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A Tavitian

Publications and source records attributed to A Tavitian.

At least 19 recordsLinked to original sources
PubMed2001

A novel group IIA phospholipase A2 interacts with v-Src oncoprotein from RSV-transformed hamster cells.

We have isolated a novel isoform of phospholipase A(2). This enzyme was designated srPLA(2) because it was discovered while analyzing the proteins interacting with different forms of the v-Src oncoproteins isolated from Rous sarcoma virus-transformed hamster cells. It contains all the functional regions of the PLA(2) group IIA proteins but differs at its C-terminal end where there is an additional stretch of 8 amino acids. The SrPLA(2) isoform was detected as a 17-kDa precursor in cells and as a mature 14-kDa form secreted in culture medium. A direct interaction of the 17-kDa precursor with the Src protein was observed in lysates of transformed cells. Both the 17- and 14-kDa forms were found to be phosphorylated on tyrosine. To our knowledge, this is the first report of a PLA(2) group II protein that is tyrosine phosphorylated. We surmise that srPLA(2) interacts with the Src protein at the cell membrane during the process of its maturation.

Amino Acid Sequence↗
PubMed1999

Phosphorylation of p125FAK and paxillin focal adhesion proteins in src-transformed cells with different metastatic capacity.

Hamster fibroblasts transformed by Rous sarcoma virus (RSV) display different metastatic potentials that are associated with specific structural features of the v-src oncoprotein. This diverse metastatic activity could be due to various tyrosine phosphorylation levels of specific src protein substrates. To check this hypothesis, phosphorylation of the FAK and paxillin proteins, involved in signal transduction pathways and known as src protein substrates, was tested. It was shown that FAK and paxillin are hyperphosphorylated in the high metastatic cell lines as compared with the phosphotyrosine level of these proteins found in the low metastatic cell lines. In addition, our data confirm that v-src protein plays a direct role in paxillin phosphorylation.

Animals↗
PubMed1998

Identification of a specific effector of the small GTP-binding protein Rap2.

Rap2 is a small GTP-binding protein that belongs to the Ras superfamily and whose function is still unknown. To elucidate Rap2 function, we searched for potential effectors by screening a mouse brain cDNA library in a yeast two-hybrid system using as a bait a Rap2A protein bearing a mutation of Gly to Val at position 12. This strategy lead to the identification of a protein that interacts specifically with Rap2A complexed with GTP, and requires an intact effector domain of Rap2A for interaction; we designated this protein Rap2-interacting protein 8 (RPIP8). Biochemical data obtained from in vitro studies with purified proteins confirmed the genetic results. Mouse RPIP8 consists of 446 amino acids, bears a coiled-coil domain between residues 265 and 313, and is expressed principally in brain. Its human counterpart, of 400 amino acids, exhibits 93.7% identity in their common region. A search for similar sequences in expressed-sequence-tags databanks revealed the presence in human and rodents of mRNAs encoding the 400-residue and 446-residue forms of RPIP8. Furthermore a doublet of 45-50 kDa, corresponding to the 400-residue and 446-residue forms of the protein, was detected by western blotting of mouse brain extracts and lysates from pheochromocytoma PC12 cells and the pancreatic beta-cell lines HIT-T15 and RIN-m5F. Using transient transfections of HIT-T15 cells it was possible to demonstrate that [Val12]Rap2 and wild-type Rap2 could be immunoprecipitated with RPIP8. These data therefore argue for RPIP8 being a specific effector of the Rap2 protein in cells exhibiting neuronal properties.

Amino Acid Sequence↗
PubMed1998

The transcription factor Spi-1/PU.1 interacts with the potential splicing factor TLS.

Spi-1/PU.1 is an Ets protein deregulated by insertional mutagenesis during the murine Friend erythroleukemia. The overexpression of the normal protein in a proerythroblastic cell prevents its terminal differentiation. In normal hematopoiesis Spi-1/PU.1 is a transcription factor that plays a key role in normal myeloid and B lymphoid differentiation. Moreover, Spi-1/PU.1 binds RNA and interferes in vitro with the splicing process. Here we report that Spi-1 interacts in vivo with TLS (translocated in liposarcoma), a RNA-binding protein involved in human tumor-specific chromosomal translocations. This interaction appears functionally relevant, since TLS is capable of reducing the abilities of Spi-1/PU.1 to bind DNA and to transactivate the expression of a reporter gene. In addition, we observe that TLS is potentially a splicing factor. It promotes the use of the distal 5' splice site during the E1A pre-mRNA splicing. This effect is counterpoised in vivo by Spi-1. These data suggest that alteration of pre-mRNA alternative splicing by Spi-1 could be involved in the transformation of an erythroblastic cell.

Alternative Splicing↗
PubMed1998

C-terminal end of v-src protein interacts with peptide coded by gadd7/adapt15-like RNA in two-hybrid system.

The significant differences in the metastatic properties of hamster fibroblasts transformed by the Rous sarcoma virus (RSV) were associated with mutations in the v-src carboxy-terminal region. To identify the capacity of this region for protein-protein interaction the two-hybrid system was used. The cDNA clone (vseap1), producing the protein specifically bound with the v-src C-terminal part in yeast cells in vivo and in GST-fusion system in vitro was isolated. Vseap1 shared 68% of homology with stressful agents induced RNA-gadd7/adapt15. Two vseap1 specific messenger RNAs were identified: 0.9-kbp RNA expressed in all transformed cells and three times less in embryo fibroblasts; 3.1-kbp transcript was deleted in the cells with suppressed v-src activity and H2O2 resistance.

Amino Acid Sequence↗
PubMed1997

A new member of the amphiphysin family connecting endocytosis and signal transduction pathways.

Src homology 3 (SH3) domains are conserved modules which participate in protein interaction by recognizing proline-rich motifs on target molecules. To identify new SH3-containing proteins, we performed a two-hybrid screen with a proline-rich region of human SOS-1. One of the specific SOS-1 interacting clones that were isolated from a mouse brain cDNA library defines a new protein that was named amphiphysin 2 because of its homology to the previously reported amphiphysin. Amphiphysin 2 is expressed in a number of mouse tissues through multiple RNA transcripts. Here, we report the amino acid sequence of a brain form of amphiphysin 2 (BRAMP2) encoded by a 2. 5-kilobase mRNA. BRAMP2 associates in vitro with elements of the endocytosis machinery such as alpha-adaptin and dynamin. On a biosensor surface, the BRAMP2/dynamin interaction appeared to be direct and partly dependent on a proline-rich sequence of dynamin. Association with dynamin was also observed in PC12 cells after cell stimulation with nerve growth factor, suggesting that amphiphysin 2 may be connected to receptor-dependent signaling pathways. This hypothesis is strengthened by the ability of BRAMP2 to interact with the p21(ras) exchange factor SOS, in vitro, as a possible point of interconnection between the endocytic and signaling pathways.

Adaptor Protein Complex alpha Subunits↗
PubMed1996

RhoGDI-3 is a new GDP dissociation inhibitor (GDI). Identification of a non-cytosolic GDI protein interacting with the small GTP-binding proteins RhoB and RhoG.

RhoB is a small GTP-binding protein highly homologous to the RhoA protein. While RhoA is known to regulate the assembly of focal adhesions and stress fibers in response to growth factors, the function of RhoB remains unknown. We have reported that the transient expression of the endogenous RhoB protein is regulated during the cell cycle, contrasting with the permanent RhoA protein expression (). Using the yeast two-hybrid system to characterize proteins interacting with RhoB, we identified a new mouse Rho GDP dissociation inhibitor, referenced as RhoGDI-3. The NH2-terminal alpha helix of RhoGDI-3 is strongly amphipatic and differs thus from that found in previously described bovine, human, and yeast RhoGDI proteins and mouse and human D4/Ly-GDIs. Contrary to the cytosolic localization of all known GDI proteins, acting on Rab or Rho, RhoGDI-3 is associated to a Triton X-100-insoluble membranous or cytoskeletal subcellular fraction. In the two-hybrid system, RhoGDI-3 interacts specifically with GDP- and GTP-bound forms of post-translationally processed RhoB and RhoG proteins, both of which show a growth-regulated expression in mammalian cells. No interaction is found with RhoA, RhoC, or Rac1 proteins. We show that GDI-3 is able to inhibit GDP/GTP exchange of RhoB and to release GDP-bound but not GTP-bound RhoB from cell membranes.

Amino Acid Sequence↗
PubMed1996

An alternatively spliced isoform of the Spi-B transcription factor.

Spi-B is an Ets transcription factor related to the oncoprotein Spi-1/PU.1 and highly expressed in B lymphoid cells. The Ets proteins share a conserved Ets domain that mediates specific DNA binding. Spi-B binds DNA sequences containing a core 5'-GGAA-3' and activates transcription through this motif. Up to date, the biological function of Spi-B remains unknown. Here, we describe the characterization of an alternatively spliced variant of Spi-B, named deltaSpi-B, which has lost the Ets domain. In B lymphoid cells, deltaspi-B and spi-B mRNAs were present simultaneously in a ratio of around 10%. DeltaSpi-B product was not able to bind DNA and was recovered in cytoplasmic cellular extracts. We raise the hypothesis that delta Spi-B might affect Spi-B function by recruiting factors involved in Spi-B activity.

Alternative Splicing↗
PubMed1996

The transcription factor Spi-1/PU.1 binds RNA and interferes with the RNA-binding protein p54nrb.

The protooncogene for Spi-1/PU.1 is an Ets-related transcription factor overexpressed during Friend erythroleukemia. The molecular basis by which Spi-1/PU.1 is involved in the erythroleukemic process remains to be elucidated. By using an immobilized protein binding assay, we have identified a 55-kDa protein as a putative partner of Spi-1/PU.1 protein. Microsequence analysis revealed that this 55-kDa protein was p54nrb (nuclear RNA-binding protein, 54 kDa) a RNA-binding protein highly similar to the splicing factor PSF (polypyrimidine tract-binding protein-associated splicing factor). In this paper, we show that Spi-1/PU.1 impedes the binding of p54nrb to RNA and alters the splicing process in vitro. Moreover, we present evidence that the transcriptional factor Spi-1/PU.1, unlike other Ets proteins, is able to bind RNA. Altogether, these results raise the intriguing possibility that the functional interference observed between Spi-1/PU.1 and RNA-binding proteins might represent a novel mechanism in malignant erythropoiesis.

Alternative Splicing↗
PubMed1996

Differential phosphorylations of Spi-B and Spi-1 transcription factors.

Spi-1/PU-1 and Spi-B are hematopoietic transcription factors, which, in vitro, display similar affinities for DNA target sequences containing the consensus binding site 5'-GGAA-3'. While the role of Spi-1 in the transcriptional regulation of B cell and myeloid specific genes has been largely demonstrated, the biological function of Spi-B still remains to be elucidated. Since Spi-B and Spi-1 are very divergent in their transactivator domain, these domains might acquire functional specificity in vivo by interacting with different co-factors and/or by undergoing different phosphorylations. First, we observed that casein kinase II phosphorylates Spi-B as well as Spi-1, in vitro. Then, by affinity chromatographies and in vitro kinase assays with fusion proteins between glutathione-S-transferase and the transactivator domain of Spi-B, two kinases were identified on their ability to interact and phosphorylate this domain; the MAP kinase ERK1 and the stress activated protein kinase JNK1. The Threonine 56 was defined as the ERK1 phosphorylation site by using phosphoamino-acid analyses and a Spi-B mutant version with the substitution T56 to A56. Strikingly, ERK1 failed to phosphorylate Spi-1, in vitro, whereas JNK1, like CK II, phosphorylated Spi-B and Spi-1. In addition, other purified Spi-B-kinase activities, unidentified as yet, display similar specificity than ERK1 for Spi-B versus Spi-1. Furthermore, the evident interaction of pRb protein with the transactivator domain of Spi-B in an unphosphorylated state disappeared when this domain was first phosphorylated in vitro either by ERK1 or by the purified Spi-B-kinase activities. Our data revealed multiple phosphorylation sites within Spi-B whose some of them appeared specific for Spi-B versus Spi-1 and which may account for differential regulation of their activities.

Amino Acid Sequence↗
PubMed1996

Spi-1/PU.1 transgenic mice develop multistep erythroleukemias.

Insertional mutagenesis of the spi-1 gene is associated with the emergence of malignant proerythroblasts during Friend virus-induced acute erythroleukemia. To determine the role of spi-1/PU.1 in the genesis of leukemia, we generated spi-1 transgenic mice. In one founder line the transgene was overexpressed as an unexpected-size transcript in various mouse tissues. Homozygous transgenic animals gave rise to live-born offspring, but 50% of the animals developed a multistep erythroleukemia within 1.5 to 6 months of birth whereas the remainder survived without evidence of disease. At the onset of the disease, mice became severely anemic. Their hematopoietic tissues were massively invaded with nontumorigenic proerythroblasts that express a high level of Spi-1 protein. These transgenic proerythroblasts are partially blocked in differentiation and strictly dependent on erythropoietin for their proliferation both in vivo and in vitro. A complete but transient regression of the disease was observed after erythrocyte transfusion, suggesting that the constitutive expression of spi-1 is related to the block of the differentiation of erythroid precursors. At relapse, erythropoietin-independent malignant proerythroblasts arose. Growth factor autonomy could be partially explained by the autocrine secretion of erythropoietin; however, other genetic events appear to be necessary to confer the full malignant phenotype. These results reveal that overexpression of spi-1 is essential for malignant erythropoiesis and does not alter other hematopoietic lineages.

Animals↗
PubMed1996

Small GTP-binding proteins in Plasmodium falciparum.

During its erythrocytic life cycle Plasmodium falciparum exchanges compounds with host cells through phagocytosis and exocytosis. In eucaryotic cells, small GTP-binding proteins of the Ras superfamily appear to be involved in different steps of membrane trafficking and in intracellular signals. In this paper, we investigate the Rab4, Rab6 and Ras-related proteins in P falciparum infected red cells. We report that P falciparum Rab and Ras-related proteins could be distinguished from their counterparts by iso-electrofocusing and immunoblotting. The localization of P falciparum Rab 4 and Rab 6 was studied by immunogold electron microscopy on ultrathin frozen sections of infected red blood cells. Rab4 parasite-related protein was found associated with the membranes of early endosome-like structures near the parasite plasma membrane. Rab6-related protein was associated with the Golgi/trans Golgi network, as already suggested by immunofluorescence microscopy studies and Ras-related protein was cytoplasmic and plasma membrane-associated. These results are in accordance with their mammalian counterparts and support the implication of Rab-related proteins in vesicular trafficking in Plasmodium.

Animals↗
PubMed1995

Bridging Ral GTPase to Rho pathways. RLIP76, a Ral effector with CDC42/Rac GTPase-activating protein activity.

Ra1A and Ra1B are GTPases of unknown function and are activated by proteins, Ra1GDS, that interact with the active form of another GTPase, Ras. To elucidate Ral function, we have searched for proteins interacting with an activated form of Ra1A using the two-hybrid method and a Jurkat cell library. We have identified a partial cDNA encoding a protein, RLIP1, which binds to activated Ra1A and this binding requires an intact effector domain of Ra1A. Biochemical data with purified Ra1A confirm the genetic results. This protein also bears a region of homology with GTPase-activating protein (GAP) domains that are involved in the regulation of GTPases of the Rho family and, indeed, RLIP1 displays a GAP activity acting upon Rac1 and CDC42, but not RhoA. This GAP region is not required for RLIP1 binding to Ra1. The whole cDNA was cloned, and it encodes a 76-kDa polypeptide, RLIP76, which also binds RalA. The Rho pathway is involved in membrane and cytoskeleton modifications after mitogenic stimulation and acts in parallel to and synergistically with the Ras pathway. We propose that these pathways are linked through a cascade composed of Ras --> Ra1GDS --> Ra1 --> RLIP76 --> CDC42/Rac1/Rho, allowing modulation of the Rho pathway by the Ras pathway.

ATP-Binding Cassette Transporters↗
PubMed1995

DNA binding specificities of Spi-1/PU.1 and Spi-B transcription factors and identification of a Spi-1/Spi-B binding site in the c-fes/c-fps promoter.

Spi-1/PU.1 and Spi-B encode hematopoietic-specific transcription factors that are the most distantly related members of the Ets family. The Ets proteins share a conserved 85 amino acids DNA binding domain, the Ets domain and recognize various DNA target sites around a common core 5'-GGAA/T-3'. The DNA binding specificities of Spi-1 and Spi-B were investigated by using the method of polymerase chain reaction (PCR)-mediated random site selection. The deduced Spi-1 and Spi-B consensus binding sites are very similar suggesting that the functional activities of Spi-1 and Spi-B cannot be distinguished on the basis of their DNA binding specificities. We identified a putative Spi-1/Spi-B binding site in the promoter region of the c-fes/c-fps protooncogene which encodes a tyrosine kinase expressed predominantly in myeloid cells. In vitro translated Spi-1 and Spi-B proteins were capable to bind this site similarly and to activate the c-fes promoter in HeLa transfected cells. We showed that Spi-1 binds the Spi-1/Spi-B binding site of c-fes in HL-60 cells suggesting that Spi-1 may be involved in the regulation of c-fes transcription in myeloid cells. Intriguingly, we detected only Spi-1 binding to this site in the Raji cell line which express both Spi-1 and Spi-B proteins. This suggests that Spi-1 and Spi-B exhibit different DNA binding activities in vivo although they share similar DNA binding specificities in vitro.

Animals↗
PubMed1995

Regulation of Ras-related RhoB protein expression during the cell cycle.

The immediate-early gene rhoB codes for a small GTP-binding protein highly homologous to the RhoA protein. While RhoA is known to regulate the assembly of focal adhesions and stress fibers in response to growth factors, the function of RhoB remains unknown. In a first attempt to elucidate its function, we examined the variation of the RhoB protein expression in response to induction of its mRNA. We report here that RhoB is an unstable protein rapidly and transiently induced by growth factors in PC12 and HeLa cells. Moreover, RhoB protein accumulation is periodic through the cell cycle. First detected at the G1/S phase transition, the level of the RhoB protein is maximal during the S phase and declines at the S/G2-M transition. This timing suggests that RhoB plays a role in the G1/S phase transition and/or in the S phase of the cell cycle. We also confirm here a vesicular and perinuclear localization of the endogenous RhoB protein induced by growth factors.

Cell Cycle↗
PubMed1995

[RAS proteins and related proteins].

Since the mid-eighties, numerous small G-proteins of the ras gene superfamily have been identified and characterized; more than sixty members are distributed into four subfamilies: ras, rho, rab and ran. Although it appears that, structurally, the products of the ras superfamily are related and implicated in various and diverse intracellular mechanisms: signal transduction, cell cycle, differentiation, cellular traffic, etc., their true and specific functions and their scope and extent are still largely unknown for many of them. Approaches to unravel their function include: determination of their intracellular localization, differential expression in various cell and tissues, phenotypic changes induced by the active forms, studies of the interacting molecules. Recent studies combining advanced mammalian cell biology/biochemistry and genetic studies in yeast might help elucidate the specific role exerted by these proteins.

Amino Acid Sequence↗
PubMed1994

EGF modulation of the ras-related rhoB gene expression in human breast-cancer cell lines.

The mRNA levels of the ras-related human rhoA, rhoB and rhoC genes were studied in human breast-cancer cell lines (HBCal), and in normal and immortalized mammary epithelial cells (HMEC) by Northern blot analysis and in situ hybridization. In contrast to the ubiquitous rhoA and rhoC gene expression, dramatic variations in the mRNA level of the rhoB gene were evidenced. The rhoB mRNA level appeared to be inversely correlated to the amounts of the epidermal-growth-factor(EGF) receptors in these cells. The rhoB transcripts were detected at high levels in ZR75-1, MCF7, HSL 53, HSL 59, HSL 90, T47D and SKBR3 HBCal, at hardly detectable levels in BT 20, MDA-MB 231 and H466B HBCal and at intermediate levels in normal and immortalized breast epithelial cells. Rapid and transient induction of the rhoB transcription was observed after EGF treatment in serum-deprived MDA-MB231, T47D and immortalized epithelial cells. In contrast, no modulation of rhoB expression by EGF could be objectified in the MCF7 and ZR75-1 cell lines. Yet a normal function of EGF receptors was evidenced, since the immediate early gene c-fos was rapidly induced, suggesting a constitutive expression of rhoB in these cell lines bypassing the regulation by EGF. In human mammary epithelial cells, rhoB mRNA is rapidly and transiently induced with EGF concentrations known to stimulate cell proliferation. This suggests that the rhoB product might be involved in a cascade that initiates or promotes cell proliferation, and plays an important role in EGF-stimulated growth of breast normal and cancer cells.

Blotting, Northern↗
PubMed1994

Phosphorylation of Rap1GAP during the cell cycle.

Rap1GAP (for Rap1 GTPase Activating Protein) is an 89 kD protein that highly stimulates the intrinsic GTPase activity of the small GTP binding protein Rap1. It has been shown that Rap1GAP is phosphorylated in vitro by purified p34cdc2 kinase, which regulates the G2/M transition of the cell cycle. In this work, we have studied the phosphorylation of Rap1GAP during the cell cycle and showed that Rap1GAP is phosphorylated in vivo in interphasic and mitotic Hela cells; the electrophoretic mobility of Rap1GAP from mitotic cells is reduced compared with that from interphasic cells, suggesting that the mitotic form of the protein is hyperphosphorylated. As the cdc2 kinase is specifically active during mitosis, we sought to investigate whether it actually phosphorylates Rap1GAP during this phase of the cell cycle. We show that p34cdc2 co-immunoprecipitated from mitotic Hela cell lysates with an anti human cyclin B1 antibody, but not from interphasic cell lysates, is able to phosphorylate efficiently wild-type Rap1GAP, but not a mutant in which the putative consensus site for phosphorylation by the cdc2 kinase (serine 484) has been altered. Moreover, depletion of p34cdc2 from mitotic extracts abolishes the phosphorylation of Rap1GAP by such lysates. These results therefore strongly suggest that Rap1GAP is indeed a substrate of the cdc2 kinase during mitosis. This phosphorylation does not affect the stimulation of the GTPase activity of Rap1 by Rap1GAP but may play a role in regulating the interaction of Rap1GAP with other proteins involved in the cellular functions regulated by Rap1 and Rap1GAP.

Amino Acid Sequence↗