The RasGAP-associated endoribonuclease G3BP assembles stress granules

The NTF2-like and the RNA-binding domains of G3BP mediate its recruitment to SGs

G3BP shows a modular organization in four domains, which will be termed ABCD, going from the NH2- to the COOH terminus of the protein (Fig. 2 A). Domain A is an NTF2-like domain, possibly mediating protein–protein interactions (Bullock et al., 1996; Kent et al., 1996); domain B is highly acidic, and contains the serum-dependent phosphorylation site Ser 149 (Gallouzi et al., 1998; Tourrière et al., 2001); domain C can potentially bind the SH3 domain of Ras-GAP; and domain D is the RNA-binding domain, which contains a classical RRM and an arginine glycine-rich box. To understand how G3BP is recruited to SGs, we fused each of its domains to GFP and expressed them in Cos cells in order to analyze their localization after treatment with arsenite. In all cases, double labeling with an oligo-dT probe was included to positively identify SGs. Domains A and D, containing the NTF2-like and RNA-binding domains, respectively, were recruited to SGs, albeit less efficiently for domain A (Fig. 2, A and B). In contrast, domains B and C showed the same behavior as GFP alone (Fig. 2, A and B), and were not recruited to SGs.

SGs contain high concentrations of RNA. Thus, recruitment of domain D to SGs could simply be mediated by its affinity for RNA. To test this hypothesis, we analyzed the localization of other RNA-binding domains. We chose the RRMs of two Drosophila proteins involved in splicing, RSF1, and dmSF2 because these were unlikely to have a functional partner in the cytoplasm of monkey cells. The RRM of RSF1 is normally present in both the cytoplasm and the nucleus, whereas the two RRMs of dmSF2 are nuclear (Allemand et al., 2001, 2002). Despite this, both RNA-binding domains became concentrated in SGs after arsenite treatment (Fig. 2 A), suggesting their ability to bind RNA may be sufficient to concentrate them in SGs. It is striking that even RNA-binding domains that are nuclear can become concentrated in SGs. These data suggest that many RNA-binding proteins will be found within SGs, even those that have a nuclear localization. A transient localization of nuclear protein to the cytoplasm could be important to adjust the nuclear metabolism of RNA to conditions of stress, and has in fact been observed for hnRNP A1 and HuR (Kedersha et al., 2000; van der Houven van Oordt et al., 2000). Alternatively, the RRMs could localize to SGs simply due to the movement of mRNAs to which they are attached.

Domain A is homologous to the Ran binding domain of NTF2 (Bullock et al., 1996; Kent et al., 1996). Thus, it was possible that recruitment of this domain to SGs was mediated through Ran or another small GTPase. To test this hypothesis, we analyzed whether G3BP could coimmunoprecipitate with Ran, RhoA, Rac1, and also eIF2α, which is involved in SG formation. Despite the efficient immunoprecipitation of G3BP, no interactions could be detected with any of these proteins, either before or after addition of arsenite (unpublished data). NTF2-like domains are also known to mediate interaction with themselves (Bullock et al., 1996; Kent et al., 1996). Thus, domain A could be recruited to SGs by dimerizing with the endogenous G3BP. To test this possibility, we transfected wild-type G3BP-GFP or a mutant lacking domain A, immunoprecipitated extracts with an antibody against GFP, and analyzed the bound proteins with an antibody against G3BP. G3BP could interact with itself, as shown by the ability of G3BP-GFP to immunoprecipitate endogenous G3BP. In contrast, G3BP-GFP lacking domain A could not interact with wild-type G3BP, suggesting that it indeed mediates protein multimerization (see Fig. 5 A).

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Figure 5.

Phosphorylation of Ser 149 prevents G3BP self-association. (A) Whole-cell extracts prepared from Cos cells transfected with GFP, GFP-G3BP wild type, and phosphorylation mutants GFP-S149A, GFP-S149E, GFP-S232A, and GFP-S232E were immunoprecipitated with anti-GFP antibodies. Proteins were revealed by immunoblot analysis using anti-G3BP antibody. S, supernatant; IP, immunoprecipitation. (B) Glutaraldehyde cross-linking analysis of purified G3BP phosphorylation mutants S149A (lanes 1–6) and S149E (lanes 7–11). Proteins (0.2 μg each) were incubated for the indicated time with glutaraldehyde (G.A.), and were then analyzed by Western blotting with an anti-G3BP. Asterisks correspond to proteolytic fragments of G3BP-GFP that can be detected by anti-GFP antibodies, whereas the band at level of G3BP is only seen with antiG3BP antibody.

The acidic RasGAP binding domain of G3BP harbors an arsenite-regulated phosphorylation site and dominantly inhibits SG formation

Having determined the role of individual G3BP domains in SG recruitment, we went on to analyze the localization of various deletion mutants (Fig. 2 A, numbers in right panels). In cells subjected to arsenite stress, the CD mutant was recruited to SGs (in 86% of transfected cells). In contrast, the BC, ABC, and BCD forms of G3BP were diffusely distributed in the cytoplasm (Fig. 3 A). Most importantly, although poly(A)⁺ RNA and endogenous G3BP accumulated in SGs of untransfected cells, they remained diffusely distributed in cells transfected with BC, ABC, or BCD mutants (Fig. 3 A). This indicates that the BC domain is able to block the recruitment of poly(A)⁺ RNA to SGs, thereby acting as a transdominant inhibitor of SG assembly. Suppression of SG assembly by BC-containing mutants did not reflect overexpression of these mutants in transfected cells because SGs were absent from cells expressing low levels of these mutants (Fig. 3 A, 1–3). These results suggested that domain BC interacts with and eventually titrates a critical regulator of SG formation, and raised the possibility that G3BP itself was involved in SG assembly.

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Figure 3.

G3BP BC domain inhibits arsenite-induced assembly of SGs. (A) Cos cells were transiently transfected with GFP-BC, GFP-ABC, or GFP-BCD, treated with arsenite before fixation, and double stained with fluorescent oligo-dT probe (red). GFP, green. Arrows indicate transfected cells expressing GFP fusion. (B) Equal amounts of whole-cell extracts prepared from metabolically labeled CCL39 cells were immunoprecipitated with anti-G3BP antibody. Proteins were revealed by immunoblot analysis using the same antibody. S, supernatant; IP, immunoprecipitation. (C) Phosphotryptic peptide mapping of immunopurified ³²P-labeled G3BP, shown in A, from untreated (left) and arsenite-treated (right) CCL39 cells. The identification of phosphorylation sites Ser 149 and Ser 232 was previously reported (Tourrière et al., 2001). (D) The intensity of each spot from untreated (blue) and arsenite-treated (red) was quantitated by densitometry scanning of the chromatography plates using ImageQuant™ software version 5.2. Error bars resulting from two independently performed experiments are shown.

Domain C corresponds to the RasGAP-interacting domain, and domain B contains the conserved Ser 149 that is phosphorylated in a RasGAP-dependent manner. Thus, we asked whether phosphorylation of Ser 149 was affected in cells treated with arsenite. For this purpose, proteins were metabolically labeled with [³²P]orthophosphate, immunopurified with anti-G3BP antibody, and subjected to phosphotryptic mapping. To achieve efficient metabolic labeling of G3BP, CCL39 cells were used instead of Cos cells. Consistent with previous work (Gallouzi et al., 1998; Tourrière et al., 2001), three labeled phosphopeptides characterized the tryptic pattern of immunopurified G3BP (Fig. 3 C). Two of them correspond to phosphorylation at Ser 149 and Ser 232 (Tourrière et al., 2001). Although the same amount of G3BP was immunopurified from arsenite-treated and untreated cells (Fig. 3 B), phosphorylation at Ser 149 was twofold less in treated cells compared with untreated cells, whereas phosphorylation of Ser 232 was unchanged (Fig. 3 D). These results clearly establish that arsenite treatment induces specific dephosphorylation of G3BP at Ser 149.

Wild-type G3BP (but not a phosphomimetic S149E mutant) dominantly assembles SGs

The dominant-negative effect of G3BP BC domain on SG formation, and the dephosphorylation of Ser 149 after arsenite treatment prompted us to ask whether phosphorylation of Ser 149 affected the recruitment of G3BP to SGs. The GFP was fused in frame to the amino terminus of several phosphorylation mutants of G3BP, and the proteins were transiently expressed in Cos cells. In all cases, arsenite stress resulted in the accumulation of G3BP mutants in SGs, including S149E (Fig. 2 A and Fig. 4 B, panel 10) and S149A (Fig. 2 A).

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Figure 4.

G3BP assembles SGs. (A) Untreated Cos cells transfected with GFP or G3BP-GFP (green) were counterstained with either fluorescent oligo-dT (2 and 5, red) or anti-HuR antibody (8, red). Untreated HeLa cells transfected with G3BP-GFP (10, green) were counterstained with anti-TIA1/R antibody (11, red). (B) GFP-S149E does not assemble SGs. Top, schematic representation of G3BP with the positions of the major phosphorylation sites. Bottom, localization of G3BP phosphorylation mutants in Cos cells. GFP fluorescence (green) of GFP-S149A, GFP-S149E, and GFP-S232 was acquired 20 h after transfection on cells fixed and counterstained with fluorescent oligo-dT to reveal SGs (red). Cells in 10–12 were transfected with GFP-S149E, and were treated with arsenite before fixation. Arrows indicate transfected cells expressing GFP fusions.

During the course of these experiments, we noticed that cells transfected with G3BP-GFP concentrated the protein in aggregates resembling SGs, even in absence of any arsenite treatment (Fig. 4 A, panel 4). Furthermore, G3BP aggregates were enriched in poly(A)⁺ RNA (Fig. 4 A, 5 and 6) and also contained HuR (Fig. 4 A, 8 and 9), and thus likely corresponded to bona fide SGs. Induction of SG assembly was not due to the transfection itself because GFP alone (Fig. 4 A, 1–3) induced formation of SGs to much lower levels (93% of the transfected cells for G3BP versus 28% for GFP, Fig. 2 A). Furthermore, this effect is specific for G3BP because truncation mutants, or RRMs of unrelated proteins, were unable to assemble SGs, although G3BP domain D had a weak effect (Fig. 2 A). However, not all G3BP-induced SGs colocalized with TIA1/R in HeLa cells, implying that the assembly of a subset of these SGs did not require TIA1/R (Fig. 4 A, 10–12). Remarkably, although all other phosphorylation mutants induced SGs as efficiently as wild-type G3BP (Fig. 2 A and Fig. 4 B, 1–3 and 7–9), the phosphomimetic S149E mutant did not induce SGs at all (Fig. 2 A and Fig. 4 B, 4–6).

Together, the data indicate that overexpression of G3BP can dominantly induce SG assembly, and that this requires dephosphorylation of Ser 149. This data fully support the proposal that G3BP is a regulated effector of SG assembly.

Phosphorylation of G3BP at Ser 149 prevents its oligomerization

Ser 149 is 20 amino acids COOH-terminal to the NTF2-like domain, which plays a key role in mediating protein–protein interactions (see previous paragraph). Thus, we tested the capacity of G3BP to form multimers and determined the effect of phosphorylation on multimerization. Extracts prepared from cells expressing GFP-G3BP or its phosphorylation mutants were subjected to immunoprecipitation with anti-GFP antibody, and G3BP was revealed by Western blot using monoclonal anti-G3BP antibody. Fig. 5 A clearly established that endogeneous G3BP was efficiently coimmunoprecipitated with GFP-G3BP (lane 4), GFP-S149A (lane 6), GFP-S332A (lane 10), and GFP-S232E (lane 12), but not GFP-S149E (lane 8). The lack of association could be due to either preferential binding of S149E with different partners, or to phosphorylation-mediated changes of the protein conformation incompatible with G3BP self-association. To distinguish between these possibilities, recombinant G3BP S149A and S149E phosphorylation mutants were purified from baculovirus-infected insect cells, and subjected to glutaraldehyde cross-linking. Untreated and cross-linked proteins were analyzed by Western blot (Fig. 5 B). Untreated samples revealed only monomeric S149A and S149E proteins (lanes 1 and 7, respectively). In contrast, glutaraldehyde cross-linked samples of S149A contained species of molecular masses corresponding to monomers, dimers, trimers, and tetramers (lanes 2–6), which accumulated with incubation time. However, with the S149E mutant, no homo-oligomers were detected (lanes 8–11). In this case, disappearance of the monomers with time may be due to the inactivation of the epitope as a result of glutaraldehyde treatment. The propensity of the S149A (but not S149E) mutant to form homo-oligomers provides direct evidences that phosphorylation at Ser 149 induces a conformational change that prevents G3BP self-association.

Cell culture, immunoprecipitations, and phosphopeptide analysis

Cos-7 cells were grown in DME containing 10% FCS, and were transfected by calcium phosphate coprecipitation as described previously (Samarsky et al., 1998). HeLa cells were grown in RPMI 1640 (GIBCO BRL) supplemented with 10% FCS. Chinese hamster lung fibroblasts (CCL39) and CCL39 derivative Ha-Ras were provided by J. Pouysségur (Université de Nice, Nice, France) and were maintained in DME supplemented with 10% FCS and antibiotics (50 U/ml penicillin and 50 μg/ml streptomycin) at 37°C in 5% CO₂/95% air (Seuwen et al., 1988). For arsenite-induced stress, cells were exposed to 0.5 mM sodium arsenite for 1 h, except as otherwise stated. Total extracts were prepared according to Gallouzi et al. (1998), and G3BP immunoprecipitations were performed with anti-G3BP antibodies (1F1; Gallouzi et al., 1998), or with anti-GFP antibodies (Roche).

Proteins were analyzed by Western blotting using dilutions of primary antibodies as follows: 1/3,000 for anti-G3BP (200 ng/ml), 1/3,000 for anti-HuR (3A2), 1/1000 for anti-Ran (Santa Cruz Biotechnology, Inc.), 1/1,000 for anti-eIf2α (Santa Cruz Biotechnology, Inc.), 1/200 for anti-Rac, and 1/200 for anti-RhoA. Anti-HuR antibody was a gift from Drs. I. Gallouzi and J. Steitz (Yale University, New Haven, CT). Anti-Rac and anti-RhoA antibodies were provided by P. Fort (Centre de Recherches de Biochimie Macromoléculaire, Montpellier, France). ³²P-labeling and phosphopeptide mapping were performed according to Gallouzi et al. (1998).

Immunofluorescence and in situ hybridization

Transfected Cos or HeLa cells were grown on coverslips and treated with 0.5 mM sodium arsenite 48 h after transfection. Cells were washed twice with PBS, fixed for 15 min at RT with 4% PFA, and permeabilized overnight in 70% ethanol/PBS. In situ hybridization was done with fluorescent oligo-dT probe synthesized and labeled with Cy3 as described previously (Samarsky et al., 1998). Immunofluorescence was done as described previously (1;2) with the following dilutions of primary antibody: 1/100 for anti-G3BP (1F1), and 1/300 for anti-HuR (3A2). For anti-TIA1/R (3E6) antibody, immunofluorescence was performed as described previously (Kedersha et al., 1999). Anti-TIA1/R antibody was a gift from Drs. N. Kedersha and P. Anderson (Brigham and Women's Hospital Hospital, Boston, MA).

We thank J. Derancourt for phosphopeptide analyses, E. Prieto for her help with the analysis of GFP mutants, and R. Hipskind for his advice and critical reading of the manuscript. A special thanks to I. Gallouzi and J. Steitz for providing anti-HuR antibodies. We are also grateful to N. Kedersha and P. Anderson for providing anti-TIA1/R antibody.

The work was supported by grants from l'Association pour la Recherche sur la Cancer (ARC) and the CNRS. H. Tourrière was supported by a graduate fellowship from the Ministère de L'Éducation Nationale, de la Recherche et de la Technologie and benefited from graduate training fellowships from the ARC and Foundation par le Recherche Médicale.