captive and free-ranging animals.

Mycobacterium bovis isolates from cattle, captive elk, and free-ranging mule deer and coyotes were examined by restriction fragment length polymorphism (RFLP) analysis. DNA extracted from each isolate was digested with restriction endonucleases AluI and PvuII. DNA probes used for Southern hybridizations were a 37-base oligonucleotide and a 123-base-pair sequence specific for the insertion sequence IS6110 and a plasmid, pTBN12, which contains a polymorphic GC-rich repetitive sequence present in several species of mycobacteria. Generally, M. bovis isolates originating from a single herd of either cattle or captive elk had identical RFLP patterns, whereas isolates from unrelated sources had distinct patterns. The RFLP patterns for M. bovis isolates from free-ranging mule deer and coyotes were identical to patterns observed for isolates from a captive elk herd that was located in the area where the free-ranging animals were found. These results indicate that the captive elk herd may have been the source of M. bovis that infected the free-ranging animals. Results of this study show that RFLP analysis is a useful tool for differentiation of M. bovis isolates and for molecular epidemiology studies to determine possible sources of infection in outbreaks of tuberculosis in animals.

Cleavage of rabaptin-5 blocks endosome fusion during apoptosis.

Cells undergoing apoptosis exhibit striking changes in membrane organization, including plasma membrane blebbing and invagination, vacuolation and fragmentation of organelles, and alterations in the surface expression of receptors. The underlying mechanisms for these changes are unknown, though alterations in vesicular fusion are likely to play a role. Using a cell-free system based on Xenopus laevis egg extracts we have found that endosome fusion is blocked during apoptosis. Inhibition of fusion is prevented by Bcl-2 or Bcl-xL, two negative regulators of apoptosis, or by specific inhibitors of members of the caspase family of apoptotic proteases. Selective cleavage of Rabaptin-5, an essential and rate-limiting component of endosome fusion, is responsible for the loss of fusion activity. Cleavage of Rabaptin-5 also occurs in cellular models for apoptosis. These results suggest that inactivation of Rabaptin-5 and inhibition of vesicle transport lead to fragmentation of endosomes and inhibition of the endocytic pathway during the execution phase of apoptosis. We propose that parallel changes to other membrane transport pathways would give rise to general membrane fragmentation in apoptotic cells. These changes are likely to play an important role in the generation of apoptotic bodies and their recognition by phagocytosing cells.

Xenopus Ran-binding protein 1: molecular interactions and effects on nuclear assembly in Xenopus egg extracts.

Ran is a nuclear GTPase implicated in nucleocytoplasmic transport, the maintenance of nuclear structure, mRNA processing, and cell cycle regulation. By two-hybrid interaction in yeast, we have identified a Xenopus homologue of Ran-binding protein 1 (RanBP1). Xenopus RanBP1 interacts specifically with the GTP-bound form of Ran and forms complexes in Xenopus egg extracts with Ran, importin-beta/karyopherin-beta and importin-alpha/karyopherin-alpha, but not p10, p120/RanBP7, RanBP2 or other nucleoporins. These complexes may play roles in the recycling of Ran and importins/karyopherins during nucleocytoplasmic transport. Increased concentrations of RanBP1 stabilise an interaction between Ran and RCC1 in egg extracts, inhibiting the exchange activity of RCC1 towards Ran. Under these conditions, the assembly of nuclei from chromatin is dramatically affected: the nuclei do not assemble a lamina and become very small with homogeneously condensed chromatin. They fail to actively import proteins and do not undergo DNA replication. By field emission in-lens scanning electron microscopy, we show that these nuclei have an intact nuclear envelope containing pore complexes, but the envelope is highly convoluted. However, RanBP1 does not directly inhibit nuclear protein import in assembled nuclei. These results suggest that RCC1 and/or Ran have a function early in nuclear assembly that is disrupted by RanBP1.

Regulation of apoptosis by BH3 domains in a cell-free system.

BACKGROUND: The Bcl-2 family of proteins plays a key role in the regulation of apoptosis. Some family members prevent apoptosis induced by a variety of stimuli, whereas others promote apoptosis. Competitive dimerisation between family members is thought to regulate their function. Homologous domains within individual proteins are necessary for interactions with other family members and for activity, although the specific mechanisms might differ between the pro-apoptotic and anti-apoptotic proteins. RESULTS: Using a cell-free system based on extracts of Xenopus eggs, we have investigated the role of the Bcl-2 homology domain 3 (BH3) from different members of the Bcl-2 family. BH3 domains from the pro-apoptotic proteins Bax and Bak, but not the BH3 domain of the anti-apoptotic protein Bcl-2, induced apoptosis in this system, as determined by the rapid activation of specific apoptotic proteases (caspases) and by DNA fragmentation. The apoptosis-inducing activity of the BH3 domains requires both membrane and cytosolic fractions of cytoplasm, involves the release of cytochrome c from mitochondria and is antagonistic to Bcl-2 function. Short peptides, corresponding to the minimal sequence of BH3 domains required to bind anti-apoptotic Bcl-2 family proteins, also trigger apoptosis in this system. CONCLUSIONS: The BH3 domains of pro-apoptotic proteins are sufficient to trigger cytochrome c release, caspase activation and apoptosis. These results support a model in which pro-apoptotic proteins, such as Bax and Bak, bind to Bcl-2 via their BH3 domains, inactivating the normal ability of Bcl-2 to suppress apoptosis. The ability of synthetic peptides to reproduce the effect of pro-apoptotic BH3 domains suggests that such peptides may provide the basis for engineering reagents to control the initiation of apoptosis.

Cleavage and inactivation of DNA-dependent protein kinase catalytic subunit during apoptosis in Xenopus egg extracts.

DNA-dependent protein kinase (DNA-PK) consists of a 460 kDa subunit that contains the catalytic domain (DNA-PKcs) complexed with two polypeptides of 70 kDa and 80 kDa (Ku70 and Ku80) which comprise the Ku autoantigen. DNA-PKcs requires association with DNA via Ku for catalytic activation and is implicated in double strand break repair, V(D)J recombination and transcription. We have utilised a cell-free system of concentrated Xenopus laevis egg extracts to investigate the regulation and possible functions of DNA-PK. Recently, we have shown that this system can reproduce events of apoptosis, including activation of an apoptotic protease that cleaves poly(ADP-ribose) polymerase. Here, we report that DNA-PK is rapidly inactivated with the onset of apoptosis in this system. Loss of activity is concomitant with cleavage of the catalytic subunit, whereas the Ku subunits are stable. Cleavage and inactivation of DNA-PKcs is prevented by prior addition of the anti-apoptotic protein Bcl-2 or inhibition of an apoptotic protease that has characteristics of the CPP-32/Ced-3 family of cysteine proteases that cleave poly(ADP-ribose) polymerase. These results suggest that cleavage and inactivation of DNA-PKcs prevents this factor from functioning in DNA repair, recombination or transcriptional regulation during apoptosis.

Abortion and placentitis in pregnant bison (Bison bison) induced by the vaccine candidate, Brucella abortus strain RB51.

OBJECTIVE: To determine the ability of Brucella abortus strain RB51 to induce placentitis and abortion in bison after SC vaccination. ANIMALS: 10 pregnant bison cows, 3 to 10 years old and at 3 to 8 months' gestation. PROCEDURE: Pregnant bison cows on a Montana ranch were vaccinated SC with 10(9) colony-forming units of B abortus strain RB51. Two cows, identified prior to the study, were euthanatized and examined 5 weeks after vaccination to obtain optimal histologic samples of placenta. Other cows were euthanatized and examined after abortion. After euthanasia, tissue specimens were collected for histologic and immunohistochemical evaluation. Tissue and fluid specimens for bacteriologic culture were also collected during necropsy. RESULTS: Of 8 cows, 2 aborted at 68 and 107 days after vaccination. Aborting cows had endometritis. Strain RB51 was isolated from reproductive tissues and supramammary lymph nodes. Fetal lesions were not seen; however, fetal bronchial lymph nodes and amniotic fluid contained strain RB51. Cows examined 5 weeks after vaccination had placentitis and endometritis, with numerous bacteria within trophoblastic epithelial cells that were immunoreactive for strain RB51 antigen. Strain RB51 was isolated from placentomes and numerous lymph nodes. Fetal lesions were not seen 5 weeks after vaccination; however, strain RB51 was isolated from numerous lymph nodes and lung, allantoic fluid, and rectal swab specimens. CONCLUSIONS: The vaccine candidate B abortus RB51 has tropism for the bison placenta, and can cause placentitis, which induces abortion in pregnant bison. The vaccine dose used was similar to that being tested in cattle, but may not be appropriate for pregnant bison.

Ran, a GTPase involved in nuclear processes: its regulators and effectors.

Ran is a small GTPase that has been implicated in a variety of nuclear processes, including the maintainance of nuclear structure, protein import, mRNA processing and export, and cell cycle regulation. There has been significant progress in determining the role of Ran in nuclear protein import. However, it has been unclear whether this role is sufficient to account for the diverse effects of disrupting Ran functions. Recently, several proteins have been identified that bind specifically to Ran and are, therefore, possible effectors. Other experiments using dominant mutants of Ran that block its GTP/GDP cycle have suggested that Ran may have multiple roles. Here, these results are summarised and discussed with respect to the action of Ran.

Bcl-2 regulates activation of apoptotic proteases in a cell-free system.

BACKGROUND: Apoptosis plays an important role in the normal development and homeostasis of metazoans. Aberrations in this process have been implicated in several major human diseases, but its molecular mechanism is poorly understood. In animals as diverse as humans and nematodes, the Bcl-2 oncoprotein prevents or delays apoptosis, whereas proteases of the interleukin-1beta-converting enzyme (ICE) family are required, suggesting that they are components of a conserved mechanism controlling the onset of apoptosis. RESULTS: A cell-free system produced from Xenopus laevis eggs reproduces nuclear events characteristic of apoptosis after a lag phase. We have used this system to define the temporal sequence of biochemical events and to examine the relationship between Bcl-2 and apoptotic proteases. Bcl-2 prevents apoptotic chromatin condensation and DNA cleavage, but only when added prior to the activation of a protease which has characteristics similar to the Ced-3 sub-family of ICE-like proteases and which cleaves poly(ADP-ribose) polymerase (PARP). Bcl-2 attenuates activation of this protease, an effect that does not require de novo protein synthesis or the presence of intact nuclei. The Ced-3-related protease CPP-32 is cleaved during the late stages of apoptosis in this system and after PARP cleavage. Generation of CPP-32-cleaving activity is inhibited by Bcl-2. CONCLUSIONS: These experiments provide direct biochemical evidence that Bcl-2 protects against apoptosis, at least in part, by regulating the activation of a series of apoptotic proteases that cleave PARP and other substrates. This cell-free system provides a useful biochemical model for analyzing the molecular mechanism controlling the activation of these proteases.

Regulation of Cdc2/cyclin B activation by Ran, a Ras-related GTPase.

During the cell cycle, a checkpoint prevents the initiation of mitosis until S-phase is completed. The molecular mechanism may involve the RCC1 protein, which catalyses guanine nucleotide exchange on the Ras-related nuclear protein, Ran (or TC4). Genetic studies have suggested that RCC1 may be involved in sensing the replication state of DNA and controlling the activation of Cdc2/cyclin B protein kinase through Ran. In this report, we present direct biochemical evidence for the post-translational control of Cdc2/cyclin B activation by Ran. In a cell-free system of concentrated Xenopus egg extracts supplemented with nuclei, a mutant form of Ran (T24N) analogous to dominant inactive mutants of other Ras-related GTPases inhibits Cdc2/cyclin B activation in the presence of replicating nuclear DNA. This role for Ran is mediated through control of the tyrosine phosphorylation state of Cdc2 and appears to be distinct from other effects on nuclear import, nuclear formation and DNA replication. When extracts were supplemented with RCC1 protein prior to addition of Ran T24N, inhibition of Cdc2/cyclin B by Ran T24N was relieved. This suggests that Ran T24N may act in a dominant manner by sequestering RCC1 in an inactive form. In contrast to Ran T24N, a mutant of Ran (Q69L) defective in GTPase activity and hence locked in the GTP-bound state has no inhibitory effect on Cdc2/cyclin B activation. In the light of these results, we propose that generation of the GTP-bound form of Ran is required for Cdc2/cyclin B activation and entry into mitosis when this process is coupled to the progression of S-phase.

Cyclin-dependent kinases. CAK-handed kinase activation.

A protein kinase that activates cyclin-dependent kinases has been identified as a related catalytic subunit in association with a novel cyclin regulatory subunit--it is itself a cyclin-dependent kinase.