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 rasGAP120 interactive region of the G3BP‐2 isoforms and show that both G3BP‐2a and G3BP‐2b use an N‐terminal NTF2‐like domain for rasGAP120 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. J. Cell. Biochem. 84: 173–187, 2002. © 2001 Wiley‐Liss, Inc.

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.

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.

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.

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.

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.

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.

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.

ABSTRACT 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.

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.

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.

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.

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.

Sam68 is the main tyrosine-phosphorylated and Src-associated protein in mitotic cells. Sam68 exhibits a conserved functional KH (hnRNPK homology) RNA binding domain and binds single strand nucleic acids. Tyrosine phosphorylation mediates the interaction of Sam68 with many SH3- and SH2-containing proteins and negatively regulates its nucleic acid binding properties. But the function and the impact of Sam68 on cell signaling and cell proliferation remains elusive. We report here the identification of a natural isoform of Sam68 with a deletion within the KH domain. This isoform, called Sam68 Delta KH, is specifically expressed at growth arrest upon confluency in normal cells. In cells that do not enter quiescence at confluency such as Src-transformed cells, no recruitment of Sam68 Delta KH is observed. Transfected Sam68 Delta KH inhibits serum-induced DNA synthesis and cyclin D1 expression. Sam68 overcomes these effects, suggesting that isoforms of Sam68 are involved, through KH domain signaling, in cell proliferation, and more precisely in G(1)/S transition.

One attractive candidate for a Ras effector protein, other than the Raf kinases, is Ras-GAP. Indeed, recent literature suggests that besides the Raf/MAP kinase cascade, additional pathways must be stimulated to elicit a full biological response to Ras. Ras binds the COOH terminal domain of Ras-GAP, while the NH2 terminal domain appears to be essential for triggering downstream signals. Since Ras-GAP itself has no obvious enzymatic function that might explain a role in processes associated with proliferation, differentiation or apoptosis, candidates for downstream Ras-GAP effectors that fulfill this role remain to be identified. The newly found GAP-SH3 domain Binding Protein (G3BP) may be one of these. This review will briefly overview the candidates Ras effectors and discuss the results that position Ras-GAP as a critical effector downstream of Ras.

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 proline-rich 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 confirmedin vitrowith GST fusion proteins andin vivoby coimmunoprecipitation experiments in NIH3T3 cells stably transfected with Grb3-3. The functional significance of this finding is discussed.

In order to design new potent inhibitors of the epidermal growth factor receptor (EGF-R) associated protein tyrosine kinase (PTK) activity as antitumor agents, several families of phenylhydroquinone derivatives were synthesized. Some of these compounds were shown to block PTK activity in vitro, but also efficiently to inhibit EGF-stimulated DNA synthesis in ER 22 cells (CCL 39 hamster fibroblasts transfected with EGF-R) with IC50 values in the range 1-10 microM. In some cases, a correlation between the two sets of data was observed, allowing structure-activity relationships to be established. However, inhibitors which had in vitro specificity with regard to other kinases were not specific in the cellular test. Similar effects on DNA synthesis were observed after stimulation by various activating agents, suggesting that our compounds may also act against other cellular targets involved in the EGF-dependent pathways leading to cell proliferation.

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