Stress granules (SGs) are formed in the cytoplasm in response to various toxic agents, and are believed to play a critical role in the regulation of mRNA metabolism during stress. In SGs, mRNAs are stored in an abortive translation initiation complex that can be routed to either translation initiation or degradation. Here, we show that G3BP, a phosphorylation-dependent endoribonuclease that interacts with RasGAP, is recruited to SGs in cells exposed to arsenite. G3BP may thus determine the fate of mRNAs during cellular stress. Remarkably, SG assembly can be either dominantly induced by G3BP overexpression, or on the contrary, inhibited by expressing a central domain of G3BP. This region binds RasGAP and contains serine 149, whose dephosphorylation is induced by arsenite treatment. Critically, a phosphomimetic mutant (S149E) fails to oligomerize and to assemble SGs, whereas a nonphosphorylatable G3BP mutant (S149A) does both. These results suggest that G3BP is an effector of SG assembly, and that Ras signaling contributes to this process by regulating G3BP dephosphorylation.

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...

Mitogen activation of mRNA decay pathways likely involves specific endoribonucleases, such as G3BP, a phosphorylation-dependent endoribonuclease that associates with RasGAP in dividing but not quiescent cells. G3BP exclusively cleaves between cytosine and adenine (CA) after a specific interaction with RNA through the carboxyl-terminal RRM-type RNA binding motif. Accordingly, G3BP is tightly associated with a subset of poly(A)؉ mRNAs containing its high-affinity binding sequence, such as the c-myc mRNA in mouse embryonic fibroblasts. Interestingly, c-myc mRNA decay is delayed in RasGAP-deficient fibroblasts, which contain a defective isoform of G3BP that is not phosphorylated at serine 149. A G3BP mutant in which this serine is changed to alanine remains exclusively cytoplasmic, whereas a glutamate for serine substitution that mimics the charge of a phosphorylated serine is translocated to the nucleus. Thus, a growth factor-induced change in mRNA decay may be modulated by the nuclear localization of a site-specific endoribonuclease such as G3BP.