Abstract
The Rheb1 and Rheb2 small GTPases and their effector mTOR are aberrantly activated in human cancer and are attractive targets for anti-cancer drug discovery. Rheb is targeted to endomembranes via its C-terminal CAAX (C=cysteine, A=aliphatic, X=terminal amino acid) motif, a substrate for posttranslational modification by a farnesyl isoprenoid. After farnesylation, Rheb undergoes two additional CAAX-signaled processing steps, Ras converting enzyme 1 (Rce1)-catalyzed cleavage of the AAX residues and isoprenylcysteine carboxyl methyltransferase (Icmt)-mediated carboxylmethylation of the farnesylated cysteine. However, whether these postprenylation processing steps are required for Rheb signaling through mTOR is not known. We found that Rheb1 and Rheb2 localize primarily to the endoplasmic reticulum and Golgi apparatus. We determined that Icmt and Rce1 processing is required for Rheb localization, but is dispensable for Rheb-induced activation of the mTOR substrate p70 S6 kinase (S6K). Finally, we evaluated whether farnesylthiosalicylic acid (FTS) blocks Rheb localization and function. Surprisingly, FTS prevented S6K activation induced by a constitutively active mTOR mutant, indicating that FTS inhibits mTOR at a level downstream of Rheb. We conclude that inhibitors of Icmt and Rce1 will not block Rheb function, but FTS could be a promising treatment for Rheb- and mTOR-dependent cancers.
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References
Aspuria PJ, Tamanoi F . (2004). The Rheb family of GTP-binding proteins. Cell Signal 16: 1105–1112.
Astrinidis A, Henske EP . (2005). Tuberous sclerosis complex: linking growth and energy signaling pathways with human disease. Oncogene 24: 7475–7481.
Basso AD, Mirza A, Liu G, Long BJ, Bishop WR, Kirschmeier P . (2005). The farnesyl transferase inhibitor (FTI) SCH66336 (lonafarnib) inhibits Rheb farnesylation and mTOR signaling. Role in FTI enhancement of taxane and tamoxifen anti-tumor activity. J Biol Chem 280: 31101–31108.
Blum R, Cox AD, Kloog Y . (2008). Inhibitors of chronically active ras: potential for treatment of human malignancies. Recent Pat Anticancer Drug Discov 3: 31–37.
Bodemann BO, White MA . (2008). Ral GTPases and cancer: linchpin support of the tumorigenic platform. Nat Rev Cancer 8: 133–140.
Buerger C, DeVries B, Stambolic V . (2006). Localization of Rheb to the endomembrane is critical for its signaling function. Biochem Biophys Res Commun 344: 869–880.
Castro AF, Rebhun JF, Clark GJ, Quilliam LA . (2003). Rheb binds tuberous sclerosis complex 2 (TSC2) and promotes S6 kinase activation in a rapamycin- and farnesylation-dependent manner. J Biol Chem 278: 32493–32496.
Chakraborty S, Mohiyuddin SA, Gopinath KS, Kumar A . (2008). Involvement of TSC genes and differential expression of other members of the mTOR signaling pathway in oral squamous cell carcinoma. BMC Cancer 8: 163.
Chenette EJ, Mitin NY, Der CJ . (2006). Multiple sequence elements facilitate Chp Rho GTPase subcellular location, membrane association, and transforming activity. Mol Biol Cell 17: 3108–3121.
Chou MM, Blenis J . (1996). The 70 kDa S6 kinase complexes with and is activated by the Rho family G proteins Cdc42 and Rac1. Cell 85: 573–583.
Choy E, Chiu VK, Silletti J, Feoktistov M, Morimoto T, Michaelson D et al. (1999). Endomembrane trafficking of ras: the CAAX motif targets proteins to the ER and Golgi. Cell 98: 69–80.
Clark GJ, Kinch MS, Rogers-Graham K, Sebti SM, Hamilton AD, Der CJ . (1997). The Ras-related protein Rheb is farnesylated and antagonizes Ras signaling and transformation. J Biol Chem 272: 10608–10615.
Cox AD, Der CJ . (2002). Farnesyltransferase inhibitors: promises and realities. Curr Opin Pharmacol 2: 388–393.
Cully M, You H, Levine AJ, Mak TW . (2006). Beyond PTEN mutations: the PI3K pathway as an integrator of multiple inputs during tumorigenesis. Nat Rev Cancer 6: 184–192.
Drenan RM, Liu X, Bertram PG, Zheng XF . (2004). FKBP12-rapamycin-associated protein or mammalian target of rapamycin (FRAP/mTOR) localization in the endoplasmic reticulum and the Golgi apparatus. J Biol Chem 279: 772–778.
Du G, Altshuller YM, Vitale N, Huang P, Chasserot-Golaz S, Morris AJ et al. (2003). Regulation of phospholipase D1 subcellular cycling through coordination of multiple membrane association motifs. J Cell Biol 162: 305–315.
Gau CL, Kato-Stankiewicz J, Jiang C, Miyamoto S, Guo L, Tamanoi F . (2005). Farnesyltransferase inhibitors reverse altered growth and distribution of actin filaments in Tsc-deficient cells via inhibition of both rapamycin-sensitive and -insensitive pathways. Mol Cancer Ther 4: 918–926.
Gromov PS, Madsen P, Tomerup N, Celis JE . (1995). A novel approach for expression cloning of small GTPases: identification, tissue distribution and chromosome mapping of the human homolog of Rheb. FEBS Lett 377: 221–226.
Haklai R, Weisz MG, Elad G, Paz A, Marciano D, Egozi Y et al. (1998). Dislodgment and accelerated degradation of Ras. Biochemistry 37: 1306–1314.
Heo WD, Meyer T . (2003). Switch-of-function mutants based on morphology classification of Ras superfamily small GTPases. Cell 113: 315–328.
Huang J, Manning BD . (2008). The TSC1-TSC2 complex: a molecular switchboard controlling cell growth. Biochem J 412: 179–190.
Inoki K, Corradetti MN, Guan KL . (2005). Dysregulation of the TSC-mTOR pathway in human disease. Nat Genet 37: 19–24.
Jiang H, Vogt PK . (2008). Constitutively active Rheb induces oncogenic transformation. Oncogene 27: 5729–5740.
Kloog Y, Cox AD . (2004). Prenyl-binding domains: potential targets for Ras inhibitors and anti-cancer drugs. Semin Cancer Biol 14: 253–261.
Konstantinopoulos PA, Karamouzis MV, Papavassiliou AG . (2007). Post-translational modifications and regulation of the RAS superfamily of GTPases as anticancer targets. Nat Rev Drug Discov 6: 541–555.
Law BK, Norgaard P, Moses HL . (2000). Farnesyltransferase inhibitor induces rapid growth arrest and blocks p70s6k activation by multiple stimuli. J Biol Chem 275: 10796–10801.
Long X, Lin Y, Ortiz-Vega S, Yonezawa K, Avruch J . (2005). Rheb binds and regulates the mTOR kinase. Curr Biol 15: 702–713.
Ma D, Bai X, Guo S, Jiang Y . (2008). The switch I region of Rheb is critical for its interaction with FKBP38. J Biol Chem 283: 25963–25970.
Maehama T, Tanaka M, Nishina H, Murakami M, Kanaho Y, Hanada K . (2008). RalA functions as an indispensable signal mediator for the nutrient-sensing system. J Biol Chem 283: 35053–35059.
Marom M, Haklai R, Ben-Baruch G, Marciano D, Egozi Y, Kloog Y . (1995). Selective inhibition of Ras-dependent cell growth by farnesylthiosalisylic acid. J Biol Chem 270: 22263–22270.
Mavrakis KJ, Zhu H, Silva RL, Mills JR, Teruya-Feldstein J, Lowe SW et al. (2008). Tumorigenic activity and therapeutic inhibition of Rheb GTPase. Genes Dev 22: 2178–2188.
McMahon LP, Yue W, Santen RJ, Lawrence Jr JC . (2005). Farnesylthiosalicylic acid inhibits mammalian target of rapamycin (mTOR) activity both in cells and in vitro by promoting dissociation of the mTOR-raptor complex. Mol Endocrinol 19: 175–183.
Michaelson D, Ali W, Chiu VK, Bergo M, Silletti J, Wright L et al. (2005). Postprenylation CAAX processing is required for proper localization of Ras but not Rho GTPases. Mol Biol Cell 16: 1606–1616.
Michaelson D, Silletti J, Murphy G, D’Eustachio P, Rush M, Philips MR . (2001). Differential localization of Rho GTPases in live cells: regulation by hypervariable regions and RhoGDI binding. J Cell Biol 152: 111–126.
Nakase Y, Fukuda K, Chikashige Y, Tsutsumi C, Morita D, Kawamoto S et al. (2006). A defect in protein farnesylation suppresses a loss of Schizosaccharomyces pombe tsc2+, a homolog of the human gene predisposing to tuberous sclerosis complex. Genetics 173: 569–578.
Nardella C, Chen Z, Salmena L, Carracedo A, Alimonti A, Egia A et al. (2008). Aberrant Rheb-mediated mTORC1 activation and Pten haploinsufficiency are cooperative oncogenic events. Genes Dev 22: 2172–2177.
Peterson TR, Laplante M, Thoreen CC, Sancak Y, Kang SA, Kuehl WM et al. (2009). DEPTOR is an mTOR inhibitor frequently overexpressed in multiple myeloma cells and required for their survival. Cell 137: 873–886.
Roberts PJ, Mitin N, Keller PJ, Chenette EJ, Madigan JP, Currin RO et al. (2008). Rho Family GTPase modification and dependence on CAAX motif-signaled posttranslational modification. J Biol Chem 283: 25150–25163.
Rotblat B, Ehrlich M, Haklai R, Kloog Y . (2008). The ras inhibitor farnesylthiosalicylic acid (salirasib) disrupts the spatiotemporal localization of active ras: a potential treatment for cancer. Methods Enzymol 439: 467–489.
Rowell CA, Kowalczyk JJ, Lewis MD, Garcia AM . (1997). Direct demonstration of geranylgeranylation and farnesylation of Ki-Ras in vivo. J Biol Chem 272: 14093–14097.
Saito K, Araki Y, Kontani K, Nishina H, Katada T . (2005). Novel role of the small GTPase Rheb: its implication in endocytic pathway independent of the activation of mammalian target of rapamycin. J Biochem (Tokyo) 137: 423–430.
Sancak Y, Peterson TR, Shaul YD, Lindquist RA, Thoreen CC, Bar-Peled L et al. (2008). The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1. Science 320: 1496–1501.
Sato T, Umetsu A, Tamanoi F . (2008). Characterization of the Rheb-mTOR signaling pathway in mammalian cells: constitutive active mutants of Rheb and mTOR. Methods Enzymol 438: 307–320.
Sebti SM, Der CJ . (2003). Opinion: searching for the elusive targets of farnesyltransferase inhibitors. Nat Rev Cancer 3: 945–951.
Takahashi K, Nakagawa M, Young SG, Yamanaka S . (2005). Differential membrane localization of ERas and Rheb, two Ras-related proteins involved in the phosphatidylinositol 3-kinase/mTOR pathway. J Biol Chem 280: 32768–32774.
Urano J, Comiso MJ, Guo L, Aspuria PJ, Deniskin R, Tabancay Jr AP et al. (2005). Identification of novel single amino acid changes that result in hyperactivation of the unique GTPase, Rheb, in fission yeast. Mol Microbiol 58: 1074–1086.
Urano J, Sato T, Matsuo T, Otsubo Y, Yamamoto M, Tamanoi F . (2007). Point mutations in TOR confer Rheb-independent growth in fission yeast and nutrient-independent mammalian TOR signaling in mammalian cells. Proc Natl Acad Sci USA 104: 3514–3519.
Weisz B, Giehl K, Gana-Weisz M, Egozi Y, Ben-Baruch G, Marciano D et al. (1999). A new functional Ras antagonist inhibits human pancreatic tumor growth in nude mice. Oncogene 18: 2579–2588.
Whyte DB, Kirschmeier P, Hockenberry TN, Nunez-Oliva I, James L, Catino JJ et al. (1997). K- and N-Ras are geranylgeranylated in cells treated with farnesyl protein transferase inhibitors. J Biol Chem 272: 14459–14464.
Wienecke R, Maize Jr JC, Shoarinejad F, Vass WC, Reed J, Bonifacino JS et al. (1996). Co-localization of the TSC2 product tuberin with its target Rap1 in the Golgi apparatus. Oncogene 13: 913–923.
Winter-Vann AM, Casey PJ . (2005). Post-prenylation-processing enzymes as new targets in oncogenesis. Nat Rev Cancer 5: 405–412.
Wood LD, Parsons DW, Jones S, Lin J, Sjoblom T, Leary RJ et al. (2007). The genomic landscapes of human breast and colorectal cancers. Science 318: 1108–1113.
Yue W, Fan P, Wang J, Li Y, Santen RJ . (2007). Mechanisms of acquired resistance to endocrine therapy in hormone-dependent breast cancer cells. J Steroid Biochem Mol Biol 106: 102–110.
Yue W, Wang J, Li Y, Fan P, Santen RJ . (2005). Farnesylthiosalicylic acid blocks mammalian target of rapamycin signaling in breast cancer cells. Int J Cancer 117: 746–754.
Acknowledgements
We thank Yoel Kloog, Saïd Sebti, and Andrew Hamilton for inhibitors, Lawrence Quilliam and Mark Philips for providing plasmid constructs, Stephen Young for MEF cell lines deficient in Rce1 and Icmt, and the UNC Michael Hooker Microscopy Facility for imaging assistance. This research was supported by grants from the National Institutes of Health to CJD (CA042978), ADC (CA109550), CJD and ADC (CA67771), and FT (CA41996). ABH was supported by a Department of Defense Breast Cancer Research Program predoctoral fellowship (BC061107).
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Hanker, A., Mitin, N., Wilder, R. et al. Differential requirement of CAAX-mediated posttranslational processing for Rheb localization and signaling. Oncogene 29, 380–391 (2010). https://doi.org/10.1038/onc.2009.336
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DOI: https://doi.org/10.1038/onc.2009.336
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