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Rapamycin Inhibits Cytoskeleton Reorganization and Cell Motility by Suppressing RhoA Expression and Activity*

  • Lei Liu
    Footnotes
    Affiliations
    Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130-3932
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  • Yan Luo
    Footnotes
    Affiliations
    Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130-3932
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  • Long Chen
    Affiliations
    Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130-3932
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  • Tao Shen
    Affiliations
    Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130-3932
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  • Baoshan Xu
    Affiliations
    Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130-3932
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  • Wenxing Chen
    Affiliations
    Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130-3932
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  • Hongyu Zhou
    Affiliations
    Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130-3932
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  • Xiuzhen Han
    Affiliations
    Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130-3932
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  • Shile Huang
    Correspondence
    To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, 1501 Kings Highway, Shreveport, LA 71130-3932. Tel.: 318-675-7759; Fax: 318-675-5180
    Affiliations
    Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130-3932

    Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130-3932
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grant CA115414 (to S. H.). This work was also supported by American Cancer Society Grant RSG-08-135-01-CNE (to S. H.).
    1 Both authors contributed equally to this work.
Open AccessPublished:October 11, 2010DOI:https://doi.org/10.1074/jbc.M110.141168
      The mammalian target of rapamycin (mTOR) functions in cells at least as two complexes, mTORC1 and mTORC2. Intensive studies have focused on the roles of mTOR in the regulation of cell proliferation, growth, and survival. Recently we found that rapamycin inhibits type I insulin-like growth factor (IGF-1)-stimulated lamellipodia formation and cell motility, indicating involvement of mTOR in regulating cell motility. This study was set to further elucidate the underlying mechanism. Here we show that rapamycin inhibited protein synthesis and activities of small GTPases (RhoA, Cdc42, and Rac1), crucial regulatory proteins for cell migration. Disruption of mTORC1 or mTORC2 by down-regulation of raptor or rictor, respectively, inhibited the activities of these proteins. However, only disruption of mTORC1 mimicked the effect of rapamycin, inhibiting their protein expression. Ectopic expression of rapamycin-resistant and constitutively active S6K1 partially prevented rapamycin inhibition of RhoA, Rac1, and Cdc42 expression, whereas expression of constitutively hypophosphorylated 4E-BP1 (4EBP1-5A) or down-regulation of S6K1 by RNA interference suppressed expression of the GTPases, suggesting that both mTORC1-mediated S6K1 and 4E-BP1 pathways are involved in protein synthesis of the GTPases. Expression of constitutively active RhoA, but not Cdc42 and Rac1, conferred resistance to rapamycin inhibition of IGF-1-stimulated lamellipodia formation and cell migration. The results suggest that rapamycin inhibits cell motility at least in part by down-regulation of RhoA protein expression and activity through mTORC1-mediated S6K1 and 4E-BP1-signaling pathways.

      Introduction

      The mammalian target of rapamycin (mTOR),
      The abbreviations used are: mTOR
      mammalian target of rapamycin
      4E-BP1
      eukaryotic initiation factor 4E binding protein 1
      IGF-1
      type I insulin-like growth factor
      mTORC1/2
      mTOR complex 1/2
      raptor
      regulatory-associated protein of mTOR
      rictor
      rapamycin (Rapa)-insensitive companion of mTOR
      S6K1
      ribosomal p70 S6 kinase 1.
      a member of the phosphoinositide 3′-kinase-related kinase family, is a central controller of cell proliferation, growth, and survival (
      • Guertin D.A.
      • Sabatini D.M.
      ). Rapamycin can form a complex with FK506 binding protein 12 and then bind mTOR, selectively inhibiting its kinase activity and function (
      • Guertin D.A.
      • Sabatini D.M.
      ). Recently, two mTOR complexes (mTORC1 and mTORC2) have been identified in mammalian cells (
      • Guertin D.A.
      • Sabatini D.M.
      ). mTORC1 is composed of mTOR, mLST8 (also termed G-protein β-subunit-like protein, GβL, a yeast homolog of LST8), PRAS40 (proline-rich Akt substrate 40 kDa), and raptor (regulatory-associated protein of mTOR) and is rapamycin-sensitive (
      • Fonseca B.D.
      • Smith E.M.
      • Lee V.H.
      • MacKintosh C.
      • Proud C.G.
      ,
      • Hara K.
      • Maruki Y.
      • Long X.
      • Yoshino K.
      • Oshiro N.
      • Hidayat S.
      • Tokunaga C.
      • Avruch J.
      • Yonezawa K.
      ,
      • Kim D.H.
      • Sarbassov D.D.
      • Ali S.M.
      • King J.E.
      • Latek R.R.
      • Erdjument-Bromage H.
      • Tempst P.
      • Sabatini D.M.
      ,
      • Kim D.H.
      • Sarbassov D.D.
      • Ali S.M.
      • Latek R.R.
      • Guntur K.V.
      • Erdjument-Bromage H.
      • Tempst P.
      • Sabatini D.M.
      ,
      • Loewith R.
      • Jacinto E.
      • Wullschleger S.
      • Lorberg A.
      • Crespo J.L.
      • Bonenfant D.
      • Oppliger W.
      • Jenoe P.
      • Hall M.N.
      ,
      • Sancak Y.
      • Thoreen C.C.
      • Peterson T.R.
      • Lindquist R.A.
      • Kang S.A.
      • Spooner E.
      • Carr S.A.
      • Sabatini D.M.
      ,
      • Vander Haar E.
      • Lee S.I.
      • Bandhakavi S.
      • Griffin T.J.
      • Kim D.H.
      ). In response to growth factors and nutrients, mTORC1 regulates cell proliferation and growth by modulating many processes, including protein synthesis and ribosome biogenesis through downstream effectors like 4E-BP1 (eukaryotic initiation factor 4E-binding protein 1) and S6K1 (ribosomal p70 S6 kinase 1) (
      • Guertin D.A.
      • Sabatini D.M.
      ,
      • Fasolo A.
      • Sessa C.
      ). mTORC2 consists of mTOR, mLST8, mSin1 (mammalian stress-activated protein kinase-interacting protein 1), rictor (rapamycin-insensitive companion of mTOR), and protor (protein observed with rictor; also named PRR5 (proline-rich protein 5)), and is rapamycin-insensitive (
      • Frias M.A.
      • Thoreen C.C.
      • Jaffe J.D.
      • Schroder W.
      • Sculley T.
      • Carr S.A.
      • Sabatini D.M.
      ,
      • Jacinto E.
      • Loewith R.
      • Schmidt A.
      • Lin S.
      • Rüegg M.A.
      • Hall A.
      • Hall M.N.
      ,
      • Jacinto E.
      • Facchinetti V.
      • Liu D.
      • Soto N.
      • Wei S.
      • Jung S.Y.
      • Huang Q.
      • Qin J.
      • Su B.
      ,
      • Pearce L.R.
      • Huang X.
      • Boudeau J.
      • Pawłowski R.
      • Wullschleger S.
      • Deak M.
      • Ibrahim A.F.
      • Gourlay R.
      • Magnuson M.A.
      • Alessi D.R.
      ,
      • Sarbassov D.D.
      • Ali S.M.
      • Kim D.H.
      • Guertin D.A.
      • Latek R.R.
      • Erdjument-Bromage H.
      • Tempst P.
      • Sabatini D.M.
      ,
      • Yang Q.
      • Inoki K.
      • Ikenoue T.
      • Guan K.L.
      ,
      • Woo S.Y.
      • Kim D.H.
      • Jun C.B.
      • Kim Y.M.
      • Haar E.V.
      • Lee S.I.
      • Hegg J.W.
      • Bandhakavi S.
      • Griffin T.J.
      • Kim D.H.
      ). mTORC2 phosphorylates Akt and PKC, signals to small GTPases (RhoA and Rac1), and controls cytoskeleton organization (
      • Jacinto E.
      • Loewith R.
      • Schmidt A.
      • Lin S.
      • Rüegg M.A.
      • Hall A.
      • Hall M.N.
      ,
      • Sarbassov D.D.
      • Guertin D.A.
      • Ali S.M.
      • Sabatini D.M.
      ,
      • Dada S.
      • Demartines N.
      • Dormond O.
      ,
      • Facchinetti V.
      • Ouyang W.
      • Wei H.
      • Soto N.
      • Lazorchak A.
      • Gould C.
      • Lowry C.
      • Newton A.C.
      • Mao Y.
      • Miao R.Q.
      • Sessa W.C.
      • Qin J.
      • Zhang P.
      • Su B.
      • Jacinto E.
      ,
      • Ikenoue T.
      • Inoki K.
      • Yang Q.
      • Zhou X.
      • Guan K.L.
      ). Most recently, mTORC2 has been reported to phosphorylate SGK1 (serum and glucocorticoid-inducible kinase 1) (
      • García-Martínez J.M.
      • Alessi D.R.
      ), although this remains controversial (
      • Hong F.
      • Larrea M.D.
      • Doughty C.
      • Kwiatkowski D.J.
      • Squillace R.
      • Slingerland J.M.
      ). Both mTORC1 and mTORC2 interact with a negative regulator DEPTOR (
      • Peterson T.R.
      • Laplante M.
      • Thoreen C.C.
      • Sancak Y.
      • Kang S.A.
      • Kuehl W.M.
      • Gray N.S.
      • Sabatini D.M.
      ).
      Clinical trials have demonstrated that rapamycin and its analogs (CCI-779, RAD001, and AP23573) (termed rapalogs) are promising anticancer drugs. They share the same mechanism and specifically block the function of mTOR, inhibiting growth of numerous solid tumors (renal, breast, prostate, colon, and brain cancers) with only mild side effects (
      • Fasolo A.
      • Sessa C.
      ). Intensive studies have focused on the crucial roles of mTOR in controlling cell proliferation, growth, and survival. Recently this laboratory and others have further revealed its pivotal role in regulation of tumor cell migration and cancer metastasis (
      • Dada S.
      • Demartines N.
      • Dormond O.
      ,
      • Liu L.
      • Chen L.
      • Chung J.
      • Huang S.
      ,
      • Liu L.
      • Li F.
      • Cardelli J.A.
      • Martin K.A.
      • Blenis J.
      • Huang S.
      ,
      • Hernández-Negrete I.
      • Carretero-Ortega J.
      • Rosenfeldt H.
      • Hernández-García R.
      • Calderón-Salinas J.V.
      • Reyes-Cruz G.
      • Gutkind J.S.
      • Vázquez-Prado J.
      ). We found that rapamycin suppresses IGF-1-stimulated F-actin reorganization and migration in various tumor cell lines by inhibiting mTORC1-mediated 4E-BP1 and S6K1 pathways (
      • Liu L.
      • Chen L.
      • Chung J.
      • Huang S.
      ). This is in part associated with rapamycin inhibition of S6K1-mediated phosphorylation of focal adhesion proteins (FAK, paxillin, and p130Cas) (
      • Liu L.
      • Li F.
      • Cardelli J.A.
      • Martin K.A.
      • Blenis J.
      • Huang S.
      ). However, how mTOR regulates F-actin reorganization and cell motility, particularly how 4E-BP1 pathway regulates cell motility, remains to be elucidated.
      RhoA, Rac1, and Cdc42 are Rho-family small GTPases that cycle between an active GTP-bound form and an inactive GDP-bound form and were identified as key regulators of actin cytoskeletal dynamics and cell motility (
      • Heasman S.J.
      • Ridley A.J.
      ). Specifically, RhoA induces formation of actin stress fibers and focal adhesions, Rac1 stimulates formation of lamellipodia and membrane ruffles, and Cdc42 promotes formation of filopodia and actin microspikes (
      • Heasman S.J.
      • Ridley A.J.
      ,
      • Tzima E.
      ). Recent studies further show that RhoA also spatiotemporally regulates tail detachment and lamellipodia formation (
      • Kurokawa K.
      • Matsuda M.
      ,
      • Pertz O.
      • Hodgson L.
      • Klemke R.L.
      • Hahn K.M.
      ). In NIH 3T3 and HeLa cells, reduced Rac1 and RhoA activity was observed by disruption of mTORC2 using siRNAs to rictor, mLST8, or mTOR (
      • Jacinto E.
      • Loewith R.
      • Schmidt A.
      • Lin S.
      • Rüegg M.A.
      • Hall A.
      • Hall M.N.
      ). Short time (30 min) rapamycin treatment does not affect Rac1 activity in HeLa cells (
      • Hernández-Negrete I.
      • Carretero-Ortega J.
      • Rosenfeldt H.
      • Hernández-García R.
      • Calderón-Salinas J.V.
      • Reyes-Cruz G.
      • Gutkind J.S.
      • Vázquez-Prado J.
      ), but prolonged rapamycin treatment (28 days) does inhibit RhoA protein expression and activity in an ex vivo organ culture model of human internal mammary arteries (
      • Guérin P.
      • Sauzeau V.
      • Rolli-Derkinderen M.
      • Al Habbash O.
      • Scalbert E.
      • Crochet D.
      • Pacaud P.
      • Loirand G.
      ). However, the role of the small GTPases in mTOR-mediated cell motility and F-actin reorganization has not been studied.
      Here we show that rapamycin inhibited protein expression and activities of RhoA, Cdc42, and Rac1 by suppressing their protein synthesis in a panel of tumor cells. Both mTORC1 and mTORC2 regulated the activities of these proteins. However, only disruption of mTORC1 mimicked the effect of rapamycin, inhibiting their protein expression. mTORC1-mediated 4E-BP1 and S6K1 pathways were essential for the expression of these small GTPases. Constitutively active RhoA, but not Cdc42 and Rac1, prevented rapamycin inhibition of IGF-1-stimulated F-actin reorganization and cell motility. The results suggest that rapamycin inhibits F-actin reorganization and cell motility at least in part by down-regulation of RhoA protein expression and activity through mTORC1-mediated S6K1- and 4E-BP1-signaling pathways.

      DISCUSSION

      Recently we demonstrated that rapamycin inhibits F-action reorganization and cell motility by inhibition of mTOR kinase activity (
      • Liu L.
      • Chen L.
      • Chung J.
      • Huang S.
      ,
      • Liu L.
      • Li F.
      • Cardelli J.A.
      • Martin K.A.
      • Blenis J.
      • Huang S.
      ). mTOR controls synthesis of a variety of proteins, such as cyclin D1, c-Myc, ornithine decarboxylase, vascular endothelial growth factor, etc. (
      • Huang S.
      • Houghton P.J.
      ). Small GTPases (RhoA, Rac1, and Cdc42) are crucial for cytoskeleton organization and cell migration (
      • Heasman S.J.
      • Ridley A.J.
      ). Therefore, we hypothesized that rapamycin may inhibit mTOR-mediated protein synthesis or activities of the small GTPases, leading to inhibition of F-action reorganization and cell motility. To test this hypothesis, we first studied the effect of rapamycin on cellular protein levels and activities of RhoA, Rac1, and Cdc42. By Western blotting and small GTPase activity assay, we found that rapamycin did inhibit the basal and IGF-1-stimulated activities and protein expression of RhoA, Rac1, and Cdc42 in Rh30 cells. The inhibitory effect of rapamycin on expression of the small GTPases was also observed in other tumor cell lines, including those derived from cervical cancer (HeLa), prostate cancer (PC-3) (Fig. 1), Ewing sarcoma (Rh1), and glioblastoma (U-373) (data not shown), suggesting that this is not cell type-dependent.
      It is well known that rapamycin selectively inhibits mTOR kinase activity and function (
      • Guertin D.A.
      • Sabatini D.M.
      ). However, studies have also shown that rapamycin inhibits differentiation of C2C12 cells in mTOR kinase activity-independent manner (
      • Erbay E.
      • Chen J.
      ), although this remains controversial (
      • Shu L.
      • Zhang X.
      • Houghton P.J.
      ). During skeletal myogenesis, mTOR regulates the production of IGF-II mRNA, which is also independent of the kinase activity of mTOR (
      • Erbay E.
      • Park I.H.
      • Nuzzi P.D.
      • Schoenherr C.J.
      • Chen J.
      ). This prompted us to study whether rapamycin inhibition of small GTPase expression is through inhibition of mTOR kinase activity. Here we found that rapamycin failed to inhibit RhoA, Cdc42, and Rac1 expression in cells expressing rapamycin-resistant mTOR (mTOR-T) but not in control cells expressing GFP or rapamycin-resistant but kinase dead mTOR (mTOR-TE), suggesting that the kinase activity of mTOR is essential for expression of RhoA, Cdc42, and Rac1. This is consistent with our previous finding that the kinase activity of mTOR is necessary for IGF-1-stimulated F-actin reorganization and cell motility (
      • Liu L.
      • Chen L.
      • Chung J.
      • Huang S.
      ,
      • Liu L.
      • Li F.
      • Cardelli J.A.
      • Martin K.A.
      • Blenis J.
      • Huang S.
      ).
      mTOR may control protein expression at transcriptional, translational, or post-translational levels (
      • Hashemolhosseini S.
      • Nagamine Y.
      • Morley S.J.
      • Desrivières S.
      • Mercep L.
      • Ferrari S.
      ). In this study we found that rapamycin did not alter mRNA levels or protein turnover of RhoA, Cdc42, and Rac1 but inhibited protein synthesis of these small GTPases, suggesting that mTOR controls protein expression of RhoA, Cdc42, and Rac1 at translational level.
      Previous studies only showed that mTORC2 regulates the activities of RhoA and Rac1 in NIH 3T3, HeLa cells, and human umbilical vein endothelial cells (
      • Jacinto E.
      • Loewith R.
      • Schmidt A.
      • Lin S.
      • Rüegg M.A.
      • Hall A.
      • Hall M.N.
      ,
      • Dada S.
      • Demartines N.
      • Dormond O.
      ,
      • Hernández-Negrete I.
      • Carretero-Ortega J.
      • Rosenfeldt H.
      • Hernández-García R.
      • Calderón-Salinas J.V.
      • Reyes-Cruz G.
      • Gutkind J.S.
      • Vázquez-Prado J.
      ). Here for the first time we demonstrate that mTOR not only controls the cellular activities of RhoA, Rac1, and Cdc42 but also regulates the cellular protein expression of these small GTPases. Our data support the notion that mTORC1 mediates protein synthesis of RhoA, Cdc42, and Rac1; mTORC2 regulates the activities of these proteins. This is supported by the findings that disruption of mTORC1 by down-regulation of raptor inhibited the expression and activities of the small GTPases, whereas disruption of mTORC2 by down-regulation of rictor only inhibited the activities of RhoA, Cdc42, and Rac1. This is consistent with the previous findings that a 20–30% decrease in GTP-bound Rac1 was observed in mTOR-, mLST8-, or mAVO3-siRNA-transfected NIH 3T3 cells and little-to-no decrease in GTP-bound Rac1 in raptor siRNA-transfected cells (
      • Jacinto E.
      • Loewith R.
      • Schmidt A.
      • Lin S.
      • Rüegg M.A.
      • Hall A.
      • Hall M.N.
      ). Recent studies have revealed that mTORC2 regulates Rac1 activation through P-Rex1 (
      • Hernández-Negrete I.
      • Carretero-Ortega J.
      • Rosenfeldt H.
      • Hernández-García R.
      • Calderón-Salinas J.V.
      • Reyes-Cruz G.
      • Gutkind J.S.
      • Vázquez-Prado J.
      ). How mTORC2 regulates RhoA and Cdc42 activity remains to be determined.
      4E-BP1 and S6K1 are the best characterized mTORC1 effectors, which were found to be involved in the regulation of F-actin reorganization and cell motility (
      • Liu L.
      • Chen L.
      • Chung J.
      • Huang S.
      ,
      • Liu L.
      • Li F.
      • Cardelli J.A.
      • Martin K.A.
      • Blenis J.
      • Huang S.
      ). Recently we observed that only S6K1 pathway regulated phosphorylation of the focal adhesion proteins (FAK, paxillin, and p130Cas) (
      • Liu L.
      • Li F.
      • Cardelli J.A.
      • Martin K.A.
      • Blenis J.
      • Huang S.
      ), which is related to F-actin reorganization and cell migration (
      • Mitra S.K.
      • Schlaepfer D.D.
      ). It was not clear how 4E-BP1/eIF4E pathway regulates F-actin reorganization and cell motility. To address this question, we used 4EBP1-5A, which is a constitutively hypophosphorylated mutant 4E-BP1 (T36A, T45A, S64A, T69A, and S82A) (
      • Mothe-Satney I.
      • Brunn G.J.
      • McMahon L.P.
      • Capaldo C.T.
      • Abraham R.T.
      • Lawrence Jr., J.C.
      ) and can tightly binds and sequester eIF4E, inhibiting Cap-dependent translation (
      • Mothe-Satney I.
      • Brunn G.J.
      • McMahon L.P.
      • Capaldo C.T.
      • Abraham R.T.
      • Lawrence Jr., J.C.
      ). We found that expression of 4EBP1-5A mimicked the effect of rapamycin, remarkably inhibiting expression of RhoA, Cdc42, and Rac1 in Rh30 cells stimulated with or without IGF-1. We also investigated the role of S6K1 pathway in the regulation of the GTPase expression. Interestingly, down-regulation of S6K1 also impaired the expression of RhoA, Cdc42, and Rac1. In contrast, expression of a rapamycin-resistant and constitutively active of S6K1 mutant (S6K1-F5A-E389-R3A, S6K1-ca) (
      • Liu L.
      • Chen L.
      • Chung J.
      • Huang S.
      ,
      • Schalm S.S.
      • Tee A.R.
      • Blenis J.
      ) was able to confer partial resistance to rapamycin inhibition of the GTPase expression. Collectively, we conclude that both 4E-BP1 and S6K1 pathways participate in the regulation of expression of RhoA, Cdc42, and Rac1.
      RhoA, Cdc42, and Rac1 are all critical molecules for cytoskeleton organization and cell migration, but they play different roles during cytoskeletal dynamics (
      • Heasman S.J.
      • Ridley A.J.
      ,
      • Tzima E.
      ). Our data indicate that although the activities and expression of RhoA, Cdc42, and Rac1 were inhibited by rapamycin, only overexpression of constitutively active RhoA (RhoA-L63), but not Cdc42 (Cdc42-L28) and Rac1 (Rac1-L61), prevented rapamycin inhibition of IGF-1 stimulated lamellipodia formation and cell motility, implying a crucial role of RhoA in mTOR-mediated cell motility. This is strongly supported by our observations that expression of constitutively active RhoA enhanced the basal level of lamellipodia formation and cell motility to a level stimulated by IGF-1, whereas expression of dominant negative RhoA (RhoA-N19) blocked the effect of IGF-1 stimulation. Interestingly, although expression of constitutively active Cdc42 or Rac1 failed to rescue rapamycin inhibition of IGF-1 stimulated F-actin reorganization and cell migration, expression of dominant negative Cdc42 (Cdc42-N17) or Rac1 (Rac1-N17), like dominant negative RhoA (RhoA N19), abolished IGF-1-stimulated cell motility. These results suggest that a certain level of RhoA, Cdc42, or Rac1 activity may be required for either cell polarization/protrusion or adhesion/de-adhesion, leading to cell migration. In this study we also noticed that expression of constitutively active Rac1 actually increased the cellular stress fibers and inhibited IGF-1-stimulated lamellipodia formation and cell motility in Rh30 cells (Fig. 6). This is in contrast to the finding in NIH 3T3 cells in which expression of constitutively active Rac1 induces formation of membrane ruffles and lamellipodia, whereas expression of constitutively active RhoA results in formation of stress fibers (
      • Jacinto E.
      • Loewith R.
      • Schmidt A.
      • Lin S.
      • Rüegg M.A.
      • Hall A.
      • Hall M.N.
      ). The discrepancy may likely be due to different cell types used. It has been described that Rac1 is essential for actin stress fiber formation in primary mouse embryonic fibroblasts (
      • Guo F.
      • Debidda M.
      • Yang L.
      • Williams D.A.
      • Zheng Y.
      ). In colon carcinoma cells (
      • O'Connor K.L.
      • Nguyen B.K.
      • Mercurio A.M.
      ), hepatocarcinoma cells (
      • Genda T.
      • Sakamoto M.
      • Ichida T.
      • Asakura H.
      • Kojiro M.
      • Narumiya S.
      • Hirohashi S.
      ), and human microvascular endothelial cells (
      • Abécassis I.
      • Olofsson B.
      • Schmid M.
      • Zalcman G.
      • Karniguian A.
      ), activation of RhoA promotes lamellipodia formation and cell migration. Therefore, the roles of the small GTPases in the regulation of F-actin reorganization and cell motility depend on cell types studied.
      In summary, here we found that rapamycin inhibited RhoA, Cdc42, and Rac1 expression and activity in an mTOR kinase activity-dependent manner. mTORC1 controls the protein synthesis, whereas mTORC2 regulates the activities of the small GTPases. Both mTORC1-mediated S6K1 and 4E-BP1/eIF4E pathways are essential for RhoA, Cdc42, and Rac1 expression. However, inhibition of RhoA activity is primarily responsible for rapamycin inhibition of IGF-1-stimulated lamellipodia formation and cell motility.

      Acknowledgments

      We thank Drs. John Blenis, Jie Chen, Peter J. Houghton, John Lawrence, Jr., David M. Sabatini, and Yi Zheng for providing cell lines or constructs.

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