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. 2014 Mar;26(3):461-7.
doi: 10.1016/j.cellsig.2013.11.035. Epub 2013 Dec 3.

RhoA modulates signaling through the mechanistic target of rapamycin complex 1 (mTORC1) in mammalian cells

Bradley S Gordon  1 , Abid A Kazi  1 , Catherine S Coleman  1 , Michael D Dennis  1 , Vincent Chau  1 , Leonard S Jefferson  1 , Scot R Kimball  2
Affiliations

RhoA modulates signaling through the mechanistic target of rapamycin complex 1 (mTORC1) in mammalian cells

Bradley S Gordon et al. Cell Signal. 2014 Mar.

Abstract

The mechanistic target of rapamycin (mTOR) in complex 1 (mTORC1) pathway integrates signals generated by hormones and nutrients to control cell growth and metabolism. The activation state of mTORC1 is regulated by a variety of GTPases including Rheb and Rags. Recently, Rho1, the yeast ortholog of RhoA, was shown to interact directly with TORC1 and repress its activation state in yeast. Thus, the purpose of the present study was to test the hypothesis that the RhoA GTPase modulates signaling through mTORC1 in mammalian cells. In support of this hypothesis, exogenous overexpression of either wild type or constitutively active (ca)RhoA repressed mTORC1 signaling as assessed by phosphorylation of p70S6K1 (Thr389), 4E-BP1 (Ser65) and ULK1 (Ser757). Additionally, RhoA·GTP repressed phosphorylation of mTORC1-associated mTOR (Ser2481). The RhoA·GTP mediated repression of mTORC1 signaling occurred independent of insulin or leucine induced stimulation. In contrast to the action of Rho1 in yeast, no evidence was found to support a direct interaction of RhoA·GTP with mTORC1. Instead, expression of caRheb, but not caRags, was able to rescue the RhoA·GTP mediated repression of mTORC1 suggesting RhoA functions upstream of Rheb to repress mTORC1 activity. Consistent with this suggestion, RhoA·GTP repressed phosphorylation of TSC2 (Ser939), PRAS40 (Thr246), Akt (Ser473), and mTORC2-associated mTOR (Ser2481). Overall, the results support a model in which RhoA·GTP represses mTORC1 signaling upstream of Akt and mTORC2.

Keywords: 4E-BP1; 70kDa ribosomal protein S6 kinase 1; Akt; PRAS40; RAPTOR; RAPTOR-independent companion of mTOR; RICTOR; Sin1; TSC; TSC2; ULK1; UTR; caRags; caRheb; caRhoA; constitutively active Rags B and C; constitutively active Rheb; constitutively active RhoA; eukaryotic initiation factor 4E binding protein 1; mTOR; mTORC; mTORC2; mechanistic target of rapamycin; mechanistic target of rapamycin complex; p70S6K1; proline-rich Akt/PKB substrate 40kDa; regulatory-associated protein of mTOR, complex 1; stress-activated map kinase-interacting protein 1; tuberous sclerosis complex; uncoordinated-51-like kinase 1; untranslated region; wild type RhoA; wtRhoA.

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Figures

Fig. 1
Fig. 1
Increasing RhoA expression and cellular content of RhoA associated with GTP represses mTORC1 signaling in mammalian cells. (A) HEK 293E cells were transfected with control (Empty), wild type RhoA plasmids —pRK7-myc-RhoA-WT (wtRhoA) or (B) plasmid expressing constitutively active RhoA (caRhoA; pRK7-myc-RhoA-Q63L). Forty-eight hours later, cells were exposed to serum/leucine free medium for 2 h prior to stimulation of mTORC1 with insulin (10 nM) and leucine (0.76 mM) for 30 min. The ratio of phosphorylated p70S6K1 (Thr389), 4E-BP1 (Ser65), and ULK1 (Ser757) to total p70S6K1, 4E-BP1, and ULK1, respectively, were assessed by Western blot analysis. (C) mTORC1 was isolated by immunoprecipitation of RAPTOR and the ratio of phosphorylated mTOR (Ser2481) to total mTOR was assessed by Western blot analysis. Representative blots are shown. Data are means ± SEM; n = 6/group (A) and n = 6/group (B) from two independent experiments. *p < 0.05 by Student's t-test.
Fig. 2
Fig. 2
RhoA·GTP mediated repression of mTORC1 occurs independent of insulin or leucine stimulation without directly binding to themTORC1 complex. (A) Rat2 cells were transiently transfected with a control plasmid (empty) or a plasmid encoding constitutively active RhoA (caRhoA) and exposed to serum/leucine free medium for 2 h. Cells were treated for 30 min with insulin (10 nM), leucine (0.76 mM), or the combination for 30 min. The phosphorylation of p70S6K1 (Thr389) was assessed by Western blot analysis, and the ratio of phosphorylated p70S6K1 (Thr389) to total p70S6K1 is depicted in the graph. Bars with different letters indicate significant difference at p < 0.05 by Two-way ANOVA. * p < 0.05 by Student's t-test. (B) HEK 293E cells were transfected with a control plasmid (Empty) or a plasmid encoding constitutively active RhoA (caRhoA). Forty-eight hours following transfection, cells were incubated in serum/leucine free medium for 2 h prior to stimulation of mTORC1with insulin (10 nM) and leucine (0.76 mM) for 30 min.mTORC1was isolated by immunoprecipitation of RAPTOR and the presence of mTOR, PRAS40, and myc-RhoA were assessed by Western blot analysis. Samples were run on the same gel but in non-contiguous lanes. Black lines denote non-contiguous lanes.
Fig. 3
Fig. 3
RhoA·GTP mediated repression of mTORC1 occurs upstream of the Rheb GTPase. HEK 293E cells were transfected with a control plasmid (Empty), or plasmids expressing either constitutively active Rheb (Myc-caRheb; pRK5-Myc-Rheb-S16H) or a combination of constitutively active RagB (pRK5-HA-GST-RagB-Q99L) and constitutively active RagC (pRK5-HA-GST-RagC-S75L) (collectively denoted as caRags B/C) alone or in combination with constitutively active RhoA (caRhoA; pRK7-myc-RhoA-Q63L). Forty-eight hours following transfection, cells were deprived of serum and leucine for 2 h prior to stimulation of mTORC1 with insulin (10 nM) or leucine (0.76 mM) for 30 min where indicated. Expression of caRhoA and caRheb were assessed by anti-Myc antibodies (Cell Signaling, Danvers, MA) while expression of caRags B/C were assessed by anti-HA antibodies (Santa Cruz Biotechnology, Dallas, TX). Representative blots are shown.
Fig. 4
Fig. 4
RhoA·GTP represses signaling upstream of mTORC1. HEK 293E cells were transfected with control plasmid (Empty) or constitutively active RhoA (caRhoA; pRK7-myc-RhoA-Q63L) and deprived of serum and leucine for 2 h prior to stimulation of mTORC1 with insulin (10 nM) and leucine (0.76 mM) for 30 min. The ratio of (A) phosphorylated Akt (Ser473) to total Akt, (B) phosphorylated PRAS40 (Thr246) to total PRAS40, and (C) phosphorylated TSC2 (Ser939) to total TSC2 was determined by Western blot analysis. (D) mTORC2 was isolated by immunoprecipitation of RICTOR and the ratio of phosphorylated mTOR (Ser2481) to total mTOR was assessed by Western blot analysis. Representative blots are shown. Data are means ± SEM; n = 4–6/group from two independent experiments. *p < 0.05 by Student's t-test.
Fig. 5
Fig. 5
Proposed model of RhoA·GTP-mediated repression of mTORC1/2 signaling in mammalian cells. The data presented within suggests that RhoA·GTP represses mTORC1 signaling by reducing the amount of Rheb·GTP. This effect likely occurs through RhoA·GTP-mediated inhibition of mTORC2 signaling to Akt, leading to repressed phosphorylation of Akt (Ser473) and TSC2 (Ser939) and ultimately to decreased Rheb GTP loading.
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