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. Author manuscript; available in PMC: 2013 Aug 23.
Published in final edited form as: Mol Cell. 2011 Apr 8;42(1):50–61. doi: 10.1016/j.molcel.2011.03.017

Rac1 Regulates the Activity of mTORC1 and mTORC2 and Controls Cellular Size

Abdelhafid Saci 1,*, Lewis C Cantley 1,*, Christopher L Carpenter 1,2
PMCID: PMC3750737  NIHMSID: NIHMS386684  PMID: 21474067

SUMMARY

Mammalian target of rapamycin (mTOR) is a serine/threonine kinase that exists in two separate complexes, mTORC1 and mTORC2, that function to control cell size and growth in response to growth factors, nutrients, and cellular energy levels. Low molecular weight GTP-binding proteins of the Rheb and Rag families are key regulators of the mTORC1 complex, but regulation of mTORC2 is poorly understood. Here, we report that Rac1, a member of the Rho family of GTPases, is a critical regulator of both mTORC1 and mTORC2 in response to growth-factor stimulation. Deletion of Rac1 in primary cells using an inducible-Cre/Lox approach inhibits basal and growth-factor activation of both mTORC1 and mTORC2. Rac1 appears to bind directly to mTOR and to mediate mTORC1 and mTORC2 localization at specific membranes. Binding of Rac1 to mTOR does not depend on the GTP-bound state of Rac1, but on the integrity of its C-terminal domain. This function of Rac1 provides a means to regulate mTORC1 and mTORC2 simultaneously.

INTRODUCTION

Mammalian target of rapamycin (mTOR) is a member of the phosphoinositide-related kinases. mTOR is part of 2 complexes, mTORC1 and mTORC2, and mediates cellular responses to a number of signals, including growth-factor stimulation, energy state, and oxygen and amino acid concentrations (reviewed by Wullschleger et al., 2006). In addition to mTOR, mTORC1 contains Raptor, mLST8, PRAS40, and Deptor. The primary function of mTORC1 is to integrate cellular signals to balance anabolism and catabolism. The best-studied substrates of mTORC1 are p70 S6 kinase and 4eBP1. Many, but not all, mTORC1 functions are inhibited by rapamycin. mTORC2 contains mTOR, mLST8, Deptor, and the unique components Rictor, mSIN1, and PRR5. The primary function of mTORC2 appears to be phosphorylation and activation of the kinases Akt (Sarbassov et al., 2005) and SGK (García-Martínez and Alessi, 2008). mTORC2 is not directly inhibited by rapamycin, but mTORC2 levels decline following prolonged cellular exposure to rapamycin (reviewed by Foster and Fingar, 2010).

mTORC1 is regulated primarily by the Rheb GTPase and by phosphorylation of mTORC1 components. The pathways regulating Rheb and phosphorylation of mTORC1 are complex, and many of them are not well understood. Rheb must be in the GTP-bound state to activate mTORC1. GTP binding of Rheb is regulated by TSC2 (tuberin), a Rheb GTPase-activating protein (GAP) (reviewed by Avruch et al., 2006). In response to growth factors, phosphorylation of TSC2 by AKT, ERK, or p90 RSK1 inhibits TSC2’s GAP activity, allowing Rheb to accumulate in the GTP-bound state and activate mTORC1 (reviewed by Tee and Blenis, 2005). Inhibition of TSC2 by Akt allows mTORC1 to be activated downstream of mTORC2. Activation of mTORC1 in response to amino acids requires additional low-molecular GTP-binding proteins in the Rag family (Sancak et al., 2008). A heterodimeric complex of Rag proteins (RagA-RagC or RagB-RagD) must be properly charged with GTP and GDP, respectively, to recruit mTORC1 to membranes, particularly lysosomes, and colocalize with GTP-bound Rheb (Sancak et al., 2008).

The mechanisms of mTORC2 activation are not known. In addition to Akt activation, mTORC2 can regulate cytoskeletal changes through the Rho family GTPases (Jacinto et al., 2004). P-Rex1 is the Rac1 guanine nucleotide exchange factor (GEF) necessary for mTOR-dependent cell migration (Hernández-Negrete et al., 2007). TSC1- and TSC2-dependent cell motility and adhesion require Rac1 (Goncharova et al., 2004). Many pathways utilize positive- and negative-feedback loops to optimize signal strength. Regulation of mTORC2 by Rho family G proteins would provide this opportunity.

Rho family GTPases regulate a wide variety of cell functions including gene transcription, proliferation, apoptosis, motility, and redox signaling (reviewed by Burridge and Wennerberg, 2004; Bustelo et al., 2007). The Rac subfamily contains Rac1, Rac2, and Rac3. Rac1 expression is ubiquitous, Rac2 is hematopoietic specific, and Rac3 is restricted primarily to neural tissue (reviewed by Wennerberg and Der, 2004). Rac1-null mice are embryonic lethal due to the absence of formation of three germ layers during gastrulation (Sugihara et al., 1998). Rac3-null mice are viable and have no obvious phenotype (Cho et al., 2005; Corbetta et al., 2005). Rac2-null mice are also viable, but have some defects in T and B cell function, gene expression in mast cells, and homing of hematopoietic progenitors (Croker et al., 2002; Gu et al., 2002; Carstanjen et al., 2005). Deletion of either Rac1 or Rac2 alone has modest effects on B and T cell development and function, but the deletion of both GTPases in B and T cells results in profound defects in cell development, proliferation, and survival, indicating that Rac1 and Rac2 have redundant functions in B and T cells (Walmsley et al., 2003; Guo et al., 2008). Rac1 and Rac2 also have distinct functions in some hematopoietic cells, such as the hematopoietic stem cell progenitors (HSC) where Rac1 is important for proliferation and entry into cell cycle, while Rac2 is important for adhesion, spreading, cytoskeletal changes, and AKT-dependent survival (Gu et al., 2003; Benvenuti et al., 2004; Cancelas et al., 2005).

Here, we present evidence that Rac1binds directlyto mTORand is a critical regulator of both mTORC1 and mTORC2. Deletion of Rac1 in MEFs, as well as in B and T cells, results in a decrease in cell size. Rac1 deletion inhibits activation of downstream targets of mTORC1 (4eBP1 and p70 S6 kinase) and mTORC2 (Akt). Rac1 colocalizes with mTORC1 and mTORC2 at specific membranes of activated MEFs and HeLa cells, and the deletion of Rac1 prevents membrane localization of mTOR. Rac1 interaction with mTORC1 and mTORC2 does not require that Rac1 be bound to GTP, but the C terminus of Rac1 binds to mTOR. These results suggest a model in which Rac1 regulates mTOR activity by facilitating the localization of both mTORC1 and mTORC2 in response to growth factors in proximity to their substrates and other activators, such as Rheb in the case of mTORC1.

RESULTS

Rac1 Deletion Reduces Cellular Size

In order to study the role of Rac1 in cell signaling, we crossed Rac1 fl/fl mice to mice carrying a Cre gene driven by the hydroxy-tamoxifen promoter (4-HT)-Cre to generate mice in which Rac1 could be conditionally deleted by 4-HT. Primary mouse embryonic fibroblasts (MEFs) were isolated from these mice, grown in 10% serum, then treated with 500 nM 4-HT for at least 48 hr to delete Rac1. Rac1 deletion in fl/fl MEFs was confirmed by western blot (Figure 1A). Following Rac1 deletion, the fl/fl MEFs exhibited a more contracted shape than the fl/+ MEFs (Figure S1). When compared to fl/+ or to untreated fl/fl MEFs, the deletion of Rac1 suppressed lamellipodia formation, cell spreading, and cell adhesion to the surface (Figure S1), as previously described by Vidali et al. (2006). Interestingly, we also noticed that cell size was reduced by about 10%, in Rac1 null MEFs but was unaffected by 4-HT treatment of the control fl/+ MEFs (Figure 1B).

Figure 1. Rac1 Deletion Reduced the Size of MEFs and Lymphocytes.

Figure 1

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(A) Fl/+ and fl/fl MEFs were grown in 10% serum in the presence and absence of 4-HT for 48 hr (1: fl/+ treated with 4-HT, 2: fl/fl treated with ethanol, 3: fl/fl treated with 4-HT). Cells were lysed and subjected to SDS-PAGE and WB to assess Rac1 deletion.

(B) Rac1 fl/+ and fl/fl MEFs were grown in 10% serum, resuspended in Trypsin, diluted in PBS, then subjected to cell size analysis on a Beckman Coulter cell sorter before and after 4-HT treatment.

(C–F) B cells (C and D) and T cells (E and F) from WT, Rac1 fl/fl, Rac2−/− and Rac1 fl/fl Rac2−/− mice were grown in RPMI with 10 % serum for 48 hr in the presence of 500 nM 4-HT. Cells were resuspended in PBS, then subjected to FACscan analysis, based on forward scatter/side scatter (FS/SSC). The histograms in (D) and (F) resulted from the quantification of the cell size on the Forward Scatter scale. Error bars in (B), (D), and (F) specify the standard deviation between four independently performed experiments. Results are representative of four experiments.

To determine whether the role of Rac1 in regulating cell size was unique to MEFs, we examined the effect of Rac1 deletion on the size of T and B lymphocytes. Primary B and T cells were isolated from wild-type mice and Rac1fl/fl, 4-HT-Cre mice and treated with 4-HT to delete Rac1, as described in theExperimental Procedures. Deletion of Rac1 induced a 20%–25% reduction in the size of both B (Figures 1C and 1D) and T cells (Figures 1E and 1F).

Rac1 and Rac2 are 95% identical (they differ primarily in the C-terminal sequence) and appear to share many, but not all, functions. Whether Rac2 (which is not expressed in MEFs) could compensate for Rac1 deletion was assessed in B and T cells, which express both proteins. B and T cells were isolated from wild-type mice, Rac2−/− mice, or Rac2−/− Rac1fl/fl, 4-HT-Cre mice then treated with 4-HT for 48 hr. Rac2−/− B and T cells were not significantly smaller than wild-type B and T cells. Deletion of Rac1 in Rac2−/− B or T cells had no additional effect on cell size, compared to deletion of Rac1 alone (Figures 1D and 1F). These data demonstrate that Rac1 specifically is critical in determining cellular size in multiple cell types.

Mechanism of Rac1-Dependent Regulation of Cellular Size

Cellular size is regulated by the mTOR pathway through mTORC1-dependent phosphorylation of S6 kinase (S6K) and 4eBP1 (Schmelzle and Hall, 2000; Fingar et al., 2002). The cell-size reduction caused by Rac1 deletion in MEFs was similar to the decrease induced by the mTOR inhibitor rapamycin in U2OS and 293 cells (Fingar et al., 2002), suggesting that deletion of Rac1 might affect mTOR signaling. To determine whether the effect of Rac1 deletion on cell size was the result of inhibition of mTORC1 activity, we compared S6K and 4eBP1 phosphorylation in wild-type (WT) MEFs and Rac1 fl/fl MEFs growing in 10% serum and treated with 500 nM 4-HT to delete Rac1. In WT MEFs treated with 4-HT, or Rac1 fl/fl MEFs (treated with ethanol), the mTOR pathway and Rac1 expression were not affected (Figures 2A and 2B). Deletion of Rac1 inhibited phosphorylation of the mTORC1 substrates 4eBP1 and S6K, as well as the S6K substrate, ribosomal subunit S6 (Figure 2B, compare lanes 1 and 2), to the same extent as rapamycin treatment (Figure 2B, lane 5). mTOR is also a component of the mTORC2 complex that phosphorylates S473 of Akt. Deletion of Rac1 inhibited the phosphorylation of Akt at S473 to nearly the same degree as the PI3K inhibitor wortmannin (Figure 2B). Deletion of Rac1 also inhibited phosphorylation of Akt T308, but to a lesser extent. ERK phosphorylation was not affected by deletion of Rac1 (Figure 2B). These data indicate that Rac1 regulates mTOR activity in both the mTORC1 and mTORC2 complexes. Chou and Blenis showed that activated Rac1 or Cdc42 directly stimulated p70 S6 kinase activity (Chou and Blenis, 1996). Our data indicate that Rac1 also functions upstream of mTOR, since mTOR phosphorylation of both 4eBP1 and p70 S6 kinase was inhibited by Rac1 deletion.

Figure 2. Rac1 Regulates mTOR Activity in MEFs and HeLa Cells.

Figure 2

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(A and B) WT (A) and Rac1 fl/fl (B) MEFs were cultured in DMEM with 10% serum. They were treated with ethanol (control) or with 500 nM 4-HT for 48 hr to delete Rac1. In some conditions, cells were treated for 1 hr with 100 nM wortmannin or 100 nM rapamycin at 37°C.

(C) Rac1 or control RNAi oligos were introduced into HeLa cells before analysis.

(D) Immortalized MEFs were infected or not with control or Rac1-expressing lentiviral particles, then incubated or not with 4-HT for 48 hr with serum-starvation for the last 14 hr. They were then stimulated or not for 10 min with 10% serum supplemented with 50 nM PDGF. The cells were then lysed and subjected to SDS-PAGE and western blot analysis. These results are representative of three experiments.

To confirm that the effect of Rac1 deletion on mTOR activity was not specific to MEFs, we knocked down Rac1 in HeLa cells using siRNA. Phosphorylation of 4eBP1, S6K, S6, and Akt S473 and T308 were all significantly inhibited, similar to what was observed in 4-HT-treated MEFs (Figure 2C). In addition, Rac1, but not Rac2, deletion significantly inhibited phosphorylation of 4eBP1 in T cells and S6K and Akt S473 in B cells (Figures S2A and S2B). Similar effects of Rac1 knockdown on S6K phosphorylation were also seen in A549 breast and Panc1 pancreatic cancer cell lines as well as in H929 myeloma cells (data not shown).

The experiments described above were all done in the presence of 10% serum. To confirm that deletion of Rac1 blocks stimulation of mTOR, we compared Akt, S6, and p70 S6 kinase phosphorylation in serum starved and stimulated Rac1 fl/fl immortalized MEFs. The MEFs were preinfected with a Rac1-expressing lentivirus (to allow confirmation that the effect is due to Rac1 deletion) or a control virus (empty vector), then treated or not with 4-HT to delete the endogenous Rac1. As shown in Figure 2D, deletion of Rac1-inhibited serum stimulated phosphorylation of Akt S473, S6, and p70 S6 kinase. Reconstitution of Rac1 expression by lentivirus infection restored serum stimulation of Akt, S6, and p70 S6 kinase phosphorylation (Figure 2D). These data show that Rac1 is critical for the regulation of mTOR activity in both the mTORC1 and mTORC2 complexes and explain the effect of Rac1 deletion on cell size.

Rac1 could regulate mTOR by any of several different pathways. In some systems Rac1 binds to and regulates PI3K activity (Tolias et al., 1995; Murga et al., 2002). The lipid products of PI3K are critical for phosphorylation of Akt at both S473 (by mTORC2) and T308 (by PDK1). Akt, when phosphorylated at both of these sites, phosphorylates TSC2, leading to mTORC1 activation. Thus, the inhibition of Akt and mTORC1 substrate phosphorylation in Rac1-deleted MEFs could be explained by PI3K inhibition. Alternatively, Rac1 could regulate both mTORC1 and mTORC2 by another, yet unknown, mechanism.

Rac1 Regulates mTOR Signaling Independently of the PI3K/Akt Pathway

To determine whether regulation of mTOR by Rac1 is mediated by PI3K, we treated Rac1fl/fl MEFs with 100 nM wortmannin (a concentration that inhibits type I PI3K with little effect on mTOR [Bain et al., 2007]). As expected, wortmannin blocked phosphorylation of Akt at S473 and was much more effective than Rac1 deletion in blocking phosphorylation at T308 (Figure 2B). However, Rac1 deletion was more effective than wortmannin in blocking phosphorylation of the mTORC1 substrates 4eBP1 and S6K (Figure 2B, compare lanes 2 and 3), suggesting that Rac1 has a PI3K-independent effect on mTORC1 activity. To test this hypothesis, we investigated the effect of Rac1 deletion in Rac1 fl/fl MEFs on phosphorylation of mTORC1 substrates stimulated by phorbol myristate acetate (PMA). PMA activates mTORC1 independently of PI3K through the MAP kinase pathway (Monfar et al., 1995; Radimerski et al., 2002). Deletion of Rac1 suppressed phosphorylation of both 4eBP1 and S6K following PMA stimulation (Figure 3A), but wortmannin did not block PMA stimulation of 4eBP1 phosphorylation or S6K phosphorylation (Figure 3A). Taken together, these results indicate that the effects of Rac1 deletion on mTORC1 targets are independent of PI3K and that Rac1 could have a direct role in the regulation of mTORC1.

Figure 3. Rac1 Regulates mTOR Activity Independently of PI3K, Binding to GTP/GDP and Actin Polymerization.

Figure 3

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(A) Primary Rac1 fl/fl MEFs were grown in 10% serum and treated or not with 4-HT for 48 hr, then serum starved (overnight). They were incubated or not with 100 nM wortmannin for 30 min then subjected to stimulation with 1 μM PMA for 15 min.

(B) Cells were grown in 10% serum and treated with ethanol (control) or with 500 nM 4-HT for 48 hr to delete Rac1. In some conditions, cells were treated for 1 hr with 100 nM wortmannin or 100 nM Rapamycin at 37°C or with 10 μM of a Rac inhibitor, NSC-23766.

(C) MEFs were treated or not with 500 nM 4-HT for 36 hr in DMEM with 10% heat-inactivated FBS to delete Rac1. Cells were then infected with recombinant vaccinia viruses, expressing Rac1 WT, V12Rac1, N17Rac1, or the empty vector pSC65 for 14 hr.

(D) HeLa cells were grown in 10% serum then serum starved for 14 hr. They were treated or not with 10 μg/ml cytochalasin D and stimulated or not with 10% serum supplemented with 50 nM PDGF. Cells were then lysed and subjected to SDS-PAGE and western blot analysis. These results are representative of three experiments.

Rac1 Regulates mTOR Activity Independently of Its Binding to GTP or GDP

Although canonical GTPase-dependent signaling pathways require GTP binding, some GTP-independent effects have been described, including binding of PRK2 to RhoA and type I PI4P-5K to Rac1 (Vincent and Settleman, 1997; Tolias et al., 2000). Therefore, we asked whether Rac1 activation is necessary to regulate mTOR activity. We treated MEFs with NSC-23766, an inhibitor of Rac activation (Gao et al., 2004), and compared mTOR activity in MEFs from which Rac1 was deleted. Compared to rapamycin treatment, inhibition of Rac1 activation by NSC-23766 did not affect mTORC1 activity, as illustrated by the intact phosphorylation of 4eBP1 and S6 (Figure 3B). Rac1 activation was significantly inhibited when cells were incubated with NSC-23766 (10 μM), as shown by a decrease in the ability of Rac1 to be pulled down by GST-PBD (Figure S2C). To confirm that Rac1 activation is not necessary for mTORC1 activity we reconstituted Rac1 expression in 4-HT-treated MEFs with WT Rac1, dominant-active (V12Rac1), or dominant-negative (N17Rac1) mutants. MEFs were treated or not for 36 hr with 4-HT, then subjected to infection with the different recombinant vaccinia viruses for about 14 hr. As shown in Figure 3C, 4eBP1, S6K, and S6 phosphorylation were decreased when Rac1 was deleted in both uninfected cells and in cells infected by the vaccinia virus empty vector, pSc65. The reconstitution with WT Rac1, V12Rac1 mutant, or N17Rac1 mutant rescued the phosphorylation of Akt, 4eBP1, S6K, and S6 (Figure 3C). These results confirm the requirement of Rac1 expression for mTORC1 activity, but indicate that GTP binding to Rac1 is not required for this function.

Subcellular Colocalization of Rac1 with mTOR

The PI3K- and the GTP-independent effect of Rac1 on mTORC1 activity led us to ask whether Rac1 could act as an adaptor protein, required for mTOR localization at specific cellular regions to ensure its optimal activity. We used immunofluorescence to determine whether Rac1 and mTOR colocalize, which would support a possible direct regulation of mTOR by Rac1. In serum-starved cells, mTOR and Rac1 colocalization was limited to the perinuclear region (Figure 4A). In serum-stimulated MEFs, the colocalization was observed in both the perinuclear region and at specific membranes, especially at the plasma membrane (Figures 4B and S3C). Colocalization at both perinuclear region in serum-starved MEFs and at the plasma membrane of activated MEFs was confirmed by confocal imaging (Figures 4C and 4D). Similar results were seen in HeLa cells, where Rac1 colocalizes with mTOR at the perinuclear region in serum-starved cells, but upon cell stimulation, this colocalization is extended to the plasma membrane (Figures S3A and S3B). Rac1 and mTOR also colocalize in Panc1 and A549 cells stimulated with 10% serum (Figures S4A and S4B).

Figure 4. Rac1 Is Colocalized with mTOR and Is Required for Subcellular Localization of mTOR.

Figure 4

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(A and B) MEFs were serum-starved (a) or serum starved then stimulated with 10% serum for 10 min (B). Cells were fixed and immunostained for Rac1 and mTOR and analyzed by immunofluorescence.

(C and D) MEFs were serum starved (C) or serum starved then stimulated with 10% serum, supplemented with 50 nM PDGF for 10 min (D). They were fixed and immunostained for Rac1 and mTOR and subjected to confocal microscopy analysis as described in the method section.

(E and F) Rac1 fl/fl MEFs were incubated with 4-HT for 2 days. They were serum starved (E) and serum-starved then stimulated for 10 min (F). Cells were fixed and stained for Rac1 and mTOR. Cells were visualized by immunofluorescence as described in the methods section. Images are representative of four experiments.

To determine whether Rac1 is necessary for mTOR localization at specific cell regions, we used Rac1 fl/fl MEFs, treated or not with 4-HT to delete Rac1, and stained for Rac1 and mTOR. Upon Rac1 deletion, the anti-Rac1 staining disappeared, as expected, and, importantly, the localization of mTOR in the perinuclear region became more diffuse and the membrane localization was lost (Figures 4E and 4F). To confirm the specificity of the Rac1 antibody and the deletion of Rac1, we mixed MEFs expressing Rac1 and MEFs from which Rac1 had been deleted. Both Rac1 staining and its colocalization with mTOR were clear in the untreated MEFs (Figure S3D, arrow), while Rac1 staining was absent in all 4-HT pretreated cells and mTOR was mislocalized, demonstrating that the antibody is specific for Rac1 and that Rac1 is deleted following 4-HT treatment (Figure S3D). To be certain that the requirement of Rac1 for mTOR localization is not cell-type specific, the effect of Rac1 knockdown by RNAi in A549, Panc1, and HeLa cells was examined. In all of these cell lines, Rac1 knockdown impaired the membrane localization of mTOR (Figures S4C, S4D, and S5B; arrows).

We next investigated whether Rac1 mediates membrane localization of mTOR bound to mTORC1, mTORC2, or both. We examined Rac1 colocalization with Raptor, Rictor, and Sin1, specific components of mTORC1 and mTORC2, respectively, in serum stimulated HeLa cells. Rac1 and Raptor colocalized (Figure 5A), as did Rac1 with Rictor and Sin1 (Figures 5B and 5C). These data support the model that Rac1 regulates both mTORC1 and mTORC2 by directing their subcellular localization.

Figure 5. Rac1 Is Colocalized with Both mTORC1 and mTORC2 Components.

Figure 5

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(A–C) HeLa cells were serum starved then stimulated with 10% serum for 20 min. They were fixed and immunostained for Rac1 and Raptor (A) or Rac1 and Rictor (B) or Rac1 and Sin1 (C), then analyzed by immunofluorescence as described in the Experimental Procedures section. Images are representative of four experiments.

Rheb is also required for the activation of mTORC1. We therefore asked whether Rac1 and Rheb were in a common complex. We could not detect Rheb in Rac1 immunoprecipitates (IPs) or Rac1 in Rheb IPs. Using immunofluorescence, we found that Rheb colocalizes with Rac1 in serum-starved HeLa cells at the perinuclear region and partly at the membrane following serum and PDGF stimulation (Figure S6B). The shared subcellular localization of Rheb with mTOR and Rac1 suggests that Rac1 and Rheb may collaborate to localize/activate mTORC1.

Rac1 activation in response to growth factors leads to actin polymerization and lamellopodia formation, raising the possibility that Rac1-dependent recruitment of mTOR could be indirect and depend on actin polymerization. We took two approaches to investigate this possibility. We disrupted the actin cytoskeleton with cytochalasin D and determined the effect on mTOR activation. As shown in Figure 3D, treatment with cytochalasin D had no effect on stimulation of Akt, S6, or 4eBP1 phosphorylation in activated HeLa cells. Others have shown that disruption of the actin cytoskeleton using latrunculin A or cytochalasin D in dendritic cells and myotubes did not affect mTOR-dependent phosphorylation of S6K (Tsakiridis et al., 1998; Berven et al., 2004). Whether Rac1-dependent actin polymerization was essential for mTOR subcellular localization was also assessed using cytochalasin D. As shown on Figures S6C and S7D, actin disruption by cytochalasin D did not prevent mTOR colocalization with Rac1 at the plasma membrane. We also knocked down expression of the Rac1-related GTPase Cdc42. Cdc42 regulates filopodia and is upstream of Rac1-dependent lamellopodia formation (Chant and Stowers, 1995). Thus, deletion of Cdc42 will impair Rac1-dependent actin cytoskeletal regulation. Cdc42 knockdown (Figure S6A) did not inhibit Rac1 and mTOR colocalization at the membrane (Figure S6B), although in the cells in which Cdc42 had been knocked down, colocalization was primarily in punctuate structures (Figure S6B). These data indicate that Rac1 regulation of mTOR was not dependent on the control of actin polymerization by Rac1.

Rac1 Interacts Directly with mTOR through the Rac1 C-Terminal Region

To determine whether endogenous Rac1 and mTOR interact, we immunoprecipitated Rac1 from HeLa cells and western blotted for mTOR. As shown in Figure 6A, mTOR was present in the Rac1 immunoprecipates. In an effort to further understand the molecular mechanism by which Rac1 regulates mTOR signaling, we investigated whether Rac1 interacts with the various mTORC1 and mTORC2 components. Either HA-tagged Rac1 or Rac2 was cotransfected with Myc-tagged mTORC constructs. Rac1 coprecipitated with mTOR, as well as with both Raptor and Rictor (Figure 6B). Rac2, expressed at much higher levels than Rac1, immunoprecipitated with overexpressed Raptor, but showed very little interaction with mTOR and Rictor (Figure 6B). We think that the Rac2-Raptor interaction is an artifact of overexpression of the two proteins. This interpretation is supported by the lack of effect of Rac2 deletion on mTORC1 activity in B and T lymphocytes.

Figure 6. Rac1 Interacts with mTORC1 and mTORC2 Proteins.

Figure 6

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(A) Fifty to sixty percent confluent 293T cells were treated with PolyEthylImine (PEI) as a control or transfected with HA-tagged Rac1 or Rac2 and the different Myc-tagged mTORC proteins using PEI, as indicated.

(B) 293T cells were treated with PEI (control) or transfected with HA-tagged WT Rac1, V12Rac1, or N17Rac1 mutants and with either mTOR, Ric tor, or Raptor as indicated. Cells were then lysed and subjected to HA IP.

(C) 293T cells were transfected with either mTOR, Rictor or Raptor. Cells were lysed and subjected to pull-down assays using GST-fusion proteins of WT and Rac1 mutants for 1 hr at 4°C. The protein complexes were washed and then analyzed by SDS-PAGE and western blot. Results are representative of three experiments.

(D) 293T cells were transfected with either mTOR, Rictor, or Raptor. Cells were lysed and subjected to pull-down assays using GST-fusion proteins of WT and Rac1 mutants for 1 hr at 4°C. The protein complexes were washed and then analyzed by SDS–PAGE and western blot. Results are representative of three experiments.

We also investigated whether Rac1 association with the mTORC components depends on its GTP/GDP binding. HA-WT Rac1, HA-V12Rac1, or HA-N17Rac1 was coexpressed with Myc-tagged components of the mTORC1 or mTORC2, and then an anti-HA IP was performed on the cell lysates. As shown on Figure 6C, both Rac1 mutants were similar to WT Rac1 in their abilities to coprecipitate with the mTORC proteins. Bacterially expressed GST-fusion proteins containing WT Rac1 or the V12 or the N17 mutants precipitated Rictor, mTOR, and Raptor (Figure 6D). These results indicate that Rac1 interacts with both mTOR complexes independently of its binding to GTP or GDP.

Since Rac1 and Rac2 are 95% identical and differ only in their C-terminal sequences, it is likely that differences in sequences in this region explain the preferential ability of Rac1 to bind to the mTORC proteins. We synthesized biotinylated forms of the C-terminal 10 amino acid peptides of Rac1, Rac2, and Cdc42. Lysates from 293T cells transfected with mTOR, Rictor, or Raptor were incubated with the various peptides and subjected to precipitation with streptavidine beads. As shown in Figure 7A, the Rac1 C-terminal peptide preferentially precipitated mTOR, Rictor, and Raptor, compared to the Rac2 and Cdc42 peptides.

Figure 7. The C-Terminal Region of Rac1 Is Required for the Direct Interaction with mTOR.

Figure 7

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(A) Fifty to sixty percent confluent 293T cells were treated with PEI (control) or transfected with mTOR, Rictor, or Raptor. Cells were then lysed and biotin-peptides of the Rac1, Rac2, and Cdc42 C-terminal domains and mutated Rac1 peptide: PPP/AAA or RKR/AAA was used to precipitate mTORC proteins for 1 hr at 4°C in the presence of streptavidin beads.

(B and C) 293T cells were transfected with Flag-mTOR. The Flag IPs were eluted by the Flag peptide. The free mTOR protein was precipitated by biotin-Rac1 C-terminal peptide or the biotin-RKR/AAA mutant, followed by streptavidin agarose. Samples were analyzed by SDS-PAGE and western blot (b) or the SDS-PAGE was silver stained (C). MW: molecular weight markers, WL: total proteins from Flag-mTOR transfected cells (1) WT biotin-Rac1 peptide; (2) biotin RKR/AAA mutant; (3) Flag IP. Data are representative of three experiments.

Two protein-protein interaction motifs have been identified in the C terminus of Rac1, but not Rac2 or Cdc42 (van Hennik et al., 2003). A motif involving three prolines binds the Crk SH3 domain and a motif involving the three basic residues, RKR, binds to type I phosphatidylinositol 4-phosphate 5-kinase (Tolias et al., 2000). These two C-terminal motifs are both required for Rac1 interaction with the CD2AP protein that links Rac1 to CapZ and cortactin (van Duijn et al., 2010). Mutation of the three prolines into alanines did not affect the binding of the Rac1 C-terminal peptide to mTOR, Rictor, or Raptor (Figure 7A), indicating that this motif was not necessary for Rac1 interaction with mTOR complexes. However, mutation of the RKR motif to AAA impaired the interaction between the Rac1 peptide and mTOR, Rictor and Raptor (Figure 7A). The Rac1 WT and Rac1 PPP/AAA peptides also precipitated endogenous mTOR and Rictor (Figure 7A). These results indicate that the same region of Rac1 that mediates activation-independent binding to type 1 PI4P 5-kinases also mediates binding to mTORC1 and mTORC2. Binding to mTORC2 suggests that Rac1-dependent phosphorylation of Akt S473 is mediated through Rac1-dependent localization of mTORC2.

Since mTORC1 and mTORC2 complexes have mTOR in common, we hypothesized that mTOR itself may mediate the direct interaction with Rac1. To test this hypothesis, we expressed Flag-tagged mTOR in 293T cells. Cells were lysed in stringent RIPA buffer (0.1% SDS, 1% Triton, and 1% DOC), known to disrupt protein-protein interactions. mTOR was precipitated with anti-Flag agarose conjugate and eluted with Flag peptide. Based on silver staining of SDS-PAGE, two bands were present, one at the molecular mass of mTOR and a second lower mass band. The lower mass band was shown by mass spectroscopy to be a proteolytic fragment of mTOR (Figure 7C). We confirmed that mTORC1 and mTORC2 were disrupted by western blotting for Rictor and Raptor, which were not detected (Figure 7B). Biotinylated WT Rac1 peptide and the RKR/AAA mutant were tested for their abilities to precipitate purified mTOR. As shown on Figure 7B, the WT Rac1 peptide precipitated mTOR, but the RKR/AAA mutant did not. These data indicate that the C terminus of Rac1 interacts directly with mTOR, to regulate both mTORC1 and mTORC2 and explain the specificity for Rac1 compared to Rac2.

DISCUSSION

We report here that Rac1 plays a critical role in mTOR signaling in both mTORC1 and mTORC2. In the absence of Rac1, both mTORC1 and mTORC2 fail to phosphorylate their downstream targets. Rac1 directly binds to mTOR and mediates the localization of mTORC1 and mTORC2 to cellular membranes. The interaction between Rac1 and mTOR is independent of the GTP/GDP-binding state of Rac1, but requires a sequence motif in the C-terminal region of Rac1. Rac1 is the only known regulator of both mTORC1 and mTORC2. This study provides a potential for future research on dual regulation of mTORC1/2 by providing means to inhibit both pathways.

Previous studies have shown that other low molecular weight GTP-binding proteins regulate mTORC1. Rheb, like Rac1, directly binds to mTOR independent of the GTP/GDP bound state (Long et al., 2005). However, stimulation of the kinase activity of mTORC1 requires that Rheb is GTP bound (reviewed by Avruch et al., 2006). The Rag GTPases also bind to mTORC1, but unlike Rac1 and Rheb, this interaction is dependent on the appropriate GTP/GDP-bound forms of these proteins, which is achieved only when essential amino acids are present at appropriate concentrations (Sancak et al., 2008). A properly charged Rag heterodimeric complex is required to facilitate localization of mTORC1 to lysosomal membranes where it can be activated by Rheb in response to amino acids (Sancak et al., 2008). The Rag GTPases are not required for initial activation of mTOR in response to growth-factor stimulation, but they are essential to transmit the complementary amino acid signal to mTOR and increase its activation (Sancak et al., 2008; Kim et al., 2008). Rheb has a large cellular distribution pattern and is found in the cytoplasm, the Golgi, lysosomes, and endoplasmic reticulum (ER) (reviewed by Wang and Proud, 2009). Rheb membrane localization is not required for S6K phosphorylation (Li et al., 2004), indicating that it is not the key adaptor protein that localizes mTORC1. Here, we provide evidence that Rac1 can regulate mTORC1 activity independently of PI3K, and show that Rac1 is required for proper localization of mTORC1 at cellular membranes, in response to growth-factor stimulation. In sum, Rac1 could cooperate with Rheb and the Rags to localize and activate mTORC1. In general, the localization of cytosolic proteins to specific membranes relies on multiple interactions to insure specificity in location, and thus it is likely that binding to Rac1 alone is not sufficient for localization of mTORC1 to membranes. Other interactions mediated by other components of the complex, such as Raptor (which binds Rheb and Rags) are necessary for sufficient avidity and to insure that activation occurs at the right location. We suggest that a spaciotemporal relationship between all three GTPases may exist for an optimal activation of mTORC1. In response to growth factors, Rac1 is involved in early mTOR localization/activation to appropriate cellular membranes. Subsequently and/or in parallel, active Rheb can directly stimulate mTORC1 activity. To maximally activate mTORC1, the GTP-bound Rags are required to complete and sustain the cell response by integrating the amino acid input.

mTORC2 has an evolutionarily conserved role in controlling the actin cytoskeleton (Jacinto et al., 2004), and our data suggest a model in which Rac1 functions both upstream and downstream of mTORC2 to mediate actin remodeling. Inhibition of actin polymerization by cytochalasin D did not prevent the phosphorylation of the mTORC1 targets, S6K, and 4eBP1 or the mTORC2 target, Akt. These findings indicate that actin polymerization is not necessary for mTORC1/2 activity and so Rac1 must act through a different pathway to regulate mTORC1 and mTORC2. Rac1-dependent effects on actin polymerization require GTP binding, which is catalyzed by GEFs. The P-Rex1 GEF binds to mTORC2 and activates Rac1 (Hernández-Negrete et al., 2007). These data lead us to suggest the following model. Upon cell stimulation by growth factors, Rac1 is released from Rho GDI, translocates to the plasma membrane, and recruits mTORC2 and along with it, P-Rex1, thereby facilitating Rac1 activation locally. GTP-Rac1 binds PI 3-kinase and the product of PI 3-kinase, PI-3,4,5-P3(PIP3), directly binds to and activates P-Rex1 (Tolias et al., 1995; Welch et al., 2002). mTORC2, functioning as a scaffold, could facilitate a positive-feedback loop between Rac1 and PI 3-kinase, producing high levels of PIP3 at the region of the plasma membrane where the Rac1/mTORC2 complex resides. Focal concentrations of PIP3 and Rac1 would recruit Akt in proximity to mTORC2, leading to phosphorylation of AKT at Ser-473 by mTORC2. Rac1 binding to mTOR via a mechanism independent of GTP loading is not unique. Although the majority of Rac1-interacting proteins depend on GTP loading of Rac1 (reviewed by Ridley, 2006), PI-4-P 5-kinases bind to Rac1 in an analogous manner to that of mTOR (Tolias et al., 2000). In summary, localization of Rac1 to the plasma membrane would result in recruitment of mTORC2/P-Rex. P-Rex would activate Rac1, leading to recruitment of PI3K and PIP3 synthesis. PIP3 would further stimulate P-Rex in a positive-feedback loop, amplifying Rac1 activation and leading to actin remodeling. Increasing levels of PIP3 would recruit Akt, leading to its activation through phosphorylation of S473 by mTORC2.

In summary, we have identified Rac1 as a regulator of both mTORC1 and mTORC2 that participates in the activation of both complexes by directly binding to mTOR and facilitating localization to cellular membranes. While this function of Rac1 does not require GTP loading, it is possible that GTP loading on the subfraction of Rac1 that is in complex with mTORC1 or mTORC2 allows recruitment of specific downstream targets of mTOR to mediate the plethora of responses attributed to these complexes.

EXPERIMENTAL PROCEDURES

Rac1 fl/fl mice were provided by D. Kwiatkowski (Brigham and Women’s Hospital; Boston, MA). Rac2−/− mice were from D. Williams (Children’s Hospital; Boston, MA). Tamoxifen-Cre mice were from Jackson Laboratory (Bar Harbor, Maine). 4-HT was from Sigma (St. Louis, MO). NSC-23766 was from Calbiochem (San Diego, CA). Antibodies to phospho-Akt T308 and S473, Akt, phospho-S6K T389, S6K, phospho-S6, mTOR, Rictor, Raptor, 4eBP1, phospho-4eBP1, ERK, and phospho-ERK were purchased from Cell Signaling Technology (Beverly, MA). Goat anti-HA, rabbit anti-Rictor for immunofluorescence and mAb anti-Myc antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse anti-Rac1 was from BD Biosciences (San Jose, CA). Rabbit anti-Raptor for immunofluorescence and β-Actin mAb were from Abcam (Cambridge, MA). Vaccinia viruses expressing Rac1 and its mutants were provided by E. Hong-Geller (Los Alamos, NM). HA-tagged Rac1 WT, V12Rac1, N17Rac1, and Rac2 constructs were from GenScript (Piscataway, NJ). Rictor, Raptor, and mTOR constructs were from Addgene (Cambridge, MA).

B and T Cell Isolation from Mice and Tissue Culture

Primary B cells were isolated from the spleens of approximately 10- to 12-week-old mice (WT, Rac1 fl/fl, Rac2−/− and double Rac1 fl/fl-Rac2−/− mice were selected from the same breeding liters), using MACS CD43 (Ly-48) microBeads as described in the manufacturer’s protocol (Miltenyl Biotech; Auburn, CA). Primary T cells were isolated from spleen, thymus, and lymph nodes of the same mice. T cells were grown by using 3 μg/ml anti-CD3/CD28 antibodies and 20 U/ml IL2 at the time of isolation. The following day, the antibodies were removed, and T cells were cultured in the presence of 10 U/ml IL2. Both B and T cells were grown for 48 hr in RPMI 1640, supplemented with 10% fetal bovine serum (FBS), 1% sodium pyruvate, 0.1% penicillin (100 U/ml), streptomycin (100 μg/ml), and 50 μM 2-mercaptoethanol in the presence of 500 nM 4-HT to induce Rac1 deletion. For western blot analysis, cells were serum starved overnight, in the presence of 4-HT, before activation and analysis.

MEF Isolation, Culture, and Analysis

E12 embryos were isolated and separated into tissue-culture plates containing PBS. The head and organs were discarded; the rest of the bodies were minced and treated with Trypsin/EDTA. The tissue was then resuspended in DMEM containing 10% FBS and cultured. Cells with the desired genotype were cultured and treated or not with 500 nM 4-HT to delete Rac1.

To analyze the effect of different inhibitors and Rac1 deletion, MEFs were seeded in 6-well plates, treated or not for at least 48 hr with 500 nM 4-HT then lysed in 1% Triton X-100-containing buffer with protease/phosphatase inhibitors. Lysates were subjected to SDS-PAGE and western blot analysis.

To generate immortalized MEFs from the previous primary 4HT-inducible MEFs, we have used the pLKo.1 lentiviral vector to knockdown p53 by shRNA. Viral particles were produced in 293T cells and used to infect MEFs. Positive cells were selected by puromicin.

The immortalized MEFs were used to express Rac1 prior to the deletion of endogenous Rac1 by 4-HT. Cells were infected by the Rac1-expressing pRSV lentiviral particles (GenTarget; San Diego, CA). Positive cells were selected using blasticidin for 3–4 days then lysed and analyzed by SDS-PAGE and western blot.

Immunoprecipitation and GST-Fusion Proteins

Control and transfected 293T cells were lysed in a CHAPS-containing buffer (120 mM NaCl, 1 mM EDTA, 40 mM HEPES, 50 mM NaF, 10 mM β-Glycero-phosphate, 0.3% CHAPS, 1 mM Na3VO4, 1 mM PMSF, 10 μg/ml leupeptin, 10 μg/ml aprotinin, and 10 mM MgCl2). Lysates were centrifugated at 15,000 X g for 10 min at 4°C. IP was performed at 4°C by adding the appropriate antibody to the lysates for 2 hr. Protein A-Sepharose was added for an additional hour. In the case of GST pull-down experiments, the GST-fusion proteins were added to the lysates for 1 to 2 hr. The protein complexes were washed, separated by SDS-PAGE, and analyzed by western blot.

Peptide Synthesis and Protein Precipitation

Biotin-labeled peptides representing the C-terminal sequences of Rac1, Rac2, Cdc42, and mutants of Rac1 were synthesized by Biomatik Corporation, by the “solid synthesis method,” on a Syro II and using Fmoc. The full synthesized sequences are WT-Rac1: GGG PPPVKKRKRK; Rac1 PPP/AAA: GGG AAAV KKRKRK; Rac1 RKR/AAA: GGG PPPVKKAAAK; WT-Rac2: GGG PQPTRQ QKRA, and WT-Cdc42: GGG EPPEPKKSRR.

Control and mTORC-transfected 293T cells were lysed as in the IP experiments. Lysates were clarified by centrifugation at 15, 000 X g for 10 min at 4°C. Supernatants were then incubated with 10 μg of each peptide for 1 hr in the presence of Streptavidin-coated beads (Sigma). Samples were collected, extensively washed in lysis buffer and subjected to analysis by SDS-PAGE and western blot.

Knockdown by siRNA and shRNA

HeLa, A549, and Panc1 cell lines were grown in DMEM with 10% serum. Rac1 and control siRNA oligos (Santa Cruz Biotechnology) were transfected using Lipofectamine-2000 (Invitrogen) for 6 hr. Cells were cultured for an additional 3–4 days and lysed. For shRNA, pLKO-expressing GFP (control), Rac1, and Cdc42 shRNA oligo (Open Biosystems; Huntsville, AL) were used to produce lentiviral particles in 293T cells and infect H929 cells and MEFs, respectively. Cells were then selected using puromycin for 3–5 days. Cells were either subjected to immunofluorescence analysis or lysed for SDS-PAGE and western blot analysis.

Immunoflourescence and Confocal Microscopy

Cells were seeded on glass coverslips and cultured in DMEM with 10% serum. They were serum-starved overnight, then stimulated with 10% serum (supplemented or not with 50 nM PDGF). Cells were then fixed with 3% PFA, washed, permeabilized and blocked for 2 hr with 3% BSA. Primary antibodies were incubated overnight at 4°C. DAPI was used to stain the nucleus. Bound antibodies were visualized using Alexa680- and Alexa488-conjugated IgGs (Invitrogen) and observed under oil immersion using a Zeiss Axiovert 200M microscope with a 63X Plan-Apochromat objective with a numerical aperture of 1.4. Images were analyzed using the Axiovision 4.5 software package.

Confocal microscopy analysis was performed on samples prepared as for Immunofluorescence (fixed slides). Observation and images were taken under oil immersion on Zeiss LSM 510 Inverted Live-Cell Confocal System with the 40× objective (60× Zeiss Apochromat W Korr, 1.2 NA, (UV) VIS IR).

Statistics

Results are expressed as means of at least four independent experiments. Error bars represent the standard deviation. Statistical significance was determined by Student’s t test (paired-data analysis). P values ≤ 0.05 (*) were considered to be statistically significant.

Supplementary Material

Document S1. Six Figures

ACKNOWLEDGMENTS

We thank Dr David Williams and David Kwiatkowski for providing the Rac2 null and Rac1 fl/fl mice, respectively. We thank S. Soltoff, P. Moreau, K. Dhamnaskar, A. Sasaki, K. Swanson, C. Benes, B. Zhang, D. Anastasiou, and M. Balastik for their help. This work was supported by NIH grant 5R01 CA113559 to C.L.C. and by P01 CA120964 and R01 GM41890 to L.C.C.

Footnotes

SUPPLEMENTAL INFORMATION Supplemental Information includes six figures and can be found with this article at doi:10.1016/j.molcel.2011.03.017.

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Supplementary Materials

Document S1. Six Figures
NIHMS386684-supplement-Total_Suppl_figures__S1_-_S6__No_S7.pdf (702.8KB, pdf)

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