Fabienne Parker

A Ras-GTPase-activating protein SH3-domain-binding protein

We report the purification of a Ras-GTPase-activating protein (GAP)-binding protein, G3BP, a ubiquitously expressed cytosolic 68-kDa protein that coimmunoprecipitates with GAP. G3BP physically associates with the SH3 domain of GAP, which previously had been shown to be essential for Ras signaling. The G3BP cDNA revealed that G3BP is a novel 466-amino-acid protein that shares several features with heterogeneous nuclear RNA-binding proteins, including ribonucleoprotein (RNP) motifs RNP1 and RNP2, an RG-rich domain, and acidic sequences. Recombinant G3BP binds effectively to the GAP SH3 domain G3BP coimmunoprecipitates with GAP only when cells are in a proliferating state, suggesting a recruitment of a GAP-G3BP complex when Ras is in its activated conformation.

A Novel Phosphorylation-Dependent RNase Activity of GAP-SH3 Binding Protein: a Potential Link between Signal Transduction and RNA Stability

ABSTRACT 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.

Ras-GTPase Activating Protein (GAP): A Putative Effector for Ras

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.

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.

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.

Upregulation of the RAS-GTPase activating protein (GAP)-binding protein (G3BP) in proliferating RPE cells.

Cultured human retinal pigment epithelial (RPE) cells of different passages (P0 and P3) were used as a model system to examine changes in gene expression in proliferating RPE cells by polymerase chain reaction (PCR)‐based differential expressed mRNA analysis (DEmRNA‐PCR). DEmRNA‐PCR showed enhanced expression of a specific RNA in P3 compared with P0. Sequence alignment displayed its identity with the 3′‐end of the coding sequence of the human RAS‐GTPase activating protein (GAP)‐binding protein (G3BP). Confirmation of the induced expression of G3BP was performed by gene‐specific reverse transcription‐polymerase chain reaction (RT‐PCR) of freshly prepared human RPE cells and of cultured cells of P0, P3 and P8 and by immunohistochemistry of cultivated retinal pigment epithelial cells in an artificial lesion assay. The expression of G3BP mRNA increased with the number of passages. G3BP protein expression increased in cells repopulating the artificial lesion. DEmRNA‐PCR in RPE cells with subsequent sequence analysis led to the characterization of dedifferentiation‐ and proliferation‐dependent expression of a previously undetected gene product in cultured RPE cells with a possible role in modifying signal transduction responses that may have implications on the treatment of proliferative vitreoretinopathy. J. Cell. Biochem. 74:194–201, 1999.

Local constrained shifty pseudopeptides inhibitors of rasfarnesyl transferase

Abstract Pseudopeptide analogues related to the C-terminal tetrapeptide of ras-protein (Cys-Val-X-Met) were synthesized and evaluated for inhibition of ras farnesyl transferase (FTase). We demonstrate that the introduction of a shifty amino acid related to Cys instead of Cys-Val and a tetrahydroisoquinoline carboxylic acid (TIC) instead of Phe lead to potent inhibitors of FTase on isolated enzyme or on cell based tests. One of the pseudopeptides, conceived as a prodrug, suppressed specifically the ability of ras transformed cells to form colonies in soft agar.