Biopolym. Cell. 2015; 31(6):422-428.
Structure and Function of Biopolymers
Association of Rictor with chromosomes during mitosis in MCF7 cells
1Malanchuk O. M.
- Institute of Molecular Biology and Genetics, NAS of Ukraine
150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680
Abstract
The mTOR kinase forms two multicomponent protein complexes (mTORC1 and mTORC2), where protein Rictor is a unique component of mTORC2. These two complexes differ both structurally and functionally and transmit signals to distinct downstream substrates. In contrast to mTORC1, comparatively little is known about functioning and regulation of mTORC2 and its components. Aim. To analyze of the particularity of Rictor subcellular localization in MCF7 cells. Methods. Immunofluorescence assay using confocal microscopy was applied. Results. Analysis of Rictor localization with anti N-terminal mAb revealed a different pattern of immunostaining in cells that was depended on cell cycle phase. For the first time it was shown that Rictor is at the exposed edge of condensed chromatin pool in MCF7 cells. Furthermore Z-stacking of cells revealed that character of association depends on mitosis phases. Conclusions. Our novel findings add to our understanding of functioning of both Rictor and mTORC2 in cell cycle progression.
Keywords: Rictor, mTORC2, immunofluorescence, subcellular localization, mitosis
Full text: (PDF, in English)
References
[2]
Filonenko VV. PI3K/mTOR/S6K signaling pathway – new players and new functional links. Biopolym Cell. 2013;29(3):207-14.
[3]
Laplante M, Sabatini DM. mTOR signaling in growth control and disease. Cell. 2012;149(2):274-93.
[4]
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.
[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]
Jacinto E, Facchinetti V, Liu D, Soto N, Wei S, Jung SY, Huang Q, Qin J, Su B. SIN1/MIP1 maintains rictor-mTOR complex integrity and regulates Akt phosphorylation and substrate specificity. Cell. 2006;127(1):125-37.
[10]
Shiota C, Woo JT, Lindner J, Shelton KD, Magnuson MA. Multiallelic disruption of the rictor gene in mice reveals that mTOR complex 2 is essential for fetal growth and viability. Dev Cell. 2006;11(4):583-9.
[11]
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.
[12]
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.
[13]
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.
[14]
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.
[15]
Gao D, Wan L, Wei W. Phosphorylation of Rictor at Thr1135 impairs the Rictor/Cullin-1 complex to ubiquitinate SGK1. Protein Cell. 2010;1(10):881-5.
[16]
Hagan GN, Lin Y, Magnuson MA, Avruch J, Czech MP. A Rictor-Myo1c complex participates in dynamic cortical actin events in 3T3-L1 adipocytes. Mol Cell Biol. 2008;28(13):4215-26.
[17]
McDonald PC, Oloumi A, Mills J, Dobreva I, Maidan M, Gray V, Wederell ED, Bally MB, Foster LJ, Dedhar S. Rictor and integrin-linked kinase interact and regulate Akt phosphorylation and cancer cell survival. Cancer Res. 2008;68(6):1618-24.
[18]
Zhang F, Zhang X, Li M, Chen P, Zhang B, Guo H, Cao W, Wei X, Cao X, Hao X, Zhang N. mTOR complex component Rictor interacts with PKCzeta and regulates cancer cell metastasis. Cancer Res. 2010;70(22):9360-70.
[19]
Malanchuk OM. Development of monoclonal antibody against protein Rictor, a component of the mTORC2 complex. Biopolym Cell. 2015; 31(5):379-86.
[20]
Johnson, D. How to Do Everything: Digital Camera [Online]; McGraw Hill Professional: New York, 2008; 336 p.
[22]
Zhou X, Clister TL, Lowry PR, Seldin MM, Wong GW, Zhang J. Dynamic Visualization of mTORC1 Activity in Living Cells. Cell Rep. 2015 Mar 11. pii: S2211-1247(15)00177-1.
[23]
Kosach VR, Cherednyk OV, Khoruzhenko AI. Characteristic of mTOR signaling and its involvement in the regulation of cell movements through remodeling the cytoskeleton architecture. Biopolym Cell. 2015; 31(1):5-14.
[24]
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.
[25]
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.
[26]
Rosner M, Hengstschläger M. mTOR protein localization is cell cycle-regulated. Cell Cycle. 2011;10(20):3608-10.
[27]
Rosner M, Hengstschläger M. Detection of cytoplasmic and nuclear functions of mTOR by fractionation. Methods Mol Biol. 2012;821:105-24.
[28]
Kazyken D, Kaz Y, Kiyan V, Zhylkibayev AA, Chen CH, Agarwal NK, Sarbassov dos D. The nuclear import of ribosomal proteins is regulated by mTOR. Oncotarget. 2014;5(20):9577-93.
[29]
Andrade MA, Perez-Iratxeta C, Ponting CP. Protein repeats: structures, functions, and evolution. J Struct Biol. 2001;134(2-3):117-31.
[30]
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.
[31]
Gwinn DM, Asara JM, Shaw RJ. Raptor is phosphorylated by cdc2 during mitosis. PLoS One. 2010;5(2):e9197.
[32]
Ramírez-Valle F, Badura ML, Braunstein S, Narasimhan M, Schneider RJ. Mitotic raptor promotes mTORC1 activity, G(2)/M cell cycle progression, and internal ribosome entry site-mediated mRNA translation. Mol Cell Biol. 2010;30(13):3151-64.
[33]
Vazquez-Martin A, Cufí S, Oliveras-Ferraros C, Menendez JA. Raptor, a positive regulatory subunit of mTOR complex 1, is a novel phosphoprotein of the rDNA transcription machinery in nucleoli and chromosomal nucleolus organizer regions (NORs). Cell Cycle. 2011;10(18):3140-52.
[34]
Vazquez-Martin A, Sauri-Nadal T, Menendez OJ, Oliveras-Ferraros C, Cufí S, Corominas-Faja B, López-Bonet E, Menendez JA. Ser2481-autophosphorylated mTOR colocalizes with chromosomal passenger proteins during mammalian cell cytokinesis. Cell Cycle. 2012;11(22):4211-21.
