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Purification of protein A-tagged yeast Ran reveals association with a novel karyopherin β family member, Pdr6p

JOURNAL OF BIOLOGICAL CHEMISTRY 2000年第1期275卷 467-471页

作者： Lau, D Künzler, M Braunwarth, A Hellmuth, R Podtelejnikov, A Mann, M Hurt, E Zentrum Biochem Heidelberg D-69120 Heidelberg Germany Odense Univ Ctr Expt Bioinformat DK-5230 Odense Denmark

The small gtpase Ran (encoded by GSP1 and GSP2 in yeast) plays a central role in nucleocytoplasmic transport. GSP1 and GSP2 were tagged with protein A and functionally expressed in a gsp1 null mutant. After affinity purification of protein A-tagged Gsp1p or Gsp2p by IgG-Sepharose chromatography, known karyopherin beta transport receptors (e.g. Kap121p and Kap123p) and a novel member of this protein family, Pdr6p, were found to be associated with yeast Ran. Subsequent tagging of Pdr6p with green fluorescent protein revealed association with the nuclear pore complexes in vivo, Thus, functional tagging of yeast Ran allowed the study of its in vivo distribution and interaction with known and novel Ran-binding proteins.

关键词：细胞区室化绿色荧光蛋白质类发光蛋白质类/遗传学单体gtp结合蛋白质类/遗传学单体gtp结合蛋白质类/分离和提纯单体gtp结合蛋白质类/代谢核被膜/化学核蛋白质类/遗传学核蛋白质类/分离和提纯核蛋白质类/代谢蛋白质结合酿酒酵母蛋白质类 β核胞浆转运蛋白类 rangtp结合蛋白质/遗传学 rangtp结合蛋白质/分离和提纯 rangtp结合蛋白质/代谢

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Search for UV-responsive genes in human cells by differential mRNA display: Involvement of human Ras-related gtp-binding protein, Rheb, in UV susceptibility

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 2000年第3期274卷 859-864页

作者： Kita, K Wu, YP Sugaya, S Moriya, T Nomura, J Takahashi, S Yamamori, H Nakajima, N Suzuki, N Chiba Univ Sch Med Dept Biochem Chuo Ku Chiba 260 Japan Chiba Univ Sch Med Dept Surg Chuo Ku Chiba 260 Japan

The search for genes responsible for the sensitivity of human cells to cell-killing effects of UV is an important area of biological research. To identify candidate genes responsible for UV sensitization, levels of mRNA expression were compared between W-sensitive RSa cells and UV-resistant variant UVr-1 cells, using a differential display method and Northern blot analysis. Messenger RNA expression levels of human Ras homologue enriched in brain (Rheb) and/or a Rheb-like gene were up-regulated and slightly decreased in UV-irradiated RSa and UVr-1 cells, compared to in mock-irradiated cells, respectively. RSa and UVr-1 cells, both of which were treated with antisense oligonucleotides for Rheb RNA, exhibited an increased resistance to UV cell-killing. It remains unclear why UVr-1 cells are resistant to UV yet express Rheb mRNA at high levels, However, the results of antisense experiments together with the up-regulation in W-irradiated RSa cells, suggest that Rheb is involved in the UV sensitization of both cells to UV cell-killing. (C) 2000 Academic Press.

关键词：细胞死亡/遗传学细胞死亡/辐射效应基因表达谱基因表达调控/辐射效应基因文库基因 ras 单体gtp结合蛋白质类/遗传学神经肽类/遗传学 RNA 信使/分析 RNA 信使/遗传学紫外线人类

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Immunocytochemical analyses and targeted gene disruption of gtpBP1

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MOLECULAR AND CELLULAR BIOLOGY 2000年第17期20卷 6195-6200页

作者： Senju, S Iyama, K Kudo, H Aizawa, S Nishimura, Y Kumamoto Univ Grad Sch Med Sci Dept Neurosci & Immunol Div Immunogenet Kumamoto 8600811 Japan Kumamoto Univ Sch Med Dept Surg Pathol Kumamoto 860 Japan Kumamoto Univ Sch Med Dept Morphogenesis Ins Mol Embryol & Genet Kumamoto 860 Japan

We previously identified a gene encoding a putative gtpase, gtpBP1, which is structurally related to elongation factor lot, a key component of protein biosynthesis machinery. The primary structure of gtpBP1 is highly conserved between human and mouse (97% identical at the amino acid level). Expression of this gene is enhanced by gamma interferon in a monocytic cell line, THP-1, Although counterparts of this molecule in Caenorhabditis elegans and Ascaris suum have also been identified, the function of this molecule remains to be clarified. In the present study, our immunohistochemical analyses on mouse tissues revealed that gtpBP1 is expressed in some neurons and smooth muscle cells of various organs as well as macrophages, Immunofluorescence analyses revealed that gtpBP1 is localized exclusively in cytoplasm and shows a diffuse granular network forming a gradient from the nucleus to the periphery of the cells in smooth muscle cell lines and macrophages, To investigate the physiological role of gtpBP1, we used targeted gene disruption in embryonic stem cells to generate gtpBP1-deficient mice. The mutant mice were born at the expected Mendelian frequency, developed normally, and were fertile. No manifest anatomical or behavioral abnormality was observed in the mutant mice. Functions of macrophages, including chemotaxis, phagocytosis, and nitric oxide production, in mutant mice were equivalent to those seen in wild-type mice. No significant difference was observed in the immune response to protein antigen between mutant mice and wild-type mice, suggesting normal function of antigen-presenting cells of the mutant mice. The absence of an eminent phenotype in gtpBP1 deficient mice may he due to functional compensation by gtpBP2, a molecule we recently identified which is similar to gtpBP1 in structure and tissue distribution.

关键词：脑/代谢支气管/代谢 COS细胞细胞黏附细胞培养的大脑皮质/代谢细胞质/代谢 DNA 互补/代谢上皮细胞/代谢荧光抗体技术基因文库免疫印迹法免疫组织化学巨噬细胞腹膜/代谢小鼠转基因模型遗传学单体gtp结合蛋白质类/生物合成单体gtp结合蛋白质类/遗传学单体gtp结合蛋白质类/生理学肌平滑血管/代谢诱变定点吞噬作用表型胸腺/代谢组织分布转染动物女(雌)性男(雄)性小鼠

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The Bub2p spindle checkpoint links nuclear migration with mitotic exit

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MOLECULAR CELL 2000年第1期6卷 1-10页

作者： Pereira, G Höfken, T Grindlay, J Manson, C Schiebel, E Beatson Inst Canc Res CRC Beatson Labs Glasgow G61 1BD Lanark Scotland

Bfa1p and Bub2p are spindle checkpoint proteins that likely have gtpase activation activity and are associated with the budding yeast spindle pole body (SPB). Here, we show that Bfa1p and Bub2p bind the Raslike gtpase Tem1p, a component of the mitotic exit network, to the cytoplasmic face of the SPB that enters the bud, whereas the GDP/gtp exchange factor Lte1p is associated with the cortex of the bud. Migration of the SPB into the bud probably allows activation of Tem1p through Lte1p, thereby linking nuclear migration with mitotic exit. Since components of the Bub2p checkpoint are conserved in other organisms, we propose that the position of the SPB or mammalian centrosome controls the timing of mitotic exit.

关键词：细胞周期细胞周期蛋白质类细胞核/代谢细胞支架蛋白质类真菌蛋白质类/遗传学真菌蛋白质类/代谢鸟嘌呤核苷酸交换因子类显微镜检查免疫电子微管/代谢有丝分裂有丝分裂纺锤体/代谢单体gtp结合蛋白质类/遗传学单体gtp结合蛋白质类/代谢运动(Movement) 酵母菌酿酒/细胞学酵母菌酿酒/遗传学酵母菌酿酒/代谢酿酒酵母蛋白质类姐妹染色单体交换

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The human nm23-H4 gene product is a mitochondrial nucleoside diphosphate kinase

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JOURNAL OF BIOLOGICAL CHEMISTRY 2000年第19期275卷 14264-14272页

作者： Milon, L Meyer, P Chiadmi, M Munier, A Johansson, M Karlsson, A Lascu, I Capeau, J Janin, J Lacombe, ML Univ Paris 06 INSERM U402 F-75012 Paris France Univ Paris Sud Lab Enzymol & Biochim Struct CNRS UPR 9063 F-91198 Gif Sur Yvette France Huddinge Univ Hosp Karolinska Inst Dept Clin Virol F68 S-14186 Huddinge Sweden Univ Bordeaux 2 Inst Biochim & Genet Cellulaires CNRS UMR 5095 F-33077 Bordeaux France

We demonstrate here the catalytic activity and subcellular localization of the Nm23-H4 protein, product of nm23-H4, a new member of the human nm23/nucleoside diphosphate (NDP) kinase gene family (Milon, L., Rousseau-Merck, M., Munier, A., Erent, M., Lascu, I., Capeau, J., and Lacombe, M. L. (1997) Hum. Genet. 99, 550-557). Nm3-H4 was synthesized in escherichia coli as the full-length protein and as a truncated form missing the N-terminal extension characteristic of mitochondrial targeting. The truncated form possesses NDP kinase activity, whereas the full-length protein is inactive, suggesting that the extension prevents enzyme folding and/or activity. X-ray crystallographic analysis was performed on active truncated Nm23-H4. Like other eukaryotic NDP kinases, it is a hexamer. Nm23-H4 naturally possesses a serine residue at position 129, equivalent to the li-pn mutation of the Drosophila NDP kinase. The x-ray structure shows that the presence of Ser(129) has local structural effects that weaken subunit interactions. Site-directed mutagenesis shows that the serine is responsible for the lability of Nm23-H4 to heat and urea treatment, because the S129P mutant is greatly stabilized. Examination of human embryonic kidney 293 cells transfected with green fluorescent protein fusions by confocal microscopy shows a specific mitochondrial localization of Nm23-H4 that was also demonstrated by Western blot analysis of subcellular fractions of these cells. Import into mitochondria is accompanied by cleavage of the N-terminal extension that results in NDP kinase activity. Submitochondrial fractionation indicates that Nm23-H4 is associated with mitochondrial membranes, possibly to the contact sites between the outer and inner membranes.

关键词：碱基序列细胞系转化 DNA引物 DNA 互补显微镜检查共焦线粒体/酶学模型分子分子序列数据单体gtp结合蛋白质类/化学单体gtp结合蛋白质类/遗传学单体gtp结合蛋白质类/分离和提纯 NM23核苷二磷酸激酶类二磷酸核苷激酶D 核苷二磷酸激酶/化学核苷二磷酸激酶/遗传学核苷二磷酸激酶/分离和提纯转录因子/化学转录因子/遗传学转录因子/分离和提纯人类

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Cyclin D1 is required for transformation by activated Neu and is induced through an E2F-dependent signaling pathway

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MOLECULAR AND CELLULAR BIOLOGY 2000年第2期20卷 672-683页

作者： Lee, RJ Albanese, C Fu, MF D'Amico, M Lin, B Watanabe, G Haines, GK Siegel, PM Hung, MC Yarden, Y Horowitz, JM Muller, WJ Pestell, RG Albert Einstein Coll Med Albert Einstein Canc Ctr Dept Med Bronx NY 10461 USA Albert Einstein Coll Med Albert Einstein Canc Ctr Dept Dev & Mol Biol Bronx NY 10461 USA N Carolina State Univ Coll Vet Med Dept Anat Physiol Sci & Radiol Raleigh NC 27606 USA Northwestern Univ Sch Med Dept Pathol Chicago IL 60611 USA McMaster Univ Dept Pathol Hamilton ON L8S 4K1 Canada Univ Texas MD Anderson Canc Ctr Dept Tumor Biol Houston TX 77030 USA Weizmann Inst Sci Dept Bioregulat IL-76100 Rehovot Israel

The neu (c-erbB-2) proto-oncogene encodes a tyrosine kinase receptor that is overexpressed in 20 to 30% of human breast tumors. Herein, cyclin D1 protein levels were increased in mammary tumors induced by overexpression of wild-type Neu or activating mutants of Neu in transgenic mice and in MCF7 cells overexpressing transforming Neu, Analyses of 12 Neu mutants in MCF7 cells indicated important roles for specific C-terminal autophosphorylation sites and the extracellular domain in cyclin D1 promoter activation. Induction of cyclin D1 by NeuT involved Ras, Rac, Rho, extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38, but not phosphatidylinositol 3-kinase, NeuT induction of the cyclin D1 promoter required the E2F and Spl DNA binding sites and was inhibited by dominant negative E2F-1 or DP-1, Neu-induced transformation was inhibited by a cyclin D1 antisense or dominant negative E2F-1 construct in Rat-1 cells. Growth of NeuT-transformed mammary adenocarcinoma cells in nude mice was blocked by the cyclin D1 antisense construct. These results demonstrate that E2F-1 mediates a Neu-signaling cascade to cyclin D1 and identify cyclin D1 as a critical downstream target of neu-induced transformation.

关键词：结合部位载体蛋白质类细胞周期蛋白质类细胞转化肿瘤/病理学细胞周期蛋白D1/拮抗剂和抑制剂细胞周期蛋白D1/遗传学细胞周期蛋白D1/代谢 DNA结合蛋白质类/代谢 E2F转录因子类 E2F1转录因子 JNK丝裂原活化蛋白激酶类 MAP激酶信号系统乳腺动物/代谢乳腺动物/病理学小鼠裸小鼠转基因丝裂原激活蛋白激酶类/拮抗剂和抑制剂丝裂原激活蛋白激酶类/代谢单体gtp结合蛋白质类/遗传学单体gtp结合蛋白质类/代谢突变/遗传学启动区遗传/遗传学 RNA 反义/遗传学 RNA 反义/生理学受体 erbB-2/拮抗剂和抑制剂受体 erbB-2/遗传学受体 erbB-2/代谢视网膜母细胞瘤结合蛋白1 Sp1转录因子/代谢 Sp3转录因子转录因子DP1 转录因子/代谢转录激活肿瘤细胞培养的动物人类小鼠

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Interaction of the Ras-related protein associated with diabetes Rad and the putative tumor metastasis suppressor NM23 provides a novel mechanism of gtpase regulation

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PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 1999年第26期96卷 14911-14918页

作者： Zhu, JH Tseng, YH Kantor, JD Rhodes, CJ Zetter, BR Moyers, JS Kahn, CR Harvard Univ Sch Med Dept Med Joslin Diabet CtrRes Div Boston MA 02215 USA Harvard Univ Childrens Hosp Sch Med Boston MA 02115 USA

Rad is the prototypic member of a new class of Ras-related gtpases, Purification of the gtpase-activating protein (CAP) for Rad revealed nm23, a putative tumor metastasis suppressor and a development gene in Drosophila. Antibodies against nm23 depleted Rad-CAP activity from human skeletal muscle cytosol, and bacterially expressed nm23 reconstituted the activity. The GAP activity of nm23 was specific for Rad, was absent with the S105N putative dominant negative mutant of Rad, and was reduced with mutations of nm23, In the presence of ATP, GDP Rad was also reconverted to CTP Rad by the nucleoside diphosphate (NDP) kinase activity of nm23. Simultaneously, Rad regulated nm23 by enhancing its NDP kinase activity and decreasing its autophosphorylation. Melanoma cells transfected with wild-type Rad, but not the S105N-Rad, showed enhanced DNA synthesis in response to serum;this effect was lost with coexpression of nm23, Thus, the interaction of nm23 and Rad provides a potential novel mechanism for bidirectional, bimolecular regulation in which nm23 stimulates both CTP hydrolysis and gtp loading of Rad whereas Rad regulates activity of nm23. This interaction may play important roles in the effects of Rad on glucose metabolism and the effects of nm23 on tumor metastasis and developmental regulation.

关键词： DNA/生物合成糖尿病/代谢酶激活 gtp磷酸水解酶类/代谢基因肿瘤抑制葡萄糖/代谢鸟苷三磷酸/代谢即早蛋白质类/代谢模型生物学单体gtp结合蛋白质类/遗传学单体gtp结合蛋白质类/代谢 NM23核苷二磷酸激酶类肿瘤转移/遗传学核苷二磷酸激酶/遗传学核苷二磷酸激酶/代谢蛋白质结合大鼠 Sprague-Dawley 转录因子/遗传学转录因子/代谢 ras蛋白质类/代谢动物人类大鼠

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Saccharomyces cerevisiae BUB2 prevents mitotic exit in response to both spindle and kinetochore damage

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GENETICS 2000年第2期156卷 489-500页

作者： Krishnan, R Pangilinan, F Lee, C Spencer, F Johns Hopkins Univ Sch Med Inst Med Genet Baltimore MD 21205 USA

The spindle assembly checkpoint-mediated mitotic arrest depends on proteins that signal the presence of one or more unattached kinetochores and prevents the onset of anaphase in the presence of kinetochore or spindle damage. In the presence of either damage, bub2 cells initiate a preanaphase delay but do not maintain it. Inappropriate sister chromatid separation in nocodazole-treated bub2 cells is prevented when mitotic exit is blocked using a conditional tem1(c) mutant, indicating that the preanaphase failure in bub2 cells is a consequence of events downstream of TEM1 in the mitotic exit pathway. Using a conditional bub2(tsd) mutant, we demonstrate that the continuous presence of Bub2 protein is required for maintaining spindle damage-induced arrest. BUB2 is not required to maintain a DNA damage checkpoint arrest, revealing a specificity for spindle assembly checkpoint function. In a yeast two-hybrid assay and in vitro, Bub2 protein interacts with the septin protein Cdc3, which is essential for cytokinesis. These data support the view that the spindle assembly checkpoint encompasses regulation of distinct mitotic steps, including a MAD2-directed block to anaphase initiation and a BUB2-directed block to TEM1-dependent exit.

关键词：细胞周期/遗传学细胞周期蛋白质类 DNA损伤真菌蛋白质类/遗传学真菌蛋白质类/代谢基因型染色粒/超微结构有丝分裂/遗传学有丝分裂纺锤体/遗传学有丝分裂纺锤体/超微结构单体gtp结合蛋白质类/遗传学单体gtp结合蛋白质类/代谢诺考达唑/药理学酵母菌酿酒/细胞学酵母菌酿酒/药物作用酵母菌酿酒/遗传学酿酒酵母蛋白质类

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nm23:: Unraveling its biological function in cell differentiation

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JOURNAL OF CELLULAR PHYSIOLOGY 2000年第2期182卷 144-149页

作者： Lombardi, D Lacombe, ML Paggi, MG Ctr Ric Spermentale Lab Metab Cellulare & Farmacocinet Ist Regina Elena I-00158 Rome Italy Univ Aquila Dipartimento Med Sperimentale I-67100 Laquila Italy Univ Paris 06 IGM INSERM U402 Paris France

Tumor suppressor genes have a pivotal role in normal cells regulating cell cycle processes negatively. Furthermore, the inhibition of cell proliferation is a crucial step in the achievement of cell differentiation. Increasing evidence suggests that the nm23 genes, initially documented as suppressors of the invasive phenotype in some cancer types, are involved in the control of normal development and differentiation. In this review, we summarize some data concerning the involvement of the nm23 genes in development and differentiation, attempting to delineate an overall view of many facets of their biological role. (C) 2000 Wiley-Liss, Inc.

关键词：衰老/代谢细胞分化/生理学造血干细胞/细胞学单体gtp结合蛋白质类/遗传学单体gtp结合蛋白质类/生理学多基因族核苷二磷酸激酶序列同源性转录因子/遗传学转录因子/生理学动物人类

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Mutational analysis of the NM23.H1 gene in human breast cancer

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CANCER GENETICS AND CYTOGENETICS 2000年第2期121卷 181-185页

作者： Cipollini, G Moretti, A Ghimenti, C Viacava, P Bevilacqua, G Caligo, MA Univ Pisa Dept Oncol Div Pathol Mol Genet SectLab Mol Genet I-56126 Pisa Italy

NM23.H1 is a protein connected with tumor progression. Loss of heterozygosity and reduced expression of the gene have been associated with poor prognosis and increased incidence of metastases in many epithelial tumors. The aim of this study was to detect the presence of NM23.H1 point mutations or small deletions in hum an breast carcinomas by using the single-strand-conformation polymorphism (SSCP) technique. Mutational analysis was performed on 76 breast tumors, 10 of which had allelic deletion of the gene. The NM23.H1 mRNA content also was evaluated in each sample. Only a C-to-A transversion leading to a stop codon was found ill the 5' untranslated region of exon 1. A polymorphic SSCP pattern was identified in exon 1;direct sequencing showed a C-to-T transition 30 nucleotides upstream from the 5' splice site flanking exon 1. None of the tumors analyzed presented both alleles inactivated. Our results suggest that NM23.H1 is rarely inactivated by point mutations. (C) 2000 Elsevier Science inc. All rights reserved.

关键词：乳腺肿瘤/遗传学乳腺肿瘤/病理学 DNA突变分析杂合子丢失单体gtp结合蛋白质类/遗传学 NM23核苷二磷酸激酶类核苷二磷酸激酶多态现象单链构象 RNA 信使/遗传学转录因子/遗传学人类中年人

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