The Experts below are selected from a list of 2298 Experts worldwide ranked by ideXlab platform
Takeharu Nishimoto - One of the best experts on this subject based on the ideXlab platform.
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caffeine mimics adenine and 2 deoxyadenosine both of which inhibit the guanine nucleotide exchange activity of RCC1 and the kinase activity of atr
Genes to Cells, 2003Co-Authors: Hitoshi Nishijima, Hideo Nishitani, Noriko Saito, Takeharu NishimotoAbstract:BACKGROUND: Both caffeine and the inactivation of RCC1, the guanine-nucleotide exchange factor (GEF) of Ran, induce premature chromatin condensation (PCC) in hamster BHK21 cells arrested in the S-phase, suggesting that RCC1 is a target for caffeine. RESULTS: Caffeine inhibited the Ran-GEF activity of RCC1 by preventing the binary complex formation of Ran-RCC1. Inhibition of the Ran-GEF activity of RCC1 by caffeine and its derivatives was correlated with their ability to induce PCC. Since caffeine is a derivative of xanthine, the bases and nucleosides were screened for their ability to inhibit RCC1. Adenine, adenosine, and all of the 2'-deoxynucleosides inhibited the Ran-GEF activity of RCC1; however, only adenine and 2'-deoxyadenosine (2'-dA) induced PCC. A factor(s) other than RCC1, should therefore be involved in PCC-induction. We found that both adenine and 2'-dA, but none of the other 2'-deoxynucleosides, inhibited the kinase activity of ATR, similar to that of caffeine. The ATR pathway was also abrogated by the inactivation of RCC1 in tsBN2 cells. CONCLUSION: The effect of caffeine on cell-cycle control mimics the biological effect of adenine and 2'-dA, both of which inhibit ATR. dATP, a final metabolite of adenine and 2'-dA, is suggested to inhibit ATR, resulting in PCC.
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premature chromatin condensation caused by loss of RCC1
Progress in cell cycle research, 2000Co-Authors: Hitoshi Nishijima, Takashi Seki, Hideo Nishitani, Takeharu NishimotoAbstract:Hamster rccl mutant, tsBN2, prematurely enter mitosis during S phase. RCC1 is a guanine nucleotide exchanging factor for a small G protein Ran and localised on the chromatin, whereas RanGTPase activating protein is in the cytoplasm. Consistently, Ran shuttles between the nucleus and the cytoplasm, carrying out nucleus-cytosol exchange of macromolecules, which regulates the cell cycle. The finding that loss of RCC1 which disturbs nuclear protein export due to loss of RanGTP, abrogates the check point control suggests that RCC1 senses the status of the chromatin, such as replication, and couples it to the cell cycle progression through Ran.
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model of the ran RCC1 interaction using biochemical and docking experiments
Journal of Molecular Biology, 1999Co-Authors: Yoshiaki Azuma, Takeharu Nishimoto, Louis Renault, Juan Antonio Garciaranea, Alfonso Valencia, Alfred WittinghoferAbstract:Abstract RCC1, the regulator of chromosome condensation, is the guanine nucleotide exchange factor (GEF) for the nuclear Ras-like GTP-binding protein Ran. Its structure was solved by X-ray crystallography and revealed a seven-bladed β-propeller, one side of which was proposed to be the interaction site with Ran. To gain more insight into this interaction, alanine mutagenesis studies were performed on conserved residues on the surface of the structure. Purified mutant proteins were analysed by steady-state kinetic analysis of their GEF activities towards Ran. A number of residues were identified whose mutation affected either the K M or k cat of the overall reaction, or had no effect. Mutants were further analysed by plasmon surface resonance in order to get more information on individual steps of the complex reaction pathway. Ran-GDP was coupled to the sensor chip and reacted with RCC1 mutants to categorise them into different groups, demonstrating the usefulness of plasmon surface resonance in the study of complex multi-step kinetic processes. A docking solution of Ran-RCC1 structures in combination with sequence analysis allows prediction of the site of interaction between RCC1 and Ran and proposes a model for the Ran-RCC1 structure which corresponds to and extends the biochemical data. Three invariant residues which most severely affect the k cat of the reaction, D128, D182 and H304, are located in the centre of the Ran-RCC1 interface and interfere with switch II and the phosphate binding area. The structural model suggests that different guanine nucleotide exchange factors use a similar interaction site on their respective GTP-binding proteins, but that the molecular mechanisms for the release of nucleotides are likely to be different.
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inhibition by anti RCC1 monoclonal antibodies of RCC1 stimulated guanine nucleotide exchange on ran gtpase
Journal of Biochemistry, 1997Co-Authors: Yoshiaki Azuma, Takahisa Hachiya, Takeharu NishimotoAbstract:: Nine monoclonal antibodies to RCC1, the guanine nucleotide exchange factor on Ran GTPase, were obtained using recombinant RCC1 as the antigen. Epitopes of three monoclonal antibodies, which did not inhibit RCC1 function, were localized in the N-terminus outside the RCC1 repeat, while epitopes of the other 6 monoclonal antibodies were localized within the RCC1 repeat. Three of the latter 6 monoclonal antibodies, 2B6, 6C3, and 8D9, inhibited RCC1-stimulated nucleotide release. Two of them, 2B6 and 6C3, recognized the same amino acid residues in the N-terminus of the second RCC1 repeat, Tyr89, Ser90, Phe91, and Gly92, of which one, Gly92, is conserved in Saccharomyces cerevisiae and mutated in an RCC1(-) strain, mtr1-2. The monoclonal antibody 8D9 recognized two amino acid residues, Arg320 and Ala321, downstream of Gly319 in the N-terminus of the 6th RCC1 repeat, which corresponds to Gly92 in the second RCC1 repeat. The monoclonal antibodies which inhibited RCC1 function bound to RCC1 in homogeneous solution and stained cellular RCC1. We propose that the N-terminus of the RCC1 repeat is exposed at the surface of RCC1 on the coated plate or in fixed cells, and is involved in the RCC1-stimulated nucleotide exchange on the Ran GTPase.
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genetic interaction of ded1 encoding a putative atp dependent rna helicase with srm1 encoding a mammalian RCC1 homolog in saccharomyces cerevisiae
Molecular Genetics and Genomics, 1996Co-Authors: Naoyuki Hayashi, Hiroaki Seino, Kenji Irie, M Watanabe, K L Clark, Kunihiro Matsumoto, Takeharu NishimotoAbstract:The Saccharomyces cerevisiae temperature-sensitive mutants srm1-1, mtr1-2 and prp20-1 carry alleles of a gene encoding a homolog of mammalian RCC1. In order to identify a protein interacting with RCC1, a series of suppressors of the srm1-1 mutation were isolated as cold-sensitive mutants and one of the mutants, designated ded1-21, was found to be defective in the DED1 gene. The double mutant, srm1-1 ded1-21, could grow at 35° C, but not at 37° C. A revertant of srm1-1 ded1-21 that became able to grow at 37° C acquired another mutation in the SRM1 gene, indicating the tight relationship between SRM1 and DED1. In all the RCC1- strains examined, the amount of mutated SRM1 proteins was reduced or not detectable at the nonpermissive temperature. While mutated SRM1 protein was stabilized in all of the RCC1- strains by the ded1-21 mutation, the ded1-21 mutation suppressed both srm1-1 and mtr1-2, but not the prp20-1 mutation, contrary to the previous finding that overproduction of the S. cerevisiae Ran homolog GSP1 suppresses prp20-1, but not srm1-1 or mtr1-2.
Mary Dasso - One of the best experts on this subject based on the ideXlab platform.
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ranbp1 controls the ran pathway in mammalian cells through regulation of mitotic RCC1 dynamics
Cell Cycle, 2020Co-Authors: Alexei Arnaoutov, Vasilisa Aksenova, Ross Kaufhold, Shane Chen, Mary DassoAbstract:The Ran GTPase plays critical roles in multiple cellular processes including interphase nucleocytoplasmic transport and mitotic spindle assembly. During mitosis in mammalian cells, GTP-bound Ran (Ran-GTP) is concentrated near mitotic chromatin while GDP-bound Ran (Ran-GDP) is more abundant distal to chromosomes. This pattern spatially controls spindle formation because Ran-GTP locally releases spindle assembly factors (SAFs), such as Hepatoma Up-Regulated Protein (HURP), from inhibitory interactions near chromosomes. Regulator of Chromatin Condensation 1 (RCC1) is Ran's chromatin-bound exchange factor, and RanBP1 is a conserved Ran-GTP-binding protein that has been implicated as a mitotic regulator of RCC1 in embryonic systems. Here, we show that RanBP1 controls mitotic RCC1 dynamics in human somatic tissue culture cells. In addition, we observed the re-localization of HURP in metaphase cells after RanBP1 degradation, consistent with the idea that altered RCC1 dynamics functionally modulate SAF activities. Together, our findings reveal an important mitotic role for RanBP1 in human somatic cells, controlling the spatial distribution and magnitude of mitotic Ran-GTP production and thereby ensuring the accurate execution of Ran-dependent mitotic events. ABBREVIATIONS AID: Auxin-induced degron; FLIP: Fluorescence loss in photobleaching; FRAP: Fluorescence recovery after photobleaching; GDP: guanosine diphosphate; GTP: guanosine triphosphate; HURP: Hepatoma Up-Regulated Protein; NE: nuclear envelope; NEBD: Nuclear Envelope Breakdown; RanBP1: Ran-binding protein 1; RanGAP1: Ran GTPase-Activating Protein 1; RCC1: Regulator of Chromatin Condensation 1; RRR complex: RCC1/Ran/RanBP1 heterotrimeric complex; SAF: Spindle Assembly Factor; TIR1: Transport Inhibitor Response 1 protein; XEE: Xenopus egg extract.
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RCC1 regulates inner centromeric composition in a ran independent fashion
Cell Cycle, 2018Co-Authors: Michael Shaofei Zhang, Maiko Furuta, Alexei Arnaoutov, Mary DassoAbstract:ABSTRACTRCC1 associates to chromatin dynamically within mitosis and catalyzes Ran-GTP production. Exogenous RCC1 disrupts kinetochore structure in Xenopus egg extracts (XEEs), but the molecular basis of this disruption remains unknown. We have investigated this question, utilizing replicated chromosomes that possess paired sister kinetochores. We find that exogenous RCC1 evicts a specific subset of inner KT proteins including Shugoshin-1 (Sgo1) and the chromosome passenger complex (CPC). We generated RCC1 mutants that separate its enzymatic activity and chromatin binding. Strikingly, Sgo1 and CPC eviction depended only on RCC1's chromatin affinity but not its capacity to produce Ran-GTP. RCC1 similarly released Sgo1 and CPC from synthetic kinetochores assembled on CENP-A nucleosome arrays. Together, our findings indicate RCC1 regulates kinetochores at the metaphase-anaphase transition through Ran-GTP-independent displacement of Sgo1 and CPC.
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the balance of ranbp1 and RCC1 is critical for nuclear assembly and nuclear transport
Molecular Biology of the Cell, 1997Co-Authors: Robert T Pu, Mary DassoAbstract:Ran is a small GTPase that is essential for nuclear transport, mRNA processing, maintenance of structural integrity of nuclei, and cell cycle control. RanBP1 is a highly conserved Ran guanine nucleotide dissociation inhibitor. We sought to use Xenopus egg extracts for the development of an in vitro assay for RanBP1 activity in nuclear assembly, protein import, and DNA replication. Surprisingly, when we used anti-RanBP1 antibodies to immunodeplete RanBP1 from Xenopus egg extracts, we found that the extracts were also depleted of RCC1, Ran’s guanine nucleotide exchange factor, suggesting that these proteins form a stable complex. In contrast to previous observations using extracts that had been depleted of RCC1 only, extracts lacking both RanBP1 and RCC1 (codepleted extracts) did not exhibit defects in assays of nuclear assembly, nuclear transport, or DNA replication. Addition of either recombinant RanBP1 or RCC1 to codepleted extracts to restore only one of the depleted proteins caused abnormal nuclear assembly and inhibited nuclear transport and DNA replication in a manner that could be rescued by further addition of RCC1 or RanBP1, respectively. Exogenous mutant Ran proteins could partially rescue nuclear function in extracts without RanBP1 or without RCC1, in a manner that was correlated with their nucleotide binding state. These results suggest that little RanBP1 or RCC1 is required for nuclear assembly, nuclear import, or DNA replication in the absence of the other protein. The results further suggest that the balance of GTP- and GDP-Ran is critical for proper nuclear assembly and function in vitro.
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the RCC1 protein interacts with ran ranbp1 hsc70 and a 340 kda protein in xenopus extracts
Journal of Biological Chemistry, 1995Co-Authors: Hisato Saitoh, Mary DassoAbstract:Abstract RCC1 is an abundant, highly conserved, chromatin-associated protein whose function is necessary for the preservation of a properly ordered cell cycle. RCC1 is also necessary for numerous nuclear processes, including nuclear transport and RNA metabolism; and it functions enzymatically as a guanine nucleotide exchange factor for a small, ras-related GTPase called Ran. Studies in several organisms suggest that RCC1 may be part of a large complex containing multiple proteins. There is also evidence that RCC1 associates with chromatin through other proteins and that the binding of the complex to chromatin varies within the cell cycle. In order to characterize this putative complex, we have identified a number of other proteins as candidate components of the complex by their association with a GST-RCC1 fusion protein. Three of these proteins have previously been identified (Ran, RanBP1, and hsc70). The fourth protein is novel and has a molecular mass of 340 kDa. In this report, we discuss a preliminary characterization of the interactions between these proteins.
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a mutant form of the ran tc4 protein disrupts nuclear function in xenopus laevis egg extracts by inhibiting the RCC1 protein a regulator of chromosome condensation
The EMBO Journal, 1994Co-Authors: Mary Dasso, Yoshiaki Azuma, T Seki, T Ohba, T NishimotoAbstract:Abstract The Ran protein is a small GTPase that has been implicated in a large number of nuclear processes including transport. RNA processing and cell cycle checkpoint control. A similar spectrum of nuclear activities has been shown to require RCC1, the guanine nucleotide exchange factor (GEF) for Ran. We have used the Xenopus laevis egg extract system and in vitro assays of purified proteins to examine how Ran or RCC1 could be involved in these numerous processes. In these studies, we employed mutant Ran proteins to perturb nuclear assembly and function. The addition of a bacterially expressed mutant form of Ran (T24N-Ran), which was predicted to be primarily in the GDP-bound state, profoundly disrupted nuclear assembly and DNA replication in extracts. We further examined the molecular mechanism by which T24N-Ran disrupts normal nuclear activity and found that T24N-Ran binds tightly to the RCC1 protein within the extract, resulting in its inactivation as a GEF. The capacity of T24N-Ran-blocked interphase extracts to assemble nuclei from de-membranated sperm chromatin and to replicate their DNA could be restored by supplementing the extract with excess RCC1 and thereby providing excess GEF activity. Conversely, nuclear assembly and DNA replication were both rescued in extracts lacking RCC1 by the addition of high levels of wild-type GTP-bound Ran protein, indicating that RCC1 does not have an essential function beyond its role as a GEF in interphase Xenopus extracts.
Yoshiaki Azuma - One of the best experts on this subject based on the ideXlab platform.
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model of the ran RCC1 interaction using biochemical and docking experiments
Journal of Molecular Biology, 1999Co-Authors: Yoshiaki Azuma, Takeharu Nishimoto, Louis Renault, Juan Antonio Garciaranea, Alfonso Valencia, Alfred WittinghoferAbstract:Abstract RCC1, the regulator of chromosome condensation, is the guanine nucleotide exchange factor (GEF) for the nuclear Ras-like GTP-binding protein Ran. Its structure was solved by X-ray crystallography and revealed a seven-bladed β-propeller, one side of which was proposed to be the interaction site with Ran. To gain more insight into this interaction, alanine mutagenesis studies were performed on conserved residues on the surface of the structure. Purified mutant proteins were analysed by steady-state kinetic analysis of their GEF activities towards Ran. A number of residues were identified whose mutation affected either the K M or k cat of the overall reaction, or had no effect. Mutants were further analysed by plasmon surface resonance in order to get more information on individual steps of the complex reaction pathway. Ran-GDP was coupled to the sensor chip and reacted with RCC1 mutants to categorise them into different groups, demonstrating the usefulness of plasmon surface resonance in the study of complex multi-step kinetic processes. A docking solution of Ran-RCC1 structures in combination with sequence analysis allows prediction of the site of interaction between RCC1 and Ran and proposes a model for the Ran-RCC1 structure which corresponds to and extends the biochemical data. Three invariant residues which most severely affect the k cat of the reaction, D128, D182 and H304, are located in the centre of the Ran-RCC1 interface and interfere with switch II and the phosphate binding area. The structural model suggests that different guanine nucleotide exchange factors use a similar interaction site on their respective GTP-binding proteins, but that the molecular mechanisms for the release of nucleotides are likely to be different.
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inhibition by anti RCC1 monoclonal antibodies of RCC1 stimulated guanine nucleotide exchange on ran gtpase
Journal of Biochemistry, 1997Co-Authors: Yoshiaki Azuma, Takahisa Hachiya, Takeharu NishimotoAbstract:: Nine monoclonal antibodies to RCC1, the guanine nucleotide exchange factor on Ran GTPase, were obtained using recombinant RCC1 as the antigen. Epitopes of three monoclonal antibodies, which did not inhibit RCC1 function, were localized in the N-terminus outside the RCC1 repeat, while epitopes of the other 6 monoclonal antibodies were localized within the RCC1 repeat. Three of the latter 6 monoclonal antibodies, 2B6, 6C3, and 8D9, inhibited RCC1-stimulated nucleotide release. Two of them, 2B6 and 6C3, recognized the same amino acid residues in the N-terminus of the second RCC1 repeat, Tyr89, Ser90, Phe91, and Gly92, of which one, Gly92, is conserved in Saccharomyces cerevisiae and mutated in an RCC1(-) strain, mtr1-2. The monoclonal antibody 8D9 recognized two amino acid residues, Arg320 and Ala321, downstream of Gly319 in the N-terminus of the 6th RCC1 repeat, which corresponds to Gly92 in the second RCC1 repeat. The monoclonal antibodies which inhibited RCC1 function bound to RCC1 in homogeneous solution and stained cellular RCC1. We propose that the N-terminus of the RCC1 repeat is exposed at the surface of RCC1 on the coated plate or in fixed cells, and is involved in the RCC1-stimulated nucleotide exchange on the Ran GTPase.
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dis3 implicated in mitotic control binds directly to ran and enhances the gef activity of RCC1
The EMBO Journal, 1996Co-Authors: Eishi Noguchi, Yoshiaki Azuma, Takashi Seki, Naoyuki Hayashi, Masafumi Nakamura, Nobutaka Nakashima, Mitsuhiro Yanagida, Xiangwei He, U Mueller, S SazerAbstract:Using the two-hybrid method, we isolated a Saccharomyces cerevisiae cDNA encoding a protein homologous to Schizosaccharomyces pombe protein Dis3sp, using as bait, human GTPase Ran. The DIS3 gene is essential for viability and complements S.pombe mutant dis3-54 which is defective in mitosis. Although Dis3sc has no homology to RanBP1, it bound directly to Ran and the S.cerevisiae Ran homologue Cnr1, but not to the S.cerevisiae RCC1 homologue Srm1. Upon binding to Ran with a 1:1 molar ratio, Dis3sc enhanced a nucleotide-releasing activity of RCC1 on Ran. In the presence of Dis3sc, the K(m) of RCC1 on Ran decreased by half, while the kcat was unchanged. In vivo, Dis3sp was present as oligomers of M(r) 670-200 kDa as previously reported, and the 200 kDa oligomer of Dis3sp was found to include Spi1 and Pim1, the S.pombe homologues of Ran and RCC1, respectively. Although the biological function of the heterotrimeric oligomer consisting of Dis3, Spi1 and Pim1 is unknown, our results indicate that Dis3 is a component of the RCC1-Ran pathway.
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conserved histidine residues of RCC1 are essential for nucleotide exchange on ran
Journal of Biochemistry, 1996Co-Authors: Yoshiaki Azuma, Hiroaki Seino, Satoru Uzawa, Takashi Seki, Christian Klebe, Tomoyuki Ohba, Alfred Wittinghofer, Naoyuki Hayashi, Takeharu NishimotoAbstract:: Charged amino acid residues of human RCC1 were converted to alanine and mutants which were unable to complement tsBN2 cells (a temperature-sensitive RCC1- mutant of the hamster BHK21 cell line) were selected. These RCC1 mutants were analyzed for the ability to inhibit premature chromatin condensation by microinjection into tsBN2 cells, and their steady-state kinetic parameters for guanine nucleotide exchange reaction were measured. Examined RCC1 mutants were unstable in tsBN2 cells at the restrictive temperature, yet they significantly inhibited premature chromatin condensation. Mutants located on the N-terminus of the RCC1 repeat showed an increased K(m), while their kcat values were comparable to that of wild-type RCC1. In contrast, mutants containing the conserved histidine residues in the C-terminus of the RCC1 repeat showed a value of K(m) similar to that of wild-type RCC1, while the kcat values of these mutants were reduced, depending upon the RCC1 repeats on which the mutation was located. These steady-state kinetic parameters of mutants indicate that the N-terminus and the C-terminus of RCC1 repeats play different roles in guanine nucleotide exchange on Ran. The comparison of kcat among the histidine mutants suggests that those histidine residues which are conserved in the RCC1 repeats and also through evolution comprise the catalytic site for the guanine nucleotide exchange reaction.
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premature chromatin condensation induced by loss of RCC1 is inhibited by gtp and gtpγs ran but not gdp ran
Journal of Biological Chemistry, 1996Co-Authors: Tomoyuki Ohba, Takashi Seki, Yoshiaki Azuma, Takeharu NishimotoAbstract:Abstract RCC1 is a guanine nucleotide exchanging factor acting on nuclear G protein Ran. Premature chromatin condensation occurs in the temperature-sensitive RCC1− mutant of the BHK21 cell line, tsBN2, at the restrictive temperature. This observation can be explained if the premature activation of MPF is normally inhibited by GTP-Ran. In the absence of RCC1, GDP-Ran predominates, resulting in MPF activation. However, experiments with Ran mutants to determine whether GTP- or GDP-Ran prevents activation of MPF have yielded conflicting results. In order to clarify this point, we have microinjected nucleotide-bound Ran, instead of mutated Ran, into the nuclei of tsBN2 cells treated to reduce RCC1-mediated guanine nucleotide exchange. GTP-Ran, GTPγS-Ran, and GDP-Ran all inhibited chromatin condensation. However, the inhibition of chromatin condensation by GDP-Ran could be completely abolished by co-injection with GDP, but not GTP. Thus, we conclude that GTP-Ran blocks the activation of MPF and that hydrolysis of GTP is not required to prevent MPF activation.
T Nishimoto - One of the best experts on this subject based on the ideXlab platform.
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a mutant form of the ran tc4 protein disrupts nuclear function in xenopus laevis egg extracts by inhibiting the RCC1 protein a regulator of chromosome condensation
The EMBO Journal, 1994Co-Authors: Mary Dasso, Yoshiaki Azuma, T Seki, T Ohba, T NishimotoAbstract:Abstract The Ran protein is a small GTPase that has been implicated in a large number of nuclear processes including transport. RNA processing and cell cycle checkpoint control. A similar spectrum of nuclear activities has been shown to require RCC1, the guanine nucleotide exchange factor (GEF) for Ran. We have used the Xenopus laevis egg extract system and in vitro assays of purified proteins to examine how Ran or RCC1 could be involved in these numerous processes. In these studies, we employed mutant Ran proteins to perturb nuclear assembly and function. The addition of a bacterially expressed mutant form of Ran (T24N-Ran), which was predicted to be primarily in the GDP-bound state, profoundly disrupted nuclear assembly and DNA replication in extracts. We further examined the molecular mechanism by which T24N-Ran disrupts normal nuclear activity and found that T24N-Ran binds tightly to the RCC1 protein within the extract, resulting in its inactivation as a GEF. The capacity of T24N-Ran-blocked interphase extracts to assemble nuclei from de-membranated sperm chromatin and to replicate their DNA could be restored by supplementing the extract with excess RCC1 and thereby providing excess GEF activity. Conversely, nuclear assembly and DNA replication were both rescued in extracts lacking RCC1 by the addition of high levels of wild-type GTP-bound Ran protein, indicating that RCC1 does not have an essential function beyond its role as a GEF in interphase Xenopus extracts.
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A mutant form of the Ran/TC4 protein disrupts nuclear function in Xenopus laevis egg extracts by inhibiting the RCC1 protein, a regulator of chromosome condensation.
The EMBO Journal, 1994Co-Authors: Mary Dasso, Yoshiaki Azuma, T Seki, T Ohba, T NishimotoAbstract:Abstract The Ran protein is a small GTPase that has been implicated in a large number of nuclear processes including transport. RNA processing and cell cycle checkpoint control. A similar spectrum of nuclear activities has been shown to require RCC1, the guanine nucleotide exchange factor (GEF) for Ran. We have used the Xenopus laevis egg extract system and in vitro assays of purified proteins to examine how Ran or RCC1 could be involved in these numerous processes. In these studies, we employed mutant Ran proteins to perturb nuclear assembly and function. The addition of a bacterially expressed mutant form of Ran (T24N-Ran), which was predicted to be primarily in the GDP-bound state, profoundly disrupted nuclear assembly and DNA replication in extracts. We further examined the molecular mechanism by which T24N-Ran disrupts normal nuclear activity and found that T24N-Ran binds tightly to the RCC1 protein within the extract, resulting in its inactivation as a GEF. The capacity of T24N-Ran-blocked interphase extracts to assemble nuclei from de-membranated sperm chromatin and to replicate their DNA could be restored by supplementing the extract with excess RCC1 and thereby providing excess GEF activity. Conversely, nuclear assembly and DNA replication were both rescued in extracts lacking RCC1 by the addition of high levels of wild-type GTP-bound Ran protein, indicating that RCC1 does not have an essential function beyond its role as a GEF in interphase Xenopus extracts.
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loss of RCC1 leads to suppression of nuclear protein import in living cells
Journal of Biological Chemistry, 1994Co-Authors: Taro Tachibana, T Nishimoto, Naoko Imamoto, H Seino, Yoshihiro YonedaAbstract:Abstract The role of RCC1-Ran/TC4 in nuclear protein import was examined in living cells using a temperature-sensitive RCC1 mutant cell line, tsBN2, and tsBN2 transformed with a RCC1 cDNA lacking the nuclear localization sequence domain, delta 8-29. Substrate, containing a small number of SV40 T antigen nuclear localization sequence peptides, injected into the cytoplasm of tsBN2 cells cultured at the non-permissive temperature of 39.5 degrees C did not accumulate efficiently in the nucleus. When the same substrate was injected into the cytoplasm of heterokaryons of tsBN2 and wild type BHK21 cells, import efficiency into the tsBN2 nuclei was not restored. Import into the BHK21 nuclei gradually decreased after fusion. In contrast, import efficiency into tsBN2 nuclei gradually recovered after fusion with tsBN2 cells transformed with delta 8-29 in which functional RCC1 was diffusely distributed in both the nuclei and cytoplasm. Substrate did not accumulate in the nuclei of digitonin-permeabilized tsBN2 cells cultured at 39.5 degrees C even in the presence of normal cytosol. These results suggest that loss of RCC1 function leads to the decline of import competence of the nucleus and accumulation of a factor in the cytoplasm that suppresses nuclear import. These results indicate that the RCC1-Ran/TC4 system may regulate nuclear import.
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RCC1 a regulator of mitosis is essential for dna replication
Molecular and Cellular Biology, 1992Co-Authors: Mary Dasso, H Nishitani, Sally Kornbluth, T Nishimoto, John W NewportAbstract:Abstract Temperature-sensitive mutants in the RCC1 gene of BHK cells fail to maintain a correct temporal order of the cell cycle and will prematurely condense their chromosomes and enter mitosis at the restrictive temperature without having completed S phase. We have used Xenopus egg extracts to investigate the role that RCC1 plays in interphase nuclear functions and how this role might contribute to the known phenotype of temperature-sensitive RCC1 mutants. By immunodepleting RCC1 protein from egg extracts, we find that it is required for neither chromatin decondensation nor nuclear formation but that it is absolutely required for the replication of added sperm chromatin DNA. Our results further suggest that RCC1 does not participate enzymatically in replication but may be part of a structural complex which is required for the formation or maintenance of the replication machinery. By disrupting the replication complex, the loss of RCC1 might lead directly to disruption of the regulatory system which prevents the initiation of mitosis before the completion of DNA replication.
Paul R Clarke - One of the best experts on this subject based on the ideXlab platform.
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the methylated n terminal tail of RCC1 is required for stabilisation of its interaction with chromatin by ran in live cells
BMC Cell Biology, 2010Co-Authors: Ekarat Hitakomate, Fiona E Hood, Helen S Sanderson, Paul R ClarkeAbstract:Regulator of chromosome condensation 1 (RCC1) is the guanine nucleotide exchange factor for Ran GTPase. Localised generation of Ran-GTP by RCC1 on chromatin is critical for nucleocytoplasmic transport, mitotic spindle assembly and nuclear envelope formation. Both the N-terminal tail of RCC1 and its association with Ran are important for its interaction with chromatin in cells. In vitro, the association of Ran with RCC1 induces a conformational change in the N-terminal tail that promotes its interaction with DNA. We have investigated the mechanism of the dynamic interaction of the α isoform of human RCC1 (RCC1α) with chromatin in live cells using fluorescence recovery after photobleaching (FRAP) of green fluorescent protein (GFP) fusions. We show that the N-terminal tail stabilises the interaction of RCC1α with chromatin and this function can be partially replaced by another lysine-rich nuclear localisation signal. Removal of the tail prevents the interaction of RCC1α with chromatin from being stabilised by RanT24N, a mutant that binds stably to RCC1α. The interaction of RCC1α with chromatin is destabilised by mutation of lysine 4 (K4Q), which abolishes α-N-terminal methylation, and this interaction is no longer stabilised by RanT24N. However, α-N-terminal methylation of RCC1α is not regulated by the binding of RanT24N. Conversely, the association of Ran with precipitated RCC1α does not require the N-terminal tail of RCC1α or its methylation. The mobility of RCC1α on chromatin is increased by mutation of aspartate 182 (D182A), which inhibits guanine-nucleotide exchange activity, but RCC1αD182A can still bind nucleotide-free Ran and its interaction with chromatin is stabilised by RanT24N. These results show that the stabilisation of the dynamic interaction of RCC1α with chromatin by Ran in live cells requires the N-terminal tail of RCC1α. α-N-methylation is not regulated by formation of the binary complex with Ran, but it promotes chromatin binding through the tail. This work supports a model in which the association of RCC1α with chromatin is promoted by a conformational change in the α-N-terminal methylated tail that is induced allosterically in the binary complex with Ran.
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RCC1 isoforms differ in their affinity for chromatin molecular interactions and regulation by phosphorylation
Journal of Cell Science, 2007Co-Authors: Fiona E Hood, Paul R ClarkeAbstract:RCC1 is the guanine nucleotide exchange factor for Ran GTPase. Generation of Ran-GTP by RCC1 on chromatin provides a spatial signal that directs nucleocytoplasmic transport, mitotic spindle assembly and nuclear envelope formation. We show that RCC1 is expressed in human cells as at least three isoforms, named RCC1α, RCC1β and RCC1γ, which are expressed at different levels in specific tissues. The β and γ isoforms contain short inserts in their N-terminal regions (NTRs) that are not present in RCC1α. This region mediates interaction with chromatin, binds importin α3 and/or importin β, and contains regulatory phosphorylation sites. RCC1γ is predominantly localised to the nucleus and mitotic chromosomes like RCC1α. However, compared to RCC1α, RCC1γ has a greatly reduced interaction with an importin α3-β and a stronger interaction with chromatin that is mediated by the extended NTR. RCC1γ is also the isoform that is most highly phosphorylated at serine 11 in mitosis. Unlike RCC1α, RCC1γ supports cell proliferation in tsBN2 cells more efficiently when serine 11 is mutated to non-phosphorylatable alanine. Phosphorylation of RCC1γ therefore specifically controls its function during mitosis. These results show that human RCC1 isoforms have distinct chromatin binding properties, different molecular interactions, and are selectively regulated by phosphorylation, as determined by their different NTRs.
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RCC1 isoforms differ in their affinity for chromatin, molecular interactions and regulation by phosphorylation.
Journal of cell science, 2007Co-Authors: Fiona E Hood, Paul R ClarkeAbstract:RCC1 is the guanine nucleotide exchange factor for Ran GTPase. Generation of Ran-GTP by RCC1 on chromatin provides a spatial signal that directs nucleocytoplasmic transport, mitotic spindle assembly and nuclear envelope formation. We show that RCC1 is expressed in human cells as at least three isoforms, named RCC1alpha, RCC1beta and RCC1gamma, which are expressed at different levels in specific tissues. The beta and gamma isoforms contain short inserts in their N-terminal regions (NTRs) that are not present in RCC1alpha. This region mediates interaction with chromatin, binds importin alpha3 and/or importin beta, and contains regulatory phosphorylation sites. RCC1gamma is predominantly localised to the nucleus and mitotic chromosomes like RCC1alpha. However, compared to RCC1alpha, RCC1gamma has a greatly reduced interaction with an importin alpha3-beta and a stronger interaction with chromatin that is mediated by the extended NTR. RCC1gamma is also the isoform that is most highly phosphorylated at serine 11 in mitosis. Unlike RCC1alpha, RCC1gamma supports cell proliferation in tsBN2 cells more efficiently when serine 11 is mutated to non-phosphorylatable alanine. Phosphorylation of RCC1gamma therefore specifically controls its function during mitosis. These results show that human RCC1 isoforms have distinct chromatin binding properties, different molecular interactions, and are selectively regulated by phosphorylation, as determined by their different NTRs.
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anchoring RCC1 by the tail
Nature Cell Biology, 2007Co-Authors: Paul R ClarkeAbstract:Generation of GTP-bound Ran on chromatin by its guanine nucleotide-exchange factor RCC1 provides a spatial signal that controls nuclear-envelope formation, nucleo-cytoplasmic transport and mitotic spindle assembly. A study now identifies an unusual post-translational modification, α-N-methylation of the amino-terminal tail of RCC1, which anchors the protein on chromosomes.
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phosphorylation regulates the dynamic interaction of RCC1 with chromosomes during mitosis
Current Biology, 2004Co-Authors: James R A Hutchins, Fiona E Hood, William J Moore, Jamie S J Wilson, Paul D Andrews, Jason R Swedlow, Paul R ClarkeAbstract:Abstract The small GTPase Ran has multiple roles during the cell division cycle, including nuclear transport, mitotic spindle assembly, and nuclear envelope formation [1, 2]. However, regulation of Ran during cell division is poorly understood. Ran-GTP is generated by the guanine nucleotide exchange factor RCC1, the localization of which to chromosomes is necessary for the fidelity of mitosis in human cells [3]. Using photobleaching techniques, we show that the chromosomal interaction of human RCC1 fused to green fluorescent protein (GFP) changes during progression through mitosis by being highly dynamic during metaphase and more stable toward the end of mitosis. The interaction of RCC1 with chromosomes involves the interface of RCC1 with Ran and requires an N-terminal region containing a nuclear localization signal. We show that this region contains sites phosphorylated by mitotic protein kinases. One site, serine 11, is targeted by CDK1/cyclin B and is phosphorylated in mitotic human cells. Phosphorylation of the N-terminal region of RCC1 inhibits its binding to importin α/β and maintains the mobility of RCC1 during metaphase. This mechanism may be important for the localized generation of Ran-GTP on chromatin after nuclear envelope breakdown and may play a role in the coordination of progression through mitosis.
