Biopolym. Cell. 2015; 31(5):379-386.
http://dx.doi.org/10.7124/bc.0008FA
Molecular and Cell Biotechnologies
Development of monoclonal antibody against protein Rictor, a component of the mTORC2 complex
1Malanchuk O. M.
  1. Institute of Molecular Biology and Genetics, NAS of Ukraine
    150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680

Abstract

Aim. Rictor is a component of the protein complex mTORC2 that is activated by growth factors and regulates cell growth, survival and migration. Here, we describe the development of the Rictor specific monoclonal antibody and characterize its suitability for various immunological assays. Methods. Hybridoma technology has been used for the monoclonal antibody production. Immunization was carried out with the recombinant N-terminal fragment of human Rictor expressed in E. coli as a GST-tagged fusion protein. Results. Specific monoclonal antibody (mAb) against Rictor has been developed. Conclusions. The generated mAb specifically recognizes the recombinant and endogenous Rictor and is suitable for Western blotting, immunoprecipitation, and immunofluorescence assays of mammalian cells. This mAb will be a useful tool for the investigations of a physiological role of Rictor as well as the function of the mTORC2 in general.
Keywords: Rictor, mTORC2, hybridoma technique, monoclonal antibody
Full text: (PDF, in English)

References

[1] Laplante M, Sabatini DM. mTOR signaling in growth control and disease. Cell. 2012;149(2):274-93.
[2] Shimobayashi M, Hall MN. Making new contacts: the mTOR network in metabolism and signalling crosstalk. Nat Rev Mol Cell Biol. 2014;15(3):155-62.
[3] Filonenko VV, Tytarenko R, Azatjan SK, Savinska LO, Gaydar YA, Gout IT, Usenko VS, Lyzogubov VV. Immunohistochemical analysis of S6K1 and S6K2 localization in human breast tumors. Exp Oncol. 2004;26(4):294-9.
[4] Lyzogubov V, Khozhaenko Y, Usenko V, Antonjuk S, Ovcharenko G, Tikhonkova I, Filonenko V. Immunohistochemical analysis of Ki-67, PCNA and S6K1/2 expression in human breast cancer. Exp Oncol. 2005;27(2):141-4.
[5] Sarbassov DD, Ali SM, Kim DH, Guertin DA, Latek RR, Erdjument-Bromage H, Tempst P, Sabatini DM. Rictor, a novel binding partner of mTOR, defines a rapamycin-insensitive and raptor-independent pathway that regulates the cytoskeleton. Curr Biol. 2004;14(14):1296-302.
[6] Pearce LR, Huang X, Boudeau J, Pawłowski R, Wullschleger S, Deak M, Ibrahim AF, Gourlay R, Magnuson MA, Alessi DR. Identification of Protor as a novel Rictor-binding component of mTOR complex-2. Biochem J. 2007;405(3):513-22.
[7] Woo SY, Kim DH, Jun CB, Kim YM, Haar EV, Lee SI, Hegg JW, Bandhakavi S, Griffin TJ, Kim DH. PRR5, a novel component of mTOR complex 2, regulates platelet-derived growth factor receptor beta expression and signaling. J Biol Chem. 2007;282(35):25604-12.
[8] Kaizuka T, Hara T, Oshiro N, Kikkawa U, Yonezawa K, Takehana K, Iemura S, Natsume T, Mizushima N. Tti1 and Tel2 are critical factors in mammalian target of rapamycin complex assembly. J Biol Chem. 2010;285(26):20109-16.
[9] Sarbassov DD, Guertin DA, Ali SM, Sabatini DM. Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science. 2005;307(5712):1098-101.
[10] Jacinto E, Loewith R, Schmidt A, Lin S, Rüegg MA, Hall A, Hall MN. Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive. Nat Cell Biol. 2004;6(11):1122-8.
[11] García-Martínez JM, Alessi DR. mTOR complex 2 (mTORC2) controls hydrophobic motif phosphorylation and activation of serum- and glucocorticoid-induced protein kinase 1 (SGK1). Biochem J. 2008;416(3):375-85.
[12] Facchinetti V, Ouyang W, Wei H, Soto N, Lazorchak A, Gould C, Lowry C, Newton AC, Mao Y, Miao RQ, Sessa WC, Qin J, Zhang P, Su B, Jacinto E. The mammalian target of rapamycin complex 2 controls folding and stability of Akt and protein kinase C. EMBO J. 2008;27(14):1932-43.
[13] Ikenoue T, Inoki K, Yang Q, Zhou X, Guan KL. Essential function of TORC2 in PKC and Akt turn motif phosphorylation, maturation and signalling. EMBO J. 2008;27(14):1919-31.
[14] Harlow E, Lane D. Using antibodies: A laboratory manual. New York: Cold Spring Harbor Lab. press, 1998. 495 p.
[15] Hurrell JGR. Monoclonal hybridoma antibodies: Techniques and application. Boca Raton: CRC press, 1982. 231 p.
[16] Boulbés DR, Shaiken T, Sarbassov dos D. Endoplasmic reticulum is a main localization site of mTORC2. Biochem Biophys Res Commun. 2011;413(1):46-52.
[17] Desai BN, Myers BR, Schreiber SL. FKBP12-rapamycin-associated protein associates with mitochondria and senses osmotic stress via mitochondrial dysfunction. Proc Natl Acad Sci U S A. 2002;99(7):4319-24.
[18] Betz C, Stracka D, Prescianotto-Baschong C, Frieden M, Demaurex N, Hall MN. Feature Article: mTOR complex 2-Akt signaling at mitochondria-associated endoplasmic reticulum membranes (MAM) regulates mitochondrial physiology. Proc Natl Acad Sci U S A. 2013;110(31):12526-34.
[19] Poston CN, Krishnan SC, Bazemore-Walker CR. In-depth proteomic analysis of mammalian mitochondria-associated membranes (MAM). J Proteomics. 2013;79:219-30.
[20] Rosner M, Hengstschläger M. Cytoplasmic/nuclear localization of tuberin in different cell lines. Amino Acids. 2007;33(4):575-9.
[21] Rosner M, Hengstschläger M. Cytoplasmic and nuclear distribution of the protein complexes mTORC1 and mTORC2: rapamycin triggers dephosphorylation and delocalization of the mTORC2 components rictor and sin1. Hum Mol Genet. 2008;17(19):2934-48.
[22] Rosner M, Hengstschläger M. mTOR protein localization is cell cycle-regulated. Cell Cycle. 2011;10(20):3608-10.
[23] Rosner M, Hengstschläger M. Detection of cytoplasmic and nuclear functions of mTOR by fractionation. Methods Mol Biol. 2012;821:105-24.
[24] Kunz J, Schneider U, Howald I, Schmidt A, Hall MN. HEAT repeats mediate plasma membrane localization of Tor2p in yeast. J Biol Chem. 2000;275(47):37011-20.
[25] Wedaman KP, Reinke A, Anderson S, Yates J 3rd, McCaffery JM, Powers T. Tor kinases are in distinct membrane-associated protein complexes in Saccharomyces cerevisiae. Mol Biol Cell. 2003;14(3):1204–20.
[26] Aronova S, Wedaman K, Anderson S, Yates J 3rd, Powers T. Probing the membrane environment of the TOR kinases reveals functional interactions between TORC1, actin, and membrane trafficking in Saccharomyces cerevisiae. Mol Biol Cell. 2007;18(8):2779–94.
[27] Sturgill TW, Cohen A, Diefenbacher M, Trautwein M, Martin DE, Hall MN. TOR1 and TOR2 have distinct locations in live cells. Eukaryot Cell. 2008;7(10):1819-30.
[28] Berchtold D, Walther TC. TORC2 plasma membrane localization is essential for cell viability and restricted to a distinct domain. Mol Biol Cell. 2009;20(5):1565-75.