Characterization of G3BPs: tissue specific expression, chromosomal localisation and rasGAP(120) binding studies.

The G3BP (ras-GTPase-Activating Protein SH3-Domain-Binding Protein) family of proteins has been implicated in both signal transduction and RNA-metabolism. We have previously identified human G3BP-1, G3BP-2, and mouse G3BP-2. Here, we report the cloning of mouse G3BP-1, the discovery of two alternatively spliced isoforms of mouse, and human G3BP-2 (G3BP-2a and G3BP-2b), and the chromosomal localisation of human G3BP-1 and G3BP-2, which map to 5q14.2-5q33.3 and 4q12-4q24 respectively. We mapped the rasGAP(120) interactive region of the G3BP-2 isoforms and show that both G3BP-2a and G3BP-2b use an N-terminal NTF2-like domain for rasGAP(120) binding rather than several available proline-rich (PxxP) motifs found in members of the G3BPs. Furthermore, we have characterized the protein expression of both G3BP-1 and G3BP-2a/b in adult mouse tissues, and show them to be both tissue and isoform specific.

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.

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.

Grb3-3 is up-regulated in HIV-1-infected T-cells and can potentiate cell activation through NFATc.

The MAPK pathway is required for T-cell activation; however, its role in modulating T-cell function following human immunodeficiency virus type 1 (HIV-1) infection is poorly understood. In this report, we investigated whether Grb3-3, an isoform of the Grb2 (growth factor receptor-bound protein-2) adaptor molecule that is associated with the MAPK pathway, could be involved. We found that Grb3-3, but not its isoform Grb2, is markedly up-regulated in CD4(+) peripheral blood mononuclear cells derived from either in vitro HIV-1-infected cultures or HIV-1-infected human subjects. Analysis of HIV-1 gene products indicated that Tat and Nef, both of which have been implicated in modulating T-cell function, can independently induce expression of Grb3-3. By using NFAT/AP-1, AP-1, or NFAT reporter assays, we found that Grb3-3 can potentiate NFAT (but not AP-1) promoter activity in Jurkat T-cells upon engagement of the T-cell receptor and CD28 co-receptor. In addition, potentiation of NFAT by Grb3-3 is substantially suppressed by MEKK1, a kinase that may play an important role in retaining NFAT in the cytoplasm, and by cyclosporin A. Finally, we also found that Grb3-3 potentiates HIV-1 long terminal (LTR) repeat promoter activity following T-cell receptor stimulation, an effect that can be largely suppressed by cyclosporin A. Taken together, this study indicates that Grb3-3 is a cellular factor that can be up-regulated by HIV-1. In addition, Grb3-3 can also function as a positive factor for T-cell activation and, in doing so, may aid in establishing an intracellular environment that can optimally support HIV-1 replication.

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.

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.

Restoration of transcriptional activity of p53 mutants in human tumour cells by intracellular expression of anti-p53 single chain Fv fragments.

We report here the production and the properties of single chain Fv fragments (scFvs) derived from the anti-p53 monoclonal antibodies PAb421 and 11D3. 11D3 is a newly generated monoclonal antibody which exhibits properties very comparable to those of PAb421. The scFvs PAb421 and 11D3 are able to stably associate with p53 and to restore the DNA binding activity of some p53 mutants in vitro. When expressed in p53 -/-human tumour cells, the scFv421 is essentially localized in the cytoplasm in the absence of p53, and in the nucleus when exogenous p53 is present. Thus, p53 is also able to stably associate with an anti-p53 scFv in cells. Cotransfection of p53 -/- human tumour cells with expression vectors encoding the His273 p53 mutant and either scFv leads to restoration of the p53 mutant deficient transcriptional activity. These data demonstrate that, in human tumour cells, these scFvs are able to restore a function essential for the tumour suppressor activity of p53 and may represent a novel class of molecules for p53-based cancer therapy.

A tumor specific single chain antibody dependent gene expression system.

The design of conditional gene expression systems restricted to given tissues or cellular types is an important issue of gene therapy. Systems based on the targeting of molecules characteristic of the pathological state of tissues would be of interest. We have developed a synthetic transcription factor by fusing a single chain antibody (scFv) directed against p53 with the bacterial tetracycline repressor as a DNA binding domain. This hybrid protein binds to p53 and can interact with a synthetic promoter containing tetracycline-operator sequences. Gene expression can now be specifically achieved in tumor cells harboring an endogenous mutant p53 but not in a wild-type p53 containing tumor cell line or in a non-transformed cell line. Thus, a functional transactivator centered on single chain antibodies can be expressed intracellularly and induce gene expression in a scFv-mediated specific manner. This novel class of transcriptional transactivators could be referred as 'trabodies' for transcription-activating-antibodies. The trabodies technology could be useful to any cell type in which a disease related protein could be the target of specific antibodies.

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.

Ras-GAP controls Rho-mediated cytoskeletal reorganization through its SH3 domain.

Proteins of the Ras superfamily, Ras, Rac, Rho, and Cdc42, control the remodelling of the cortical actin cytoskeleton following growth factor stimulation. A major regulator of Ras, Ras-GAP, contains several structural motifs, including an SH3 domain and two SH2 domains, and there is evidence that they harbor a signalling function. We have previously described a monoclonal antibody to the SH3 domain of Ras-GAP which blocks Ras signalling in Xenopus oocytes. We now show that microinjection of this antibody into Swiss 3T3 cells prevents the formation of actin stress fibers stimulated by growth factors or activated Ras, but not membrane ruffling. This inhibition is bypassed by coinjection of activated Rho, suggesting that the Ras-GAP SH3 domain is necessary for endogenous Rho activation. In agreement, the antibody blocks lysophosphatidic acid-induced neurite retraction in differentiated PC12 cells. Furthermore, we demonstrate that microinjection of full-length Ras-GAP triggers stress fiber polymerization in fibroblasts in an SH3-dependent manner, strongly suggesting an effector function besides its role as a Ras downregulator. These results support the idea that Ras-GAP connects the Ras and Rho pathways and, therefore, regulates the actin cytoskeleton through a mechanism which probably does not involve p190 Rho-GAP.

A novel phosphorylation-dependent RNase activity of GAP-SH3 binding protein: a potential link between signal transduction and RNA stability.

A potential p120 GTPase-activating protein (RasGAP) effector, G3BP (RasGAP Src homology 3 [SH3] binding protein), was previously identified based on its ability to bind the SH3 domain of RasGAP. Here we show that G3BP colocalizes and physically interacts with RasGAP at the plasma membrane of serum-stimulated but not quiescent Chinese hamster lung fibroblasts. In quiescent cells, G3BP was hyperphosphorylated on serine residues, and this modification was essential for its activity. Indeed, G3BP harbors a phosphorylation-dependent RNase activity which specifically cleaves the 3'-untranslated region of human c-myc mRNA. The endoribonuclease activity of G3BP can initiate mRNA degradation and therefore represents a link between a RasGAP-mediated signaling pathway and RNA turnover.

hUBC9 associates with MEKK1 and type I TNF-alpha receptor and stimulates NFkappaB activity.

hUBC9, an E2 ubiquitin conjugating enzyme, was identified by yeast two-hybrid screening and coprecipitation studies to interact with MEKK1 and the type I TNF-alpha receptor, respectively. Because both of these proteins regulate NFkappaB activity, the role of hUBC9 in modulating NFkappaB activity was investigated. Overexpression of hUBC9 in HeLa cells stimulated the activity of NFkappaB as determined by NFkappaB reporter and IL-6 secretion assays. hUBC9 also synergized with MEKK1 to activate NFkappaB reporter activity. Thus, hUBC9 modulates NFkappaB activity which, at least in part, can be attributed to its interaction with MEKK1 and the type I TNF-alpha receptor.

Sam68 is a Ras-GAP-associated protein in mitosis.

Sam68 is the major tyrosine-phosphorylated and Src-associated protein in mitotic cells. Sam68 stimulates G1/S transition and this effect is dependent on the integrity of its KH domain (hnRNPK Homology) which confers nucleic acid binding properties. During mitosis, Sam68 undergoes tyrosine phosphorylation, which negatively regulates its nucleic acid binding properties and mediates the interaction of Sam68 with critical SH2-containing signaling proteins such as Grb2, PLC gamma 1 and Ras-GAP. However, the interaction of Ras-GAP with Sam68 has been brought into question, based on the lack of co-immunoprecipitation between Sam68 and Ras-GAP in interphase cells. Here we show that the choice of anti-Ras-GAP antibodies is critical for the detection of Ras-GAP/Sam68 complex formation, and that this interaction is specific for G2/M transition in both NIH3T3 and Src-transformed cells. Such data reinforce the importance of the interaction of Ras-GAP with RNA binding proteins during cell proliferation through its SH2 and SH3 domains.

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.

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.

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.

CTS1: a p53-derived chimeric tumor suppressor gene with enhanced in vitro apoptotic properties.

The clinical potential of the p53 tumor suppressor gene is being evaluated currently for gene therapy of cancer. We have built a variant of wild-type p53, chimeric tumor suppressor 1 (CTS1), in which we have replaced the domains that mediate its inactivation. CTS1 presents some very interesting properties: (a) enhanced transcriptional activity; (b) resistance to the inactivation by oncogenic forms of p53; (c) resistance to the inactivation by MDM2; (d) lower sensitivity to E6-induced degradation; (e) ability to suppress cell growth; and (f ) faster induction of apoptosis. Thus, CTS1 is an improved tumor suppressor and an alternative for the treatment of wild-type p53-resistant human tumors by gene therapy.

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.

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.