Ukr.Biochem.J. 2018; Volume 90, Issue 4, Jul-Aug, pp. 25-35
doi: https://doi.org/10.15407/ubj90.04.025
The p60-S6K1 isoform of ribosomal protein S6 kinase 1 is a product of alternative mRNA translation
I. V. Zaiets, A. S. Sivchenko, A. I. Khoruzhenko, L. O. Savinska, V. V. Filonenko
Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, Kyiv;
e-mail: filonenko@imbg.org.ua
Ribosomal protein S6 kinase 1 (S6K1) is a well-known downstream effector of mTORC1 (mechanistic target of rapamycin complex 1) participating primarily in the regulation of cell growth and metabolism. Deregulation of mTOR/S6K1 signaling can promote numerous human pathologies, including cancer, neurodegeneration, cardiovascular disease, and metabolic disorders. As existing data suggest, the S6K1 gene encodes several protein isoforms, including p85-S6K1, p70-S6K1, and p60-S6K1. The two of these isoforms, p85-S6K1 and p70-S6K1, were extensively studied to date. The origin and functional significance of the p60-S6K1 isoform remains a mystery, however, it was suggested that the isoform could be a product of alternative S6K1 mRNA translation. Herein we report the generation of HEK-293 cells exclusively expressing p60-S6K1 as a result of CRISPR/Cas9-mediated inactivation of p85/p70-S6K1 translation. Moreover, the generated modified cells displayed the elevated level of p60-S6K1 expression compared to that in wild-type HEK-293 cells. Our data confirm an assumption that p60-S6K1 is alternatively translated, most probably, from the common for both p70- and p85-S6K1 mRNA transcript and reveal a link between p60-S6K1 expression and such cellular processes as cell proliferation and motility. In addition, our findings indicate that the p60-S6K1 isoform of S6K1 may undergo a mode of regulation distinct from p70- and p85-S6K1 due to the absence of mTOR-regulated p60-S6K1 phosphorylation at T389 that is important for S6K1 activation.
Keywords: Akt/mTOR/S6K1 signaling pathway, CRISPR/Cas9 technology, p60-S6 kinase 1 (p60-S6K1) isoform
References:
- Dann SG, Selvaraj A, Thomas G. mTOR Complex1-S6K1 signaling: at the crossroads of obesity, diabetes and cancer. Trends Mol Med. 2007 Jun;13(6):252-9. PubMed, CrossRef
- Magnuson B, Ekim B, Fingar DC. Regulation and function of ribosomal protein S6 kinase (S6K) within mTOR signalling networks. Biochem J. 2012 Jan 1;441(1):1-21. PubMed, CrossRef
- Zoncu R, Efeyan A, Sabatini DM. mTOR: from growth signal integration to cancer, diabetes and ageing. Nat Rev Mol Cell Biol. 2011 Jan;12(1):21-35. PubMed, PubMed, CrossRef
- Wullschleger S, Loewith R, Hall MN. TOR signaling in growth and metabolism. Cell. 2006 Feb 10;124(3):471-84. PubMed, CrossRef
- Fenton TR, Gout IT. Functions and regulation of the 70kDa ribosomal S6 kinases. Int J Biochem Cell Biol. 2011 Jan;43(1):47-59. PubMed, CrossRef
- 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 Nov;11(11):5541-50. PubMed, PubMedCentral, CrossRef
- 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 Mar;14(3):185-93. PubMed, PubMed, CrossRef
- Kim D, Akcakanat A, Singh G, Sharma C, Meric-Bernstam F. Regulation and localization of ribosomal protein S6 kinase 1 isoforms. Growth Factors. 2009 Feb;27(1):12-21. PubMed, CrossRef
- Cheatham L, Monfar M, Chou MM, Blenis J. Structural and functional analysis of pp70S6k. Proc Natl Acad Sci USA. 1995 Dec 5;92(25):11696-700. PubMed, PubMedCentral, CrossRef
- Mahalingam M, Templeton DJ. Constitutive activation of S6 kinase by deletion of amino-terminal autoinhibitory and rapamycin sensitivity domains. Mol Cell Biol. 1996 Jan;16(1):405-13. PubMed, PubMedCentral, CrossRef
- Schalm SS, Blenis J. Identification of a conserved motif required for mTOR signaling. Curr Biol. 2002 Apr 16;12(8):632-9. PubMed, CrossRef
- 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 Oct 20;281(42):31188-201. PubMed, CrossRef
- Bärlund M, Monni O, Kononen J, Cornelison R, Torhorst J, Sauter G, Kallioniemi OLLI-P, Kallioniemi A. Multiple genes at 17q23 undergo amplification and overexpression in breast cancer. Cancer Res. 2000 Oct 1;60(19):5340-4. PubMed
- van der Hage JA, van den Broek LJ, Legrand C, Clahsen PC, Bosch CJ, Robanus-Maandag EC, van de Velde CJ, van de Vijver MJ. Overexpression of P70 S6 kinase protein is associated with increased risk of locoregional recurrence in node-negative premenopausal early breast cancer patients. Br J Cancer. 2004 Apr 19;90(8):1543-50. PubMed, PubMedCentral, CrossRef
- Bostner J, Karlsson E, Eding CB, Perez-Tenorio G, Franzén H, Konstantinell A, Fornander T, Nordenskjöld B, Stål O. S6 kinase signaling: tamoxifen response and prognostic indication in two breast cancer cohorts. Endocr Relat Cancer. 2015 Jun;22(3):331-43. PubMed, CrossRef
- Kremer CL, Klein RR, Mendelson J, Browne W, Samadzedeh LK, Vanpatten K, Highstrom L, Pestano GA, Nagle RB. Expression of mTOR signaling pathway markers in prostate cancer progression. Prostate. 2006 Aug 1;66(11):1203-12. PubMed, CrossRef
- Yoshida S, Matsumoto K, Arao T, Taniguchi H, Goto I, Hanafusa T, Nishio K, Yamada Y. Gene amplification of ribosomal protein S6 kinase-1 and -2 in gastric cancer. Anticancer Res. 2013 Feb;33(2):469-75. PubMed
- Li PD, Zhang WJ, Zhang MY, Yuan LJ, Cha YL, Ying XF, Wu G, Wang HY. Overexpression of RPS6KB1 predicts worse prognosis in primary HCC patients. Med Oncol. 2012 Dec;29(5):3070-6. PubMed, CrossRef
- Ismail HM. Overexpression of s6 kinase 1 in brain tumours is associated with induction of hypoxia-responsive genes and predicts patients’ survival. J Oncol. 2012;2012:416927. PubMed, PubMedCentral, CrossRef
- 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 Jun;27(2):141-4. PubMed
- 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 Dec;26(4):294-9. PubMed
- Pérez-Tenorio G, Karlsson E, Waltersson MA, Olsson B, Holmlund B, Nordenskjöld B, Fornander T, Skoog L, Stål O. Clinical potential of the mTOR targets S6K1 and S6K2 in breast cancer. Breast Cancer Res Treat. 2011 Aug;128(3):713-23. PubMed, CrossRef
- Zaiets IV, Sivchenko AS, Khoruzhenko AI, Filonenko VV. Generation of HEK-293 stable cell lines with disrupted expression of ribosomal protein S6 kinase (S6K1) isoforms using the CRISPR/Cas9 genome editing system. Biopolym Cell. 2017;33(5):356-366. CrossRef
- Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, Li Y, Fine EJ, Wu X, Shalem O, Cradick TJ, Marraffini LA, Bao G, Zhang F. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol. 2013 Sep;31(9):827-32. PubMed, PubMedCentral, CrossRef
- 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):374-379. CrossRef
- Pogrebnoy PV, Kukharenko AP, Tykhonkova IA, Pal’chevskiy SS, Savinskaya LA, Pogrebnaya AP, Valevka TI, Markeeva NV, Soldatkina MA, Matsuka GK, Gout IT, Filonenko VV. Generation and characterization of monoclonal antibodies to p70S6kinase α. Exp Oncol. 1999;21(3-4):232-238.
- Isotani S, Hara K, Tokunaga C, Inoue H, Avruch J, Yonezawa K. Immunopurified mammalian target of rapamycin phosphorylates and activates p70 S6 kinase alpha in vitro. J Biol Chem. 1999 Nov 26;274(48):34493-8. PubMed, CrossRef
- Burnett PE, Barrow RK, Cohen NA, Snyder SH, Sabatini DM. RAFT1 phosphorylation of the translational regulators p70 S6 kinase and 4E-BP1. Proc Natl Acad Sci USA. 1998 Feb 17;95(4):1432-7. PubMed, PubMedCentral, CrossRef
- Weng QP, Kozlowski M, Belham C, Zhang A, Comb MJ, Avruch J. Regulation of the p70 S6 kinase by phosphorylation in vivo. Analysis using site-specific anti-phosphopeptide antibodies. J Biol Chem. 1998 Jun 26;273(26):16621-9. PubMed, CrossRef
- Hara K, Maruki Y, Long X, Yoshino K, Oshiro N, Hidayat S, Tokunaga C, Avruch J, Yonezawa K. Raptor, a binding partner of target of rapamycin (TOR), mediates TOR action. Cell. 2002 Jul 26;110(2):177-89. PubMed, CrossRef
- Dennis PB, Pullen N, Kozma SC, Thomas G. The principal rapamycin-sensitive p70(s6k) phosphorylation sites, T-229 and T-389, are differentially regulated by rapamycin-insensitive kinase kinases. Mol Cell Biol. 1996 Nov;16(11):6242-51. PubMed, PubMedCentral, CrossRef
- Fingar DC, Richardson CJ, Tee AR, Cheatham L, Tsou C, Blenis J. mTOR controls cell cycle progression through its cell growth effectors S6K1 and 4E-BP1/eukaryotic translation initiation factor 4E. Mol Cell Biol. 2004 Jan;24(1):200-16. PubMed, PubMedCentral, CrossRef
- Chiang GG, Abraham RT. Phosphorylation of mammalian target of rapamycin (mTOR) at Ser-2448 is mediated by p70S6 kinase. J Biol Chem. 2005 Jul 8;280(27):25485-90. PubMed, CrossRef
- Liu L, Li F, Cardelli JA, Martin KA, Blenis J, Huang S. Rapamycin inhibits cell motility by suppression of mTOR-mediated S6K1 and 4E-BP1 pathways. Oncogene. 2006 Nov 9;25(53):7029-40. PubMed, CrossRef
This work is licensed under a Creative Commons Attribution 4.0 International License.