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 serumstimulated 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.The Ras protein belongs to a family of low-molecular-weight GTPases which are essential components of multiple receptormediated signal transduction pathways controlling cell proliferation, differentiation, and cytoskeletal organization (23). Activated Ras is bound to GTP, while the GDP-bound form of Ras is inactive (27). Extracellular stimuli induce the exchange of GDP for GTP on Ras through a series of protein-protein interactions involving activated receptors, adaptor proteins (such as Grb2 or Shc), and Ras guanine nucleotide exchange factors (5,9,33,38). Mutations in the Ras gene which lock Ras in the GTP-bound form lead to cell growth in the absence of mitogenic signals and are associated with an oncogenic phenotype (17). Physiological inactivation of Ras involves interaction with GTPase-activating proteins (GAPs) (40), such as p120 (RasGAP) (41,43) or the product of the NF1 gene (neurofibromin) (26,44), which accelerate the hydrolysis of Ras-associated GTP, thereby converting Ras from an active to an inactive form. Disruption of either the RasGAP or the NF1 gene in mice results in an embryonic lethal phenotype (3, 14), indicating that Ras inactivation is a key process in normal cell signaling and development.In addition to being a negative regulator of Ras, RasGAP may also represent a downstream target of Ras (35). RasGAP is a widely expressed modular protein which comprises several structural features that likely enable it to function in the transduction cascade (29). While the carboxyl-terminal domain of RasGAP constitutes a catalytic domain (25), the N-terminal region is believed to mediate interactions with other signaling proteins (20). The N-terminal region is characterized by a Src homology 3 (SH3) domain flanked by two SH2 domains, as well as pleckstrin homology (PH) and calcium-dependent lipid binding domains (4, 34). Upon activation of many growth factor receptors, RasGAP becomes phosphorylated and associates with cytosolic proteins as well as with the autophosphorylated tyrosine kinase receptors (19). RasGAP has been shown to form a complex with G3BP (RasGAP SH3 binding protein) in a Ras-GTP-dependent manner (32). G3BP is composed of 466 amino acid and has a predicted molecular mass of 52 kDa; the carboxyl-terminal region contai...

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.

Alternative initiation of translation of the human fibroblast growth factor 2 (FGF-2) mRNA at five in-frame CUG or AUG translation initiation codons requires various RNA cis-acting elements, including an internal ribosome entry site (IRES). Here we describe the purification of a trans-acting factor controlling FGF-2 mRNA translation achieved by several biochemical purification approaches. We have identified the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) as a factor that binds to the FGF-2 5 -leader RNA and that also complements defective FGF-2 translation in vitro in rabbit reticulocyte lysate. Recombinant hnRNP A1 stimulates in vitro translation at the four IRES-dependent initiation codons but has no effect on the cap-dependent initiation codon. Consistent with a role of hnRNP A1 in the control of alternative initiation of translation, short interfering RNA-mediated knock down of hnRNP A1 specifically inhibits translation at the four IRESdependent initiation codons. Furthermore, hnRNP A1 binds to the FGF-2 IRES, implicating this interaction in the control of alternative initiation of translation.In eukaryotes, alternative translation initiation at several start codons is one of the processes by which a single mRNA gives rise to multiple proteins, and this contributes to the generation of protein diversity (1). According to the ribosomescanning model of translation, protein synthesis in eukaryotes involves the 40 S ribosomal subunit recruitment to the 5Ј-end cap structure of the mRNA, followed by its ATP-dependent linear scanning until an initiation codon in a good sequence context is encountered (2). In mRNAs containing several alternative translation initiation codons, it is proposed that the cap-proximal initiation codon is used inefficiently because of its usual weak sequence context and that some of the 40 S ribosomal subunits read through the site without recognizing it by a so-called "leaky scanning" process to initiate translation at the downstream position (3). However, this mechanism cannot occur when the RNA structure between the two (or more) initiation codons is stable and cannot be easily unwound by the eIF4A RNA helicase. Moreover, recent data demonstrate that 40% of mRNAs contain at least one upstream AUG that is in a similar or more favorable context than the actual initiator (4). Thus, the leaky scanning mechanism cannot explain the selection of internal codons in all mRNAs.The human FGF-2 1 mRNA provides a very interesting system to understand how internal codons are selected in a highly structured 5Ј-leader. Indeed, the control of alternative initiation of translation is a crucial aspect of the regulation of the human fibroblast growth factor 2 (FGF-2) gene (1). The FGF-2 protein isoforms belong to a family of 23 structurally related FGF polypeptides that play key roles in morphogenesis, development, angiogenesis, and wound healing (5). The FGF-2 mRNA contains five in-frame translation initiation codons (4 CUGs and 1 AUG) that give rise to five FGF-2 isoforms with different amino-t...

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.

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