Biopolym. Cell. 2015; 31(4):294-300.
http://dx.doi.org/10.7124/bc.0008EE
Molecular and Cell Biotechnologies
Generation and characterization of polyclonal antibodies specific to N-terminal extension of p85 isoform of ribosomal protein S6 kinase 1 (p85 S6K1)
1Savinska L. O., 1Klipa O. M., 1Khoruzenko A. I., 1Shkarina K. A., 1Garifulin O. M., 1Filonenko V. V.
  1. Institute of Molecular Biology and Genetics, NAS of Ukraine
    150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680

Abstract

Aim. Generation of polyclonal antibodies specific to the ribosomal protein S6 kinase isoform – p85S6K1 and directed to the N-terminal (1–23 aa) extension of p85S6K1. Methods. Animal immunization with synthetic (1–23 aa) peptide, ELISA, Western blot, Immunoprecipitation, immunofluorescent analysis. Results. Polyclonal antibodies have been generated, which specifically recognize only p85 but not p70 isoform of S6K1 in western blot, immunoprecipitation and immunofluorescence analysis. Conclusions. The obtained antibodies can be recommended for studies on the p85S6K1 and other S6K1 isoforms possessing the N-terminal extension – the identification of binding protein partners, analysis of subcellular localization under different physiological conditions, elucidation of the signal transduction pathways involving different S6K1 isoforms.
Keywords: Ribosomal protein S6 kinase 1 (S6K1), S6K1 isoforms, p70 S6K1, p85 S6K1, polyclonal antibodies.
Full text: (PDF, in English)

References

[1] Fenton TR, Gout IT. Functions and regulation of the 70kDa ribosomal S6 kinases. Int J Biochem Cell Biol. 2011;43(1):47-59.
[2] Mamane Y, Petroulakis E, LeBacquer O, Sonenberg N. mTOR, translation initiation and cancer. Oncogene. 2006;25(48):6416-22.
[3] Manning BD, Cantley LC. AKT/PKB signaling: navigating downstream. Cell. 2007;129(7):1261-74.
[4] Yuan TL, Cantley LC. PI3K pathway alterations in cancer: variations on a theme. Oncogene. 2008;27(41):5497-510.
[5] Bandi HR, Ferrari S, Krieg J, Meyer HE, Thomas G. Iden­tification of 40 S ribosomal protein S6 phosphorylation sites in Swiss mouse 3T3 fibroblasts stimulated with serum. J Biol Chem. 1993;268(6):4530–3.
[6] Krieg J, Hofsteenge J, Thomas G. Identification of the 40 S ribosomal protein S6 phosphorylation sites induced by cycloheximide. J Biol Chem. 1988;263(23):11473-7.
[7] Grove JR, Banerjee P, Balasubramanyam A, Coffer PJ, Price DJ, Avruch J, Woodgett JR. Cloning and expression of two human p70 S6 kinase polypeptides differing only at their amino termini. Mol Cell Biol. 1991;11(11):5541-50.
[8] Reinhard C, Fernandez A, Lamb NJ, Thomas G. Nuclear localization of p85s6k: functional requirement for entry into S phase. EMBO J. 1994;13(7):1557-65.
[9] Valovka T, Verdier F, Cramer R, Zhyvoloup A, Fenton T, Rebholz H, Wang ML, Gzhegotsky M, Lutsyk A, Matsuka G, Filonenko V, Wang L, Proud CG, Parker PJ, Gout IT. Protein kinase C phosphorylates ribosomal protein S6 kinase betaII and regulates its subcellular localization. Mol Cell Biol. 2003;23(3):852-63.
[10] Panasyuk G, Nemazanyy I, Zhyvoloup A, Bretner M, Litchfield DW, Filonenko V, Gout IT. Nuclear export of S6K1 II is regulated by protein kinase CK2 phosphorylation at Ser-17. J Biol Chem. 2006;281(42):31188-201.
[11] Karni R, de Stanchina E, Lowe SW, Sinha R, Mu D, Krainer AR. The gene encoding the splicing factor SF2/ASF is a proto-oncogene. Nat Struct Mol Biol. 2007;14(3):185-93.
[12] Ben-Hur V, Denichenko P, Siegfried Z, Maimon A, Krainer A, Davidson B, Karni R. S6K1 alternative splicing modulates its oncogenic activity and regulates mTORC1. Cell Rep. 2013;3(1):103-15.
[13] Savinska LO, Lyzogubov VV, Usenko VS, Ovcharenko GV, Gorbenko ON, Rodnin MV, Vudmaska MI, Pogribniy PV, Kyya­mova RG, Panasyuk GG, Nemazanyy IO, Malets MS, Palchevskyy SS, Gout IT, Filonenko VV. Immunohistochemical analysis of S6K1 and S6K2 expression in human breast tumors. Eksp Onkol. 2004;26(1):24–30.
[14] Lytvyn DI, Dudchenko TM, Lyzogubov VV, Usenko VS, Nespryadko SV, Vinnitskaya AB, Vorobyova LI, Pal’chevskiy SS, Filonenko VV, Pogrebnoy PV. Expression of aand b-isoform of p70S6 kinase in human endometrial tumors. Exp Oncol. 2003; 25(4):274–8.
[15] Lysogubov VV, Usenko VS, Khojaenko YuS, Lytvyn DI, Sol­datkina MA, Rodnin NV, Filonenko VV, Pogrebniy PV. Im­mu­nohistochemical analysis of p70S6 kinase a in human thyroid tissue upon pathology. Exp Oncol. 2003; 25(4): 304–6.
[16] 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.
[17] 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.
[18] Lyzogubov VV, Lytvyn DI, Dudchenko TM, Lubchenko NV, Pogrybniy PV, Nespryadko SV, Vinnitska AB, Usenko VS, Gout IT, Filonenko VV. Imunnohistochemical study of S6K1 and S6K2 expression in human endometrial adenocarcinomas. Exp Oncol. 2004; 26(4):287–93.
[19] Pogrebnoy PV, Kukharenko AP, Tykhonkova IA, Pal’chevskiy SS, Savinskaya LA, Pogrebnaya AP, Valevka TI, Markeeva NV, Soldatkina MA, Matsuka GKh, Gout IT, Filonenko VV. Generation and characterization of monoclonal antibodies to p70S6kinase. Exp Oncol. 1999; 21(4):232–8.
[20] Savinska LO, Kijamova RG, Pogrebnoy PV, Ovcharenko GV, Gout IT, Filonenko VV. Comparative characterization of S6 kinase α and β isoforms expression in mammalian tissues. Biopolym Cell. 2001; 17(5):374–79.