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Biomedical subjects

B Tocqué

Publications and source records attributed to B Tocqué.

At least 19 recordsLinked to original sources
PubMed2001

G3BP is overexpressed in human tumors and promotes S phase entry.

The expression of the human Ras-GTPase activating protein (GAP)-binding protein (G3BP) was studied in human tumors and cell lines of different origins. Northern blot analysis and immunoblotting experiments showed enhanced expression of G3BP in all tumor samples as compared to healthy tissue. The enhanced expression does not seem to be related to the tumor site or to the stage of development of the cancer. In light of the proposed functions of G3BP, its increased expression in tumors suggest that it plays a role in dedifferentiation and proliferation processes. We also show that G3BP promotes S phase entry in cultured fibroblasts deprived of serum and that this function is dependent on the presence of the RNA binding domain of the protein.

3T3 Cells↗
PubMed2000

Upregulation of the apoptosis-associated protein Grb3-3 in HIV-1-infected human CD4(+) lymphocytes.

The mechanism(s) by which HIV-1 infection contributes to depletion of CD4(+) T cell is not well understood. In this report, we investigated whether a recently identified isoform of growth factor receptor bound protein (Grb2), named Grb3-3, a signaling molecule that is associated with the MAP kinase pathway and with apoptosis could be involved. We find that Grb3-3 is markedly up-regulated following HIV-1 infection of CD4(+) peripheral blood mononuclear cells undergoing apoptosis. Although IL-2 deprived CD4(+) cells also undergo apoptosis to a similar extent, Grb3-3 upregulation is not detected under these experimental conditions. Transient overexpression of Grb3-3 in Jurkat T-cells also causes apoptosis. Upon staurosporine stimulation, Grb3-3 predisposes Sup-T1 cell to apoptosis. Finally, analysis of the HIV-1 genes responsible for Grb3-3 expression demonstrates that Tat and Nef can independently induces its expression, suggesting these two earliest viral gene products of HIV-1 may share some common pathway(s) in up-regulating Grb3-3 expression.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Ras-GTPase activating protein inhibition specifically induces apoptosis of tumour cells.

Oncogenes and tumour suppressor genes control the balance between apoptotic death and anti-apoptotic survival signals determining whether a cell proliferates or dies. Through which effectors might oncoproteins generate sensitivity to apoptosis remains to be determined. Ras GTPase activating protein (Ras-GAP) is a key element in the Ras signalling pathway, being both a negative regulator and possibly an effector of Ras. Ras-GAP acts as a regulator of transcription, and possibly connects Ras to stress-activated protein kinases. A role for Ras-GAP in cell survival has been suspected from the study of knock-out mouse embryos. In search for selective killing of tumour cells, we asked whether Ras-GAP inhibition by other means would lead to apoptosis in established cell lines. We injected a monoclonal antibody directed against the SH3 domain of Ras-GAP (mAb200) that has been shown to block Ras-GAP downstream signalling into various human normal and tumour cell lines. We show that inhibition of Ras-GAP induces apoptosis specifically in tumour, but not in normal cells, therefore pointing at a specific role for Ras-GAP in tumour cell survival. MAb200-induced apoptosis is largely prevented by coinjection of activated RhoA or Cdc42 proteins, by injection of a constitutively activated mutant of phosphoinositide 3-OH kinase (PI3-K), but not by injection of v-Raf. These results show that targeting of Ras-GAP could represent a novel anticancer approach.

Antibodies, Monoclonal↗
PubMed1999

Mapping the APP/presenilin (PS) binding domains: the hydrophilic N-terminus of PS2 is sufficient for interaction with APP and can displace APP/PS1 interaction.

Mutations in presenilin 1 and presenilin 2 (PS1 and PS2, respectively) genes cause the large majority of familial forms of early-onset Alzheimer's disease. The physical interaction between presenilins and APP has been recently described using coimmunoprecipitation. With a similar technique, we confirmed this interaction and have mapped the interaction domains on both PS2 and APP. Using several carboxy-terminal truncated forms of PS2, we demonstrated that the hydrophilic amino terminus of PS2 (residues 1 to 87, PS2NT) was sufficient for interaction with APP. Interestingly, only a construct with a leader peptide for secretion (SecPS2NT) and not its cytosolic counterpart was shown to interact with APP. For APP, we could demonstrate interaction of PS2 with the last 100 but not the last 45 amino acids of APP, including therefore the A beta region. Accordingly, SecPS2NT is capable of binding to A beta-immunoreactive species in conditioned medium. In addition, a second region in the extracellular domain of APP also interacted with PS2. Comparable results with PS1 indicate that the two presenilins share similar determinants of binding to APP. Confirming these results, SecPS2NT is able to inhibit PS1/APP interaction. Such a competition makes it unlikely that the PS/APP interaction results from nonspecific aggregation of PS in transfected cells. The physical interaction of presenilins with a region encompassing the A beta sequence of APP could be causally related to the misprocessing of APP and the production of A beta1-42.

Alzheimer Disease↗
PubMed1998

Grb2 interaction with MEK-kinase 1 is involved in regulation of Jun-kinase activities in response to epidermal growth factor.

Epidermal growth factor (EGF) receptor was shown to be involved in the activation pathway of the stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) cascade not only by EGF, but also by UV radiation or osmotic stress. This paper describes a specific interaction between the COOH-terminal SH3 domain of Grb2 and the NH2-terminal regulatory domain of MEKK1 in ER22 cells overexpressing the EGF receptor. This interaction results in the formation of a constitutive complex between Grb2 and MEKK1 in both proliferating and resting cells. EGF stimulation causes this complex to be rapidly and transiently recruited by Shc proteins. The subsequent release of the Grb2-MEKK1 complex from Shc proteins correlates with JNK activation. Transfection of the NH2-terminal regulatory domain of MEKK1 specifically inhibits EGF-dependent JNK activation indicating that Grb2 is involved in MEKK1 activation. Thus, adaptor proteins have a new role in the regulation of the SAPK/JNK cascade after EGF stimulation.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Grb2 and its apoptotic isoform Grb3-3 associate with heterogeneous nuclear ribonucleoprotein C, and these interactions are modulated by poly(U) RNA.

Grb2 is an adaptor molecule comprising one Src homology (SH) 2 and two SH3 domains. This protein has a natural isoform named Grb3-3 with a deletion within the SH2 domain. Numerous evidence points to a functional connection between SH2- and SH3-containing proteins and molecules implicated in RNA biogenesis. In this context, we have examined the binding of Grb2 and Grb3-3 to heterogeneous nuclear ribonucleoprotein (hnRNP) C. By the use of an in vivo genetic approach and through in vitro experiments, we furnish evidence that both Grb2 and Grb3-3 interact with hnRNP C proteins. Subcellular fractionation studies clearly show that Grb2 is partially localized in the nucleus. In addition, coimmunoprecipitation experiments demonstrate that Grb2.hnRNP C complexes exist in intact hematopoietic cells. The carboxyl-terminal SH3 domains of Grb2 and Grb3-3 are primarily responsible for the association with hnRNP C. However, although the proline-rich motif of hnRNP C is involved in the interaction with Grb2, it is not in the binding to Grb3-3. Furthermore, poly(U) RNA inhibits the association of Grb2 with hnRNP C, whereas it enhances the interaction between Grb3-3 and hnRNP C. These findings suggest that the Grb2/Grb3-3-hnRNP C interactions might fulfill different biological functions.

3T3 Cells↗
PubMed1998

Intracellular expression of an antibody fragment-neutralizing p21 ras promotes tumor regression.

Mutated ras genes are found in a large number of human tumors and, therefore, constitute one of the primary targets for cancer treatment. Microinjection of the neutralizing anti-Ras monoclonal antibody Y13-259 was previously reported to induce transient phenotypic reversion of ras-transformed rodent fibroblasts in vitro. We have prepared a single-chain Fv fragment (scFv) derived from Y13-259, and here, we show that intracellular expression of the scFv led to the specific inhibition of the Ras signaling pathway in Xenopus laevis oocytes and NIH3T3 fibroblasts. Moreover, neutralizing Ras with the scFv specifically promoted apoptosis in vitro in human cancer cells but not in untransformed cells. As a step toward cancer gene therapy, we finally demonstrated that intratumor transduction of HCT116 colon carcinoma cells with the anti-Ras scFv using an adenoviral vector elicited sustained tumor regression in nude mice.

Animals↗
PubMed1998

Differential interactions of the growth factor receptor-bound protein 2 N-SH3 domain with son of sevenless and dynamin. Potential role in the Ras-dependent signaling pathway.

In this paper, we show that the 36-45 surface-exposed sequence WYKAELNGKD of growth factor receptor-bound protein 2 (Grb2) N-SH3 domain inhibits the interaction between Grb2 and a 97-kDa protein identified as dynamin. Moreover, the peptide GPPPQVPSRPNR from dynamin also blocks the binding of dynamin to the proline-rich recognition platform of Grb2. Mutations in the 36-45 motif show that Glu-40 is critical for dynamin recognition. These observations were confirmed by immunoprecipitation experiments, carried out using ER 22 cells. It was also observed that the proline-rich peptide from dynamin was unable to dissociate the Grb2.Sos complex, whereas the proline-rich peptide from Son of sevenless (Sos) inhibited Grb2. dynamin interaction. A time-dependent stimulation of epidermal growth factor receptor overexpressing clone 22 (ER 22) cells by epidermal growth factor resulted in an immediate increase of the Grb2.Sos complex and a concomitant decrease in Grb2.dynamin. This suggests that the recruitment of Grb2.Sos to the membrane, triggered by epidermal growth factor stimulation, activates the Ras-dependent signaling and simultaneously enhances free dynamin levels, leading to both receptor internalization and endocytotic processes.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Intracellular expression and functional properties of an anti-p21Ras scFv derived from a rat hybridoma containing specific lambda and irrelevant kappa light chains.

A rat single-chain Fv (Y238 scFv) was derived from the Y13-238 monoclonal antibody, a non-neutralizing anti-Ras antibody. The Y13-238 hybridoma expresses two functional light chains. N-terminus microsequencing of these chains showed the presence of the Y3 Ag1.2.3 Vkappa chain derived from the rat fusion partner and of a rat Vlambda chain. Primers designed for rat Vlambda amplification allowed the cloning of a functional scFv that could bind p21Ras. The kinetics of interaction of purified Y238 scFv with the p21Ras protein was evaluated by BIAcore with a NTA sensor chip and gave an apparent affinity constant in the nanomolar range (K(D)=4.58+/-0.63 nM). Immunoprecipitation experiments of Y238 scFv expressed in Xenopus laevis oocytes confirmed the specificity of the scFv for the Ras protein. Y238 scFv could be intracellularly expressed in oocytes and in mammaliam cells without adverse effect on the Ras signalling cascade. This scFv was therefore used as control in experiments where another anti-Ras scFv (Y259 scFv, derived from the neutralizing anti-Ras mAb Y13-259) blocked the Ras pathway in vitro and led to tumor regression in a nude mouse model [Cochet, O., Kenigsberg, M., Delumeau, I., Virone-Oddos, A., Multon, M.C., Fridman, W.H., Schweighoffer, F., Teillaud, J.L., Tocqué, B., 1998. Intracellular expression of an antibody fragment-neutralizing p21 ras promotes tumor regression. Cancer Res. 58, 1170-1176.]. Finally, BIAcore analyses indicated that the epitopes recognized by Y238 and Y259 scFvs are not overlapping and allowed a more precise definition of the Y13-238 epitope.

3T3 Cells↗
PubMed1997

Adenosine deaminase is a specific partner for the Grb2 isoform Grb3-3.

Grb3-3 is an isoform of Grb2, thought to arise by alternative splicing, that lacks a functional SH2 domain but retains functional SH3 domains, which allow interaction with other proteins through binding to prolinerich sequences. Several evidences suggest that besides common partners for Grb2 and Grb3-3, specific targets could exist. In order to find specific partners for Grb3-3, we have screened a human cDNA library by the yeast two-hybrid system with Grb3-3 as a bait. We have identified adenosine deaminase, an enzyme involved in purine metabolism whose deficiency is associated with severe combined immunodeficiency, as a Grb3-3 binding protein that is not able to bind to Grb2. This interaction has been confirmed in vitro with GST fusion proteins and in vivo by coimmunoprecipitation experiments in NIH3T3 cells stably transfected with Grb3-3. The functional significance of this finding is discussed.

3T3 Cells↗
PubMed1995

A role for Grb2 in apoptosis?

Src homology type 2 (SH2) and type 3 (SH3) domains appear to have an important role in signal transduction pathways initiated by tyrosine kinases. SH2 domains allow proteins with signalling functions to interact with tyrosine kinases and tyrosine-phosphorylated proteins at the plasma membrane, whereas SH3 domains allow a distinct type of interaction through binding to proline-rich sequences. The adaptor protein Grb2 consists of one SH2 domain and two SH3 domains and connects tyrosine kinase receptors to activation of the Ras pathway. Its closely related counterpart, Grb3-3, thought to arise by alternative splicing of Grb2 transcripts, lacks a functional SH2 domain but retains functional SH3 domains. We recently presented evidence that Grb3-3 might deliver specific signals causing cells to undergo apoptosis. This review will document the mechanism of Grb3-3 function and discuss its putative involvement in several pathologies. It also further strengthens the notion that cells may use alternative splicing as a means to drive either a proliferative or a suicidal program.

Journal Article↗
PubMed1994

Cloning of a Grb2 isoform with apoptotic properties.

Growth factor receptor-bound protein 2 (Grb2) links tyrosine-phosphorylated proteins to a guanine nucleotide releasing factor of the son of sevenless (Sos) class by attaching to the former by its Src homology 2 (SH2) moiety and to the latter by its SH3 domains. An isoform of grb2 complementary DNA (cDNA) was cloned that has a deletion in the SH2 domain. The protein encoded by this cDNA, Grb3-3, did not bind to phosphorylated epidermal growth factor receptor (EGFR) but retained functional SH3 domains and inhibited EGF-induced transactivation of a Ras-responsive element. The messenger RNA encoding Grb3-3 was expressed in high amounts in the thymus of rats at an age when massive negative selection of thymocytes occurs. Microinjection of Grb3-3 into Swiss 3T3 fibroblasts induced apoptosis. These findings indicate that Grb3-3, by acting as a dominant negative protein over Grb2 and by suppressing proliferative signals, may trigger active programmed cell death.

3T3 Cells↗
PubMed1994

NMR structure of the N-terminal SH3 domain of GRB2 and its complex with a proline-rich peptide from Sos.

GRB2 is a small adaptor protein of 217 amino acids comprising one SH2 domain surrounded by two SH3 domains. GRB2 couples receptor tyrosine kinase activation to Ras signalling by interacting, through its SH3 domains, to the carboxy-terminal proline-rich regions of the guanine nucleotide exchange factor Sos. Here we report the synthesis and solution structure of the amino-terminal SH3 domain of GRB2 and of its more stable Ser 32 mutant. 1H NMR analysis of the complex between the Ser-32-GRB2-N-SH3 domain and the proline-rich peptide VPPPVPPRRR, derived from h-Sos, shows that relative to the SH3 peptide complexes described for PI3K, Fyn and Abl, the proline-rich peptide in this complex binds in the opposite orientation.

Adaptor Proteins, Signal Transducing↗
PubMed1993

Saccharomyces cerevisiae CDC25 (1028-1589) is a guanine nucleotide releasing factor for mammalian ras proteins and is oncogenic in NIH3T3 cells.

The best characterized yeast guanine nucleotide releasing factor is CDC25, which acts on RAS and thereby stimulates cAMP production in Saccharomyces cerevisiae. In order to determine if CDC25 could be a specific GDP-GTP releasing factor for the mammalian proteins Ha-ras, Ki-ras, and N-ras, its functions were studied both in vitro and in NIH3T3 cells. The 561 amino acid composing the C-terminal domain of CDC25 (CDC25 C-domain) released guanine nucleotides (both GDP and GTP) from Ha-, Ki-, and N-ras but not from Rap1A, Rab5, and Rab11. CDC25 acted on oncogenically activated Ha-ras even if the last 23 amino acids (167-189) of the Ras proteins were not present. CDC25 transformed NIH3T3 cells; its transforming capacity was enhanced by overexpression of wild-type Ha-ras. CDC25 C-domain probably exerts its effects through the activation of cellular Ras proteins. These data suggest that the CDC25 C-domain can function as an upstream activator of Ras proteins in a heterologous system and therefore could be a useful tool to study the regulation of Ras activation by growth factor receptors.

3T3 Cells↗
PubMed1993

Identification of the SH3 domain of GAP as an essential sequence for Ras-GAP-mediated signaling.

Guanosine triphosphatase activating protein (GAP) is an essential component of Ras signaling pathways. GAP functions in different cell types as a deactivator and a transmitter of cellular Ras signals. A domain (amino acids 275 to 351) encompassing the Src homology region 3 (SH3) of GAP was found to be essential for GAP signaling. A monoclonal antibody was used to block germinal vesicle breakdown (GVBD) induced by the oncogenic protein Ha-ras Lys12 in Xenopus oocytes. The monoclonal antibody, which was found to recognize the peptide containing amino acids 275 to 351 within the amino-terminal domain of GAP, did not modify the stimulation of the Ha-Ras-GTPase by GAP. Injection of peptides corresponding to amino acids 275 to 351 and 317 to 326 blocked GVBD induced by insulin or by Ha-Ras Lys12 but not that induced by progesterone. These findings confirm that GAP is an effector for Ras in Xenopus oocytes and that the SH3 domain is essential for signal transduction.

Amino Acid Sequence↗
PubMed1991

Short modified antisense oligonucleotides directed against Ha-ras point mutation induce selective cleavage of the mRNA and inhibit T24 cells proliferation.

We have used derivatized antisense oligodeoxynucleotides both in vitro and in vivo specifically to inhibit translation of the activated human oncogene Ha-ras. The oligonucleotides (5'-CCACACCGA-3') were targeted to a region of Ha-ras mRNA including the point mutation G----T at the 12th codon which leads to a Gly----Val substitution in the ras p21 protein. They were linked to an intercalating agent and/or to a hydrophobic tail, both to increase their affinity for their mRNA target and to enhance their uptake by tumor cells. A cell-free translation system was used to demonstrate an RNase H-dependent specific inhibition of activated ras protein synthesis. 50% inhibition was observed at a concentration of 0.5 microM of the most efficient oligonucleotide (5'-substitution with an acridine derivative and 3'-substitution by a dodecanol chain). This inhibitory effect stems from a point mutation-sensitive cleavage of the mRNA and it mirrors the growth inhibition obtained with T24 bladder carcinoma cells, which carry activated Ha-ras. The proliferation of HBL100 cells (non tumorigenic human mammary cell line) which carry two copies of normal Ha-ras was unaffected. This study shows that it is possible to design antisense agents that will inactivate the mutated oncogene but not the protooncogene which is generally essential to cell survival.

Animals↗
PubMed1990

Oncogenic proteins new targets for chemotherapeutic agents against cancer.

Over the past 10 years, more than 40 potentially oncogenic genes, termed protooncogenes, have been identified in the human genome. Little is known of the physiological role of the proteins encoded by these genes, but they seem to be involved in the reception and transmission of hormonal and other environmental information from the cell membrane to the nucleus. These proteins may acquire transforming properties when over-expressed or if structurally altered following partial deletions or point mutations. Cytogenetic analysis shows loss of genetic material from specific chromosomal loci in many human tumors, suggesting that the absence of a functional gene at these loci may permit tumor development. The genes involved have been termed "anti-oncogenes". Understanding the control mechanisms of cell proliferation is essential in order to understand how cancer cells escape from this control. To this end, numerous oncogenes have been cloned, permitting the production of modified forms of oncogenic proteins and identification of the regions essential for their biological activity. Availability of large amounts of protein also allows the production of specific antibody which can be used to verify whether blockage of a given protein results in reversion of the transformed phenotype. If it can be shown that the expression of an oncogenic protein is essential for transformation, it should be possible to search for molecules that inhibit its action or which mimic the effects of an anti-oncogene. This type of research is already well advanced for the oncogenic ras proteins, and models have been established that permit both screening for potential inhibitors and design of specific antagonists.

Animals↗