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Results for “Monomeric GTP-Binding Proteins”

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At least 19 recordsLinked to original sources
PubMed1992

flhF, a Bacillus subtilis flagellar gene that encodes a putative GTP-binding protein.

We describe the sequence and characterization of the Bacillus subtilis flhF gene. flhF encodes a basic polypeptide of 41 kDa that contains a putative GTP-binding motif. The sequence of FlhF reveals a structural relationship to two Escherichia coli proteins, Ffh and FtsY, as well as to other members of the SRP54 family, in a domain presumed to bind GTP. flhF is located in a large operon consisting of chemotaxis and flagellar genes. Cells deficient in flhF are nonmotile. Through the use of anti-flagellar antibodies we have established that flhF is a flagellar (fla) gene. Thus, flhF is a unique flagellar gene in that it encodes a GTP-binding protein with similarities to members of the SRP54 family of proteins. These data suggest that flagellar biosynthesis in B. subtilis requires GTP.

Amino Acid Sequence↗
PubMed2000

The mouse neurological mutant flailer expresses a novel hybrid gene derived by exon shuffling between Gnb5 and Myo5a.

Exon shuffling is thought to be an important mechanism for evolution of new genes. Here we show that the mouse neurological mutation flailer (flr) expresses a novel gene that combines the promoter and first two exons of guanine nucleotide binding protein beta 5 (Gnb5) with the C-terminal exons of the closely linked Myosin 5A (MyoVA) gene (Myo5a). The flailer protein, which is expressed predominantly in brain, contains the N-terminal 83 amino acids of Gnb5 fused in-frame with the C-terminal 711 amino acids of MyoVA, including the globular tail domain that binds organelles for intracellular transport. Biochemical and genetic studies indicate that the flailer protein competes with wild-type MyoVA in vivo, preventing the localization of smooth endoplasmic reticulum vesicles in the dendritic spines of cerebellar Purkinje cells. The flailer protein thus has a dominant-negative mechanism of action with a recessive mode of inheritance due to the dependence of competitive binding on the ratio between mutant and wild-type proteins. The chromosomal arrangement of Myo5a upstream of Gnb5 is consistent with non-homologous recombination as the mutational mechanism. To our knowledge, flailer is the first example of a mammalian mutation caused by germ line exon shuffling between unrelated genes.

Amino Acid Sequence↗
PubMed1992

Fission yeast and a plant have functional homologues of the Sar1 and Sec12 proteins involved in ER to Golgi traffic in budding yeast.

Sec12p and Sar1p are required for the formation of transport vesicles generated from the endoplasmic reticulum (ER) in the yeast Saccharomyces cerevisiae. Sec12p is an ER type II membrane protein that mediates the membrane attachment of the GTP-binding Sar1 protein. The SAR1 gene is a multi-copy suppressor of a thermosensitive sec12 mutation. In an attempt to identify functional homologues of Sec12p and Sar1p from other eukaryotic organisms, we screened cDNA expression libraries derived from the fission yeast Schizosaccharomyces pombe and from the plant Arabidopsis thaliana for complementation of the sec12ts mutation. Four individual cDNAs were isolated, two of which encode the S. pombe and A. thaliana homologues of Sar1p. The three Sar1 proteins are 67% identical on average. The two other cDNAs encode type II membrane proteins which were designated Stl1p for the S. pombe protein and Stl2p for the A. thaliana protein (Stl stands for Sec12p-like). Both proteins have NH2-terminal cytoplasmic domains which resemble that of Sec12p: they are similar in size and present a significant degree of amino acid identity with the cytoplasmic domain of Sec12p. In contrast, the lumenal domains of Sec12p, Stl1p and Stl2p are very different in size and do not show any appreciable homology. That Stl1p and Stl2p are functional homologues of Sec12p was confirmed by showing that expression of either cloned gene complements a sec12 null mutation. Our results indicate that some of the mechanisms regulating vesicle formation at the ER are conserved not only in yeasts, but also in plants.

Amino Acid Sequence↗
PubMed1992

Molecular cloning and functional expression of the second mouse nm23/NDP kinase gene, nm23-M2.

A new murine cDNA of nm23/NDP kinase was isolated. A RT-PCR product was obtained from the normal mouse liver mRNA with primers designed for the human nm23-H2 gene. The product was used as a probe to screen a cDNA library from the murine melanoma cell line, B16, and two clones containing the entire open reading frame were obtained. It was predicted that the DNA sequence encoded 152 amino acids which was 98% identical to the nm23-H2 protein. The entire nm23-M1 and -M2 gene-coding regions were translated as fusion proteins with a glutathione S-transferase. These fusion proteins displayed NDP kinase activities.

Amino Acid Sequence↗
PubMed1992

The NM23 gene maps to human chromosome band 17q22 and shows a restriction fragment length polymorphism with BglII.

The NM23-Hl gene is a putative tumor suppressor gene that may be important in the metastasic process. Recent genetic and immunological data indicate that the NM23-Hl gene encodes a protein with nucleoside diphosphate (NDP) kinase activity. The mapping of NM23-Hl by panels of rodent-human somatic cell hybrids and in situ hybridization showed that the gene is located in human chromosome band 17q22. A two-allele polymorphism with BglII was demonstrated.

Alleles↗
PubMed1992

X-ray structure of nucleoside diphosphate kinase.

The X-ray structure of a point mutant of nucleoside diphosphate kinase (NDP kinase) from Dictyostelium discoideum has been determined to 2.2 A resolution. The enzyme is a hexamer made of identical subunits with a novel mononucleotide binding fold. Each subunit contains an alpha/beta domain with a four stranded, antiparallel beta-sheet. The topology is different from adenylate kinase, but identical to the allosteric domain of Escherichia coli ATCase regulatory subunits, which bind mononucleotides at an equivalent position. Dimer contacts between NDP kinase subunits within the hexamer are similar to those in ATCase. Trimer contacts involve a large loop of polypeptide chain that bears the site of the Pro----Ser substitution in Killer of prune (K-pn) mutants of the highly homologous Drosophila enzyme. Properties of Drosophila NDP kinase, the product of the awd developmental gene, and of the human enzyme, the product of the nm23 genes in tumorigenesis, are discussed in view of the three-dimensional structure and of possible interactions of NDP kinase with other nucleotide binding proteins.

Amino Acid Sequence↗
PubMed2024

Functional overlap between the mammalian Sar1a and Sar1b paralogs in vivo.

Proteins carrying a signal peptide and/or a transmembrane domain enter the intracellular secretory pathway at the endoplasmic reticulum (ER) and are transported to the Golgi apparatus via COPII vesicles or tubules. SAR1 initiates COPII coat assembly by recruiting other coat proteins to the ER membrane. Mammalian genomes encode two SAR1 paralogs, SAR1A and SAR1B. While these paralogs exhibit ~90% amino acid sequence identity, it is unknown whether they perform distinct or overlapping functions in vivo. We now report that genetic inactivation of Sar1a in mice results in lethality during midembryogenesis. We also confirm previous reports that complete deficiency of murine Sar1b results in perinatal lethality. In contrast, we demonstrate that deletion of Sar1b restricted to hepatocytes is compatible with survival, though resulting in hypocholesterolemia that can be rescued by adenovirus-mediated overexpression of either SAR1A or SAR1B. To further examine the in vivo function of these two paralogs, we genetically engineered mice with the Sar1a coding sequence replacing that of Sar1b at the endogenous Sar1b locus. Mice homozygous for this allele survive to adulthood and are phenotypically normal, demonstrating complete or near-complete overlap in function between the two SAR1 protein paralogs in mice. These data also suggest upregulation of SAR1A gene expression as a potential approach for the treatment of SAR1B deficiency (chylomicron retention disease) in humans.

Animals↗
PubMed1992

Nucleoside diphosphate kinase/NM23 expression in breast cancer: lack of correlation with lymph-node metastasis.

The product of the nm23-H1 gene, reported to be a metastatic suppressor gene, was recently identified as the nucleoside diphosphate (NDP) kinase A, and was found to be overexpressed in several types of malignant tumors as compared with normal tissues. In order to determine whether NDP-kinase expression serves as a marker for metastatic potential and whether hyperproliferation of neoplastic cells would correlate with expression, we analyzed NDP-kinase levels and activity by immunohistochemical staining and by an enzymatic assay in 13 benign and 98 malignant breast-tissue specimens. Our results confirm that NDP-kinase expression increases in malignant cells of breast carcinomas, but cannot be considered as a biological marker of metastatic dissemination. No correlation was found between NDP-kinase activity and S phase, taken as an index of cell proliferation. Moreover, no correlation was observed between NDP-kinase activity and tumor size, histoprognostic index, estrogen receptors or progesterone receptors. The mechanism of over-expression of NDP in malignant cells and its role in tumor progression remain to be determined.

Breast Neoplasms↗
PubMed1992

Immunohistochemical analysis of nm23 gene product/NDP kinase expression in pulmonary adenocarcinoma: lack of prognostic value.

Levels of nm23 gene product/nucleoside diphosphate kinase (NDP kinase) expression have been demonstrated to correlate inversely with metastatic potential in several tumours, indicating that this could be a useful tool as a prognostic indicator. Using an antibody to NDP kinase, levels of nm23 gene product/NDP kinase expression in pulmonary adenocarcinoma were examined immunohistochemically. Of 88 patients tested, 39 (44%; Group B) showed strong immunoreactivity for NDP kinase in most of cancer cells within the tumour tissues, while 49 (56%; Group A) contained few or no NDP kinase-positive cancer cells. Nm23 gene product/NDP kinase was expressed independently of clinicopathological factors, and unexpectedly, no correlation of survival rates between both Groups could be demonstrated. Thus, in pulmonary adenocarcinoma, levels of nm23 gene product/NDP kinase expression may lack prognostic value.

Adenocarcinoma↗
PubMed1992

Cancer cell invasion and metastasis.

The most life-threatening aspect of cancer is the undetected spread of tumor cells throughout the body. Improved understanding of how these cells invade tissues is leading to new treatments.

Basement Membrane↗
PubMed1992

Expression in human hepatocellular carcinoma of nucleoside diphosphate kinase, a homologue of the nm23 gene product.

BACKGROUND: Expression of nucleoside diphosphate (NDP) kinase, which is highly homologous to the nm23 gene product in a variety of species, has been found to be inversely associated with metastatic potential in human breast cancer. PURPOSE: The present study was conducted to clarify the association of NDP kinase expression with metastatic potential in human hepatocellular carcinoma. METHODS: The immunohistochemical expression of NDP kinase was analyzed in 30 patients with histopathologically proven hepatocellular carcinoma. These patients included nine with distant metastases and 21 without distant metastases. Tissue specimens were reacted with rabbit anti-rat NDP kinase antibody and stained by the biotin-streptavidin complex method. The relative staining intensities were evaluated by comparing primary tumor sites with adjacent nontumorous liver tissue or with metastatic sites, RESULTS: The expression of NDP kinase in primary sites in patients with distant metastases was significantly less intense than that in patients without distant metastases (P = .018). NDP kinase was expressed significantly less intensely in metastatic sites than in primary sites (P = .005). The intensity of NDP kinase expression did not statistically correlate with tumor size or number of lesions in the liver, histopathological classification of tumor, associated liver diseases, hepatitis virus markers, or tumor markers. CONCLUSION: These results suggest that the reduced expression of NDP kinase is closely associated with distant metastatic potential in hepatocellular carcinoma. IMPLICATIONS: It is possible that both NDP kinase and the nm23 gene product may be active in the progression and differentiation of tumor cells and that their reduced expression induces a high metastatic potential in tumor cells. Studies using Northern blotting or in situ hybridization should be planned to confirm our findings.

Adult↗
PubMed1992

Modulators of intracellular Ca2+ and the calmodulin inhibitor W-7 alter the expression of metastasis-associated genes MTS1 and NM23 in metastatic variants of the B16 murine melanoma.

MTS1 is a metastasis-associated gene highly expressed in highly metastatic tumours. NM23 has been described as a putative metastasis suppressor gene. Here we show that thapsigargin (which raises intracellular calcium [Ca2+]i from intracellular stores) and verapamil (which blocks Ca2+ influx) both down-regulate MTS1 and NM23 gene expression in the poorly metastatic F1 and highly metastatic ML8 variants of the B16 murine melanoma without altering their metastatic behaviour. The data presented here suggest that Ca2+ released from intracellular stores could be functionally differentiated from influxed Ca2+ and could be activating different components of the Ca2+ signalling system. Many of the cellular responses to calcium are mediated through calmodulin. We have therefore further investigated the role of Ca2+ in the regulation of the MTS1 and NM23 genes using the calmodulin inhibitor W-7. Both these genes were down-regulated after treatment of the F1 and ML8 cell variants. We have shown previously that retinoic acid reduces lung colonization by the highly metastatic variant ML8 and that melanocyte stimulating hormone (MSH) enhances lung colonization by the poorly metastatic variant F1, with corresponding changes in the relative expression of NM23 and MTS1. Here we have found that verapamil and thapsigargin have no effect on lung colonization, possibly due to both genes being down-regulated. These data support the concept that NM23 and MTS1 gene expression is linked and that metastatic potential may be determined by their relative expression.

Animals↗
PubMed1992

Expression level of the nm23 gene in clonal populations of metastatic murine and human neoplasms.

The purpose of this study was to determine whether nm23 steady-state mRNA expression levels correlate with metastatic potential of mouse K-1735 melanoma cells, human KM12 colon cancer cells, and human SN12 renal cancer cells. Since neoplasms are heterogeneous and contain subpopulations of cells with different metastatic potentials, we analyzed multiple sets of nonmetastatic and metastatic clones isolated from each neoplasm. In addition, we also examined nine somatic cell hybrids produced by the fusion of nonmetastatic and metastatic K-1735 clones. In the mouse melanoma, we found heterogeneity in nm23-1 steady-state expression levels among the clones and hybrids that did not correlate with their metastatic phenotype. Clones isolated from human colon or renal carcinomas expressed similar levels of nm23-HI regardless of metastatic potential in nude mice. All of the human tumor cells were heterozygous for the nm23-HI-specific allelic DNA fragments, with no allelic deletions or gross alterations detected. Since the failure of tumor cells to produce metastasis can be due to multiple deficiencies, these data stress the importance of using independent clones with different metastatic potentials for the analysis of gene regulation of this process.

Alleles↗
PubMed1992

Bombesin receptors interact with Gi and p21ras proteins in plasma membranes from rat pancreatic acinar cells.

This study investigates interaction of bombesin receptors with heterotrimeric guanosine triphosphate (GTP)-binding proteins (G proteins) and monomeric small molecular weight GTP-binding proteins (smg proteins), respectively, in plasma membranes (PM) of rat pancreatic acinar cells. Addition of bombesin to isolated PM stimulated the incorporation of the photoaffinity analogue [alpha-32P]GTP-gamma-azidoanilide into Gi proteins of 40-41 kDa and reduced the pertussis toxin-induced ADP ribosylation of three 40-41 kDa proteins, which had been previously identified as Gi1, Gi2, and Gi3 (30). In PM isolated from bombesin-prestimulated acinar cells, binding of [alpha-32P]-GTP to PM proteins of 21-22 kDa and of a monoclonal antibody against p21ras proteins was increased. Two-dimensional separation of PM proteins revealed the presence of 18 or 19 differently charged smg proteins. The p21ras proteins could be separated into two differently charged proteins with isoelectric points of 5.58 and 5.79. In microsomal membranes (MM), [alpha-32P]GTP binding to yet unidentified 21-22 kDa smg proteins was decreased compared with membranes from unstimulated acinar cells. The data suggest that Gi proteins as well as p21ras proteins are involved in bombesin receptor-mediated signal transduction in the PM. Furthermore, 21-22 kDa smg proteins in MM might play a role in bombesin-induced stimulation of intracellular pathways that lead to enzyme secretion from pancreatic acinar cells.

Affinity Labels↗
PubMed1992

Purification and characterization of a low M(r) GTP-binding protein, ram p25, expressed by baculovirus expression system.

The ram gene was isolated from rat megakaryocyte cDNA library with an oligonucleotide probe which is specific for a low M(r) GTP-binding proteins c25KG purified from human platelets. Its gene product (ram p25) is a monomeric 25-kDa guanine nucleotide-binding protein. The protein was expressed by using baculovirus transfer vector, pAcYM1, which allowed the production at a high level of soluble recombinant ram p25 in Spodoptera frugiperda (Sf9) cells under the control of polyhedrin promoter. The expressed protein in cytosol of Sf9 cells was purified to near homogeneity by a combination of DEAE-Toyopearl 650(S) and hydroxyapatite HCA-100S column chromatography. The purified ram p25 bound approx. 0.8 +/- 0.02 mol of guanosine 5'-O-1-thiotriphosphate (GTP gamma S)/mol of protein with a Kd value of 340 +/- 4.91 nM in a reaction mixture containing 10 microM of free magnesium ions. In the presence of 5 mM Mg2+, [3H]GDP was dissociated from ram p25 at the rate of 0.015 +/- 0.0010 min-1 and the dissociation was greatly enhanced by addition of 250 mM (NH4)2SO4. The rate of [gamma-32P]GTP-hydrolysis for ram p25 was 0.010 +/- 0.0012 min-1. Thus, it was indicated that the GTP-hydrolysis reaction is a rate-limiting step in the guanine nucleotide turnover of ram p25. ram p25 shares 23 and 80% amino-acid homology with the Ha-ras p21 and c25KG protein, respectively, and is similar to them in GTP gamma S binding activity in a time- and dose-dependent manner. But it differs from ras p21 in the rate-limiting step of the guanine nucleotide turnover.

Baculoviridae↗
PubMed1992

Beta-adrenergic-receptor-mediated dissociation and membrane release of the Gs protein in S49 lymphoma-cell membranes. Dependence on Mg2+ and GTP.

We reported [Ransnäs, Svoboda, Jasper & Insel (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 7900-7903] that in intact S49 lymphoma cells the beta-adrenergic-receptor agonist isoprenaline dissociates the stimulatory guanine-nucleotide-binding protein, Gs, into its alpha s and beta gamma subunits, leading to redistribution of alpha s from plasma membranes to the cytoplasm. In the present studies we investigated the kinetics of Gs dissociation and membrane release in plasma membranes from S49 lymphoma cells. We analysed cholate extracts of membranes for alpha s levels by a competitive e.l.i.s.a. with a polyclonal antibody that selectively recognizes monomeric alpha s and we assayed supernatant fractions using both competitive e.l.i.s.a. and immunoblotting. The plasma membranes contained 19.3 +/- 1.4 pmol of alpha s/mg of membrane protein and lacked significant dissociation of Gs and activity of adenylate cyclase in the absence of guanine nucleotides. Mg2+ ions were obligatorily required for isoprenaline-induced dissociation of Gs in plasma membranes and for membrane release of alpha s. At a physiological concentration of free Mg2+ ions (100 microM), 100 microM-GTP induced a slow first-order (k = 0.038 +/- 0.004 min-1) dissociation of 17.8 +/- 1.2 pmol of Gs/mg of membrane protein. A substantial increase in the dissociation rate of Gs was achieved by addition of 1 microM-isoprenaline and 100 microM-GTP; 18.4 +/- 0.9 pmol of Gs/mg of membrane protein was dissociated, with a kappa of 1.49 +/- 0.22 min-1. The effect of isoprenaline on the dissociation rate and on membrane release of Gs was completely blocked by the beta-adrenergic receptor antagonist propranolol. The concentration-response relationship for isoprenaline-induced dissociation during the first 1 min after addition of hormone yielded a kappa act. of 16 +/- 5 nM, whereas the kappa act. for isoprenaline-induced membrane release was 10 nM. We conclude that release of alpha s from plasma membranes is likely to accompany Gs-subunit dissociation and constitutes a potentially important facet of Gs action.

Animals↗