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A Tavitian

Publications and source records attributed to A Tavitian.

At least 37 records · Page 2Linked to original sources
PubMed1994

Characterization of the Ras-related RAP2A protein expressed in the baculovirus-insect cell system: processing of the protein in insect cells and comparison with the bacterially produced unprocessed form.

The Ras-related protein Rap2A is a 21kD GTP-binding protein that exhibits 46% identity with Ras proteins and is similarly post-translationally modified by farnesyl and palmitate groups. Using a recombinant baculovirus, we expressed Rap2A in Sf9 insect cells. The protein is initially synthesized as a soluble hydrophilic precursor, that is post-translationally processed to a hydrophobic membrane-bound form (Rap2Am) that contains both isoprenoid and palmitate groups. The processed form of the protein was purified from the membranes of infected Sf9 cells, and its biochemical properties were compared with those of the unprocessed form produced in recombinant bacteria (Rap2Ab). Both proteins exhibited similar kinetics of GDP dissociation and GTP binding and displayed a weak intrinsic GTPase activity that was stimulated to the same extent by a factor present in bovine brain cytosol. We conclude that Rap2A is correctly processed in insect cells and that maturation does not alter its biochemical properties.

Animals↗
PubMed1994

A small GTP-binding protein is frequently overexpressed in peripheral blood mononuclear cells from patients with solid tumours.

ras oncoproteins and ras-related proteins constitute a large family of the small GTP-binding protein family. The rab branch of the ras superfamily is involved in the intracellular transport along the secretory and endocytic pathway in eukaryotic cells. We here demonstrate that a member of the rab branch, the rab2 protein, is frequently overexpressed in peripheral blood mononuclear cells from patients with solid neoplasms. Moreover, this expression is shown to be greatly modified during the course of therapy. Our results provide strong evidence for the implication of a small GTP-binding protein in immunological events associated with neoplastic diseases. The precise cellular population involved as well as the potential prognostic value of this process remains to be determined.

Antineoplastic Agents↗
PubMed1994

A small rab GTPase is distributed in cytoplasmic vesicles in non polarized cells but colocalizes with the tight junction marker ZO-1 in polarized epithelial cells.

Small rab/Ypt1/Sec4 GTPase family have been involved in the regulation of membrane traffic along the biosynthetic and endocytic pathways in eucaryotic cells. Polarized epithelial cells have morphologically and functionally distinct apical and basolateral surfaces separated by tight junctions. The establishment and maintenance of these structures require delivery of membrane proteins and lipids to these domains. In this work, we have isolated a cDNA clone from a human intestinal cDNA library encoding a small GTPase, rab13, closely related to the yeast Sec4 protein. Confocal microscopy analysis on polarized Caco-2 cells shows that rab13 protein colocalized with the tight junction marker ZO-1. Cryostat sections of tissues confirm that rab13 localized to the junctional complex region of a variety of epithelia, including intestine, kidney, liver, and of endothelial cells. This localization requires assembly and integrity of the tight junctions. Disruption of tight junctions by incubation in low Ca2+ media induces the redistribution of rab13. In cells devoid of tight junctions, rab13 was found associated with vesicles dispersed throughout the cytoplasm. Cell-cell contacts initiated by E-cadherin in transfected L cells do not recruit rab13 to the resulting adherens-like junction complexes. The participation of rab13 in polarized transport, in the assembly and/or the activity of tight junctions is discussed.

Amino Acid Sequence↗
PubMed1994

Cellular traffic and markers of subcellular compartments.

Cellular membranes play very important roles. They not only constitute envelopes that enclose the cytoplasm and the organelles, but they also are dynamic structures that are in a constant state of turnover, with their own substance being continuously shuffled, redistributed and renewed. These processes of intracellular. membranes, in various compartments, relate essentially, to cell traffic. Since the early work of George Palade's and Christian de Duve's groups, in which the relationship between transporting vesicles and distinct separate subcellular compartments was shown, many new facts and concepts were brought about by the use of refined techniques of immunocytochemistry, the set up of cell free systems for the reconstitution of intracellular transport and the study of secretion mutants in yeast. In more recent years, the field was considerably extended by the emergence of the G-proteins. Now, we begin to comprehend that members of various gene families of GTP-GDP binding proteins play important parts in vesicle coating, transport and targetting in the endocytic and exocytic secretory pathways: trimeric G-proteins, dynamins, ARF (ADP-ribosylating factors) and RAS-related proteins. Among all these, members of the Rab subfamily, which appear to exert identical functions in different locations, might be considered as dynamic and specific markers of subcellular compartments.

Animals↗
PubMed1994

Inhibition of Friend cells proliferation by spi-1 antisense oligodeoxynucleotides.

The spi-1 proto-oncogene encodes the transcription factor PU.1 which is normally expressed in all hematopoietic cell lineages except in T cell lines. During the murine acute erythroleukemia induced by the Friend retrovirus, SFFV, spi-1 deregulation by insertional mutagenesis results in the overexpression of Spi-1/PU.1 in the malignant proerythroblastic cell. To assess the Spi-1 role in the proliferation and the differentiation arrest of the Friend tumor cells we inhibited spi-1 gene expression in two Friend cell lines by using antisense oligodeoxyribonucleotides. Proliferation and cloning efficiency of both cell lines were significantly inhibited by spi1 antisense. This antiproliferative effect was not related to an apparent maturation of erythroleukemic cells demonstrating that repression of spi-1 expression is not sufficient per se to restore the ability of the proerythroblastic cells to spontaneously differentiate in mature erythroblasts. These data suggest that the spi-1 gene would be involved in the Friend leukemic process by promoting the proerythroblast to proliferate.

Base Sequence↗
PubMed1993

Isoprenylation of Rab proteins possessing a C-terminal CaaX motif.

Rab proteins are small GTPases highly related to the yeast Ypt1 and Sec4 proteins involved in secretion. The Rab proteins were found associated with membranes of different compartments along the secretory and endocytic pathways. They share distinct C-terminal cysteine motifs required for membrane association. Unlike the other Rab proteins, Rab8, Rab11 and Rab13 terminate with a C-terminal CaaX motif similar to those of Ras/Rho proteins. This report demonstrates that Rab8 and Rab13 proteins are isoprenylated in vivo and geranylgeranylated in vitro. Rab11 associates in vitro geranylgeranylpyrophosphate and farnesylpyrophosphate. Our study shows that the CaaX motif is required for isoprenylation.

Amino Acid Sequence↗
PubMed1993

Insulin and okadaic acid induce Rab4 redistribution in adipocytes.

Insulin stimulation of glucose transport involves the translocation of vesicles containing the glucose transporter Glut 4 to the plasma membrane. Rab proteins, which have been implicated in the regulation of vesicular traffic, were studied in adipocytes. Rab3B, Rab3C, Rab4, and Rab8 were detected, but Rab3A was not. In the absence of insulin, Rab3B and Rab3C were cytosolic, while Rab4 and Rab8 were associated with membranes. Only Rab4 distribution was modified by insulin. In unstimulated adipocytes, most of Rab4 was found in a low density microsomal fraction, which also contained the majority of Glut 4. After insulin treatment, a 50% decrease in Rab4 content was observed, concomitantly with a departure of transporters to the plasma membrane. The dose responses for the departure of Glut 4 and Rab4 from the microsomal fractions were superimposable, half-maximal effects being obtained with 0.1 nM insulin. Rab4 was redistributed to the cytosol and its movement was reversed by insulin withdrawal. When Glut 4-containing vesicles were immunopurified with antibodies to Glut 4, Rab4 was found in the immune pellets, suggesting that Rab4 was tightly associated with the vesicles. Okadaic acid, an inhibitor of phosphatases 1 and 2A that is known to stimulate Glut 4 translocation, caused the same movement of Rab4 from low density microsomal fraction to the cytosol, while the phorbol ester 12-O-tetradecanoylphorbol-13-acetate had no effect. We suggest that insulin and okadaic acid induce a cycling of Rab4 from a vesicular fraction containing the Glut 4 transporter to the cytosol and that this cycling may participate in the insulin stimulatory action on glucose transporter translocation.

Adipose Tissue↗
PubMed1993

Identification of small GTP-binding rab proteins in human platelets: thrombin-induced phosphorylation of rab3B, rab6, and rab8 proteins.

The activation of platelets by specific agonists is a tightly regulated mechanism that leads to the secretion of the dense- and alpha-granule contents. Platelets have been shown to possess small GTP-binding proteins thought to be involved in central biological processes; however, no rab proteins, which may regulate the exocytic process at different stages, have been reported. This study has shown that rab1, rab3B, rab4, rab6, and rab8 proteins, but not rab3A protein, were present in platelets and in endothelial cells. To probe their functional significance in platelets, rab3B, rab6, and rab8 proteins were further characterized with regard to their intracellular localization and their phosphorylation properties. Whereas rab3B protein was found to be mainly cytosolic, rab6 and rab8 proteins were preferentially targeted to the plasma membrane and to the alpha granules. The activation of platelets by thrombin, a potent inducer of secretion, resulted in the phosphorylation of rab3B, rab6, and rab8 proteins, whereas no phosphorylation was observed in the presence of prostaglandin E1, which stimulates cAMP-dependent protein kinase and inhibits the secretion process. These findings provide evidence that members of the subfamily of rab proteins, rab6 and rab8, are localized in platelets to one type of specific secretory vesicle, the alpha granule, and would suggest their possible implication in the secretion process through phosphorylation mechanisms.

Amino Acid Sequence↗
PubMed1993

A possible role for the Ras-related Rab2 protein in the immunological events associated with hematological malignancies.

The Rab branch of the Ras-related guanine nucleotide (GTP/GDP)-binding proteins currently includes at least thirty related members which are involved in the intracellular vesicular transport along the secretory and endocytic pathways in eukaryotic cells. We have demonstrated the overexpression of the Rab2 protein in peripheral blood mononuclear cells from patients exhibiting Sézary syndromes and other lymphoid and myeloid malignancies. Several lines of evidence suggest that the Rab2 overexpression can be related not to leukemic cells but to a subset of peripheral lymphocytes with a CD2+ phenotype. Our results provide strong evidences for the implication of a small GDP/GTP binding protein in immunological events associated with neoplastic states. The precise cellular population involved in this process remains to be determined.

GTP-Binding Proteins↗
PubMed1992

Regulation of the GTPase activity of the ras-related rap2 protein.

The small GTP-binding protein rap2A exhibits a high level of identity with rap1 and ras proteins (60% and 46%, respectively). Nevertheless, its intrinsic GTPase activity is not stimulated by ras-GAP, and unlike the rap1A protein, it cannot compete with ras proteins for their interaction with ras-GAP. In addition, rap1-GAPm that is highly active on the GTPase activity of the rap1A product, also stimulates the GTPase activity of the rap2A protein but with a 30-40-fold lower efficiency. An activity that greatly stimulated the GTPase activity of the rap2 protein (rap2-GAP) was found in bovine brain cytosol and purified. However, it copurified with the cytosolic form of rap1-GAP and was more efficient at stimulating the GTPase activity of the rap1 protein; this 55 kD polypeptide, that is recognized by an antibody raised against rap1-GAPm, likely represents a degraded and soluble form of the full size 89 kD molecule. In bovine brain membranes, a weak GAP activity toward the rap2A protein was also detected; however, it was also attributable to the membrane-associated rap1-GAPm. Thus, it appears that a single rap-GAP protein, complete or degraded, is able to stimulate the GTPase activity of both rap1 and rap2 proteins.

Animals↗
PubMed1992

Localization of the ras-like rab3A protein in the adult rat brain.

Rab3A is a small GTP-binding synaptic vesicle protein, shown to dissociate from synaptic vesicle membranes upon depolarization-induced exocytosis. Using an antiserum raised against rab3A, we found that the antigen was localized to the neuropil of specific brain regions, but was not present in major fiber tracts or most cell bodies. For example, the neuropil of several thalamic nuclei (i.e., dorsal lateral geniculate nucleus, lateral posterior nucleus, ventroposterior nucleus), cerebral cortex, upper layers of the superior colliculus and matrix zones of the neostriatum, were strongly immunoreactive, while the anterior commissure, corpus callosum, optic tract and internal capsule were devoid of staining. The hippocampus, regions of cerebral cortex and the cerebellum exhibited striking laminar distributions of rab3A immunoreactivity. In the hippocampus, dark staining was observed in the stratum oriens, stratum radiatum and molecular layer of the dentate gyrus, while the pyramidal, stratum lacunosum moleculare and dentate granule layers were not stained. In cerebellum the molecular layer and to a lesser extent, the underlying granule cell layer showed enhanced immunoreactivity. Seven days after excitotoxic lesions of the cerebral cortex, rab3A immunoreactivity was diminished in the mirror locus in the contralateral cortical hemisphere and in certain thalamic nuclei ipsilateral to the injection site. These results show that rab3A is localized to a number of specific regions. Its absence from other areas suggests that this synaptic vesicle protein is not universal to all neuronal terminals and pathways. In addition, our lesion studies indicate that for some brain regions, much of the antigen originates in cortical neurons and is distributed within specific axonal projections.

Animals↗
PubMed1992

Overexpression of the ras-related rab2 gene product in peripheral blood mononuclear cells from patients with hematological and solid neoplasms.

The rab2 gene product belongs to the rab branch of the ras-related GTP-binding protein family. The rab2 gene mRNA analysis in 70 tumor samples from various origins showed no obvious difference between malignant tissues and their normal counterparts. However, an over-expression has been observed at the RNA or protein levels in peripheral blood mononuclear cells from the nine patients with Sézary syndromes studied. Subsequent investigations in mycosis fungoides and solid tumor patients allowed us to link these high protein levels to immune reactive rather than to malignant cells. Circulating monocyte and lymphocyte populations from cancer patients are under analysis to correlate the Rab2 protein overexpression to a peculiar subset of cells.

Adult↗
PubMed1992

Evidence for a role of rap1 protein in the regulation of human platelet Ca2+ fluxes.

The relationship between the 22-24 kDa cyclic AMP (cAMP)-dependent phosphoprotein previously described as being involved in the regulation of human platelet membrane Ca2+ transport and a GTP-binding protein of low molecular mass (ras-like protein) was investigated. After isolation of plasma membranes and intracellular membranes, it was found that guanosine 5'-[gamma-thio]triphosphate (GTP[S]) bound to plasma membrane proteins ranging in molecular mass from 22 to 29 kDa, but not to intracellular membranes. The major GTP-binding protein appeared as a 24 kDa protein under reduced conditions and a 22 kDa protein under non-reduced conditions. A similar membrane location and electrophoretic mobility were found for both the cAMP phosphoprotein and the protein recognized by a specific anti-rap1 antibody. The identity between the cAMP phosphoprotein and the rap1 GTP-binding protein was further examined by studying the functional effect of GTP on plasma membrane Ca2+ transport. A maximal GTP[S] concentration of 40 microM was found to: (1) inhibit to the same degree (40%) both Ca(2+)-ATPase activity and the Ca2+ transport function mediated by the Ca(2+)-ATPase; (2) inhibit the phosphorylation of the 22-24 kDa protein by the catalytic subunit of the cAMP-dependent protein kinase (C.Sub.); and (3) abolish the stimulation of Ca2+ uptake induced by C.Sub. It is concluded that the platelet cAMP phosphoprotein is indeed the rap1 GTP-binding protein, and that it regulates plasma membrane Ca2+ transport, thus providing evidence for a new role of a ras-related protein.

Biological Transport↗
PubMed1992

Characterization of Spi-B, a transcription factor related to the putative oncoprotein Spi-1/PU.1.

We have cloned a human cDNA from a new gene, spi-B, on the basis of its homology with the DNA-binding domain of the Spi-1/PU.1 putative oncogene product. spi-B codes for a protein of 262 amino acids presenting 43% overall identity with Spi-1. Its highly basic carboxy-terminal region exhibits 34% sequence identity with the DNA-binding domain of the Ets-1 protein. We showed that the Spi-B protein is able to bind the purine-rich sequence (PU box) recognized by Spi-1/PU.1 and to activate transcription of a reporter plasmid containing PU boxes. Chromosome in situ hybridization allowed us to map spi-B to the 19q13.3-19q13.4 region of the human genome. spi-B, like spi-1, was found to be expressed in various murine and human hematopoietic cell lines except T lymphoid cell lines.

Amino Acid Sequence↗
PubMed1991

Effects of the ras-related rap2 protein on cellular proliferation.

Ras oncogenes encode 21-kDa (p21s) GTP binding proteins that are capable of transforming immortalized cells in culture. The ras-related rap1A/Krev-1/smgp21A protein, that exhibits a similar structural organization and contains the same effector domain as ras proteins, antagonizes ras-transformation. In order to investigate whether the closely related (61% identical) rap2 protein had similar capacities, the corresponding cDNA was inserted into constitutive as well as inducible mammalian expression vectors. Neither the wild-type, nor an "activated" mutant carrying a glycine-to-valine substitution at position 12, had any transforming activity. Several independent lines of evidence demonstrate that the rap2 protein exhibits neither growth-promoting nor growth-inhibitory effects, and that its over-expression does not interfere with ras-induced transformation. Thus, in spite of their great similarities, the rap1A/Krev-1/smgp21A and rap2 proteins have distinct physiological properties.

Animals↗
PubMed1991

The small GTP-binding protein rab4 is associated with early endosomes.

Small GTP-binding proteins of the rab family have been implicated as playing important roles in controlling membrane traffic on the biosynthetic and endocytic pathways. We demonstrate that a distinct rab protein, rab4p, is associated with the population of early endosomes involved in transferrin-receptor recycling. An antibody to human rab4p was found to detect a doublet of approximately 24-kDa proteins on immunoblots from various cell types. Seventy-five percent of these proteins were tightly membrane bound and could be released only by detergent treatment. Upon isolation of early endosomes, late endosomes, and lysosomes, by free-flow electrophoresis and Percoll density-gradient centrifugation, most (70%) of the rab4p was found to cofractionate with early endosomes and endocytic vesicles containing 125I-labeled transferrin. The rab proteins previously localized to the endoplasmic reticulum and/or Golgi apparatus were not found in these fractions. We also localized rab4p to transferrin-receptor-containing early endosomes by immunofluorescence after expression of rab4 cDNA. The association of rab4p with early endosomes and other vesicles involved in the intracellular transport of transferrin receptor suggests that rab4p may play a role in regulating the pathway of receptor recycling.

Animals↗