• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of biochemjBJ Latest papers and much more!
Biochem J. Sep 1, 2003; 374(Pt 2): 297–306.
PMCID: PMC1223624

Insulin-induced Drosophila S6 kinase activation requires phosphoinositide 3-kinase and protein kinase B.

Abstract

An important mechanism by which insulin regulates cell growth and protein synthesis is through activation of the p70 ribosomal S6 protein kinase (S6K). In mammalian cells, insulin-induced PI3K (phosphoinositide 3-kinase) activation, generates the lipid second messenger PtdIns(3,4,5) P (3), which is thought to play a key role in triggering the activation of S6K. Although the major components of the insulin-signalling pathway are conserved in Drosophila, recent studies suggested that S6K activation does not require PI3K in this system. To investigate further the role of dPI3K (Drosophila PI3K) in dS6K (Drosophila S6K) activation, we examined the effect of two structurally distinct PI3K inhibitors on insulin-induced dS6K activation in Kc167 and S2 Drosophila cell lines. We found that both inhibitors prevented insulin-stimulated phosphorylation and activation of dS6K. To investigate further the role of the dPI3K pathway in regulating dS6K activation, we also used dsRNAi (double-stranded RNA-mediated interference) to decrease expression of dPI3K and the PtdIns(3,4,5) P (3) phosphatase dPTEN ( Drosophila phosphatase and tensin homologue deleted on chromosome 10) in Kc167 and S2 cells. Knock-down of dPI3K prevented dS6K activation, whereas knock-down of dPTEN, which would be expected to increase PtdIns(3,4,5) P (3) levels, stimulated dS6K activity. Moreover, when the expression of the dPI3K target, dPKB (Drosophila protein kinase B), was decreased to undetectable levels, we found that insulin could no longer trigger dS6K activation. This observation provides the first direct demonstration that dPKB is required for insulin-stimulated dS6K activation. We also present evidence that the amino-acid-induced activation of dS6K in the absence of insulin, thought to be mediated by dTOR (Drosophila target of rapamycin), which is unaffected by the inhibition of dPI3K by wortmannin. The results of the present study support the view that, in Drosophila cells, dPI3K and dPKB, as well dTOR, are required for the activation of dS6K by insulin.

Full Text

The Full Text of this article is available as a PDF (404K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Vanhaesebroeck B, Leevers SJ, Ahmadi K, Timms J, Katso R, Driscoll PC, Woscholski R, Parker PJ, Waterfield MD. Synthesis and function of 3-phosphorylated inositol lipids. Annu Rev Biochem. 2001;70:535–602. [PubMed]
  • Avruch J, Belham C, Weng Q, Hara K, Yonezawa K. The p70 S6 kinase integrates nutrient and growth signals to control translational capacity. Prog Mol Subcell Biol. 2001;26:115–154. [PubMed]
  • Brazil DP, Hemmings BA. Ten years of protein kinase B signalling: a hard Akt to follow. Trends Biochem Sci. 2001 Nov;26(11):657–664. [PubMed]
  • Lawlor MA, Alessi DR. PKB/Akt: a key mediator of cell proliferation, survival and insulin responses? J Cell Sci. 2001 Aug;114(Pt 16):2903–2910. [PubMed]
  • Toker A, Newton AC. Cellular signaling: pivoting around PDK-1. Cell. 2000 Oct 13;103(2):185–188. [PubMed]
  • Alessi DR. Discovery of PDK1, one of the missing links in insulin signal transduction. Colworth Medal Lecture. Biochem Soc Trans. 2001 May;29(Pt 2):1–14. [PubMed]
  • Thomas G, Hall MN. TOR signalling and control of cell growth. Curr Opin Cell Biol. 1997 Dec;9(6):782–787. [PubMed]
  • Chung J, Grammer TC, Lemon KP, Kazlauskas A, Blenis J. PDGF- and insulin-dependent pp70S6k activation mediated by phosphatidylinositol-3-OH kinase. Nature. 1994 Jul 7;370(6484):71–75. [PubMed]
  • Reif K, Burgering BM, Cantrell DA. Phosphatidylinositol 3-kinase links the interleukin-2 receptor to protein kinase B and p70 S6 kinase. J Biol Chem. 1997 May 30;272(22):14426–14433. [PubMed]
  • Weng QP, Andrabi K, Klippel A, Kozlowski MT, Williams LT, Avruch J. Phosphatidylinositol 3-kinase signals activation of p70 S6 kinase in situ through site-specific p70 phosphorylation. Proc Natl Acad Sci U S A. 1995 Jun 6;92(12):5744–5748. [PMC free article] [PubMed]
  • Navé BT, Ouwens M, Withers DJ, Alessi DR, Shepherd PR. Mammalian target of rapamycin is a direct target for protein kinase B: identification of a convergence point for opposing effects of insulin and amino-acid deficiency on protein translation. Biochem J. 1999 Dec 1;344(Pt 2):427–431. [PMC free article] [PubMed]
  • Sekulić A, Hudson CC, Homme JL, Yin P, Otterness DM, Karnitz LM, Abraham RT. A direct linkage between the phosphoinositide 3-kinase-AKT signaling pathway and the mammalian target of rapamycin in mitogen-stimulated and transformed cells. Cancer Res. 2000 Jul 1;60(13):3504–3513. [PubMed]
  • Marygold Steven J, Leevers Sally J. Growth signaling: TSC takes its place. Curr Biol. 2002 Nov 19;12(22):R785–R787. [PubMed]
  • McManus Edward J, Alessi Dario R. TSC1-TSC2: a complex tale of PKB-mediated S6K regulation. Nat Cell Biol. 2002 Sep;4(9):E214–E216. [PubMed]
  • Jaeschke Anja, Hartkamp Joerg, Saitoh Masao, Roworth Wendy, Nobukuni Takahiro, Hodges Angela, Sampson Julian, Thomas George, Lamb Richard. Tuberous sclerosis complex tumor suppressor-mediated S6 kinase inhibition by phosphatidylinositide-3-OH kinase is mTOR independent. J Cell Biol. 2002 Oct 28;159(2):217–224. [PMC free article] [PubMed]
  • Inoki Ken, Li Yong, Zhu Tianquan, Wu Jun, Guan Kun-Liang. TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling. Nat Cell Biol. 2002 Sep;4(9):648–657. [PubMed]
  • Manning Brendan D, Tee Andrew R, Logsdon M Nicole, Blenis John, Cantley Lewis C. Identification of the tuberous sclerosis complex-2 tumor suppressor gene product tuberin as a target of the phosphoinositide 3-kinase/akt pathway. Mol Cell. 2002 Jul;10(1):151–162. [PubMed]
  • Tee Andrew R, Fingar Diane C, Manning Brendan D, Kwiatkowski David J, Cantley Lewis C, Blenis John. Tuberous sclerosis complex-1 and -2 gene products function together to inhibit mammalian target of rapamycin (mTOR)-mediated downstream signaling. Proc Natl Acad Sci U S A. 2002 Oct 15;99(21):13571–13576. [PMC free article] [PubMed]
  • Hara Kenta, Maruki Yoshiko, Long Xiaomeng, Yoshino Ken-ichi, Oshiro Noriko, Hidayat Sujuti, Tokunaga Chiharu, Avruch Joseph, Yonezawa Kazuyoshi. Raptor, a binding partner of target of rapamycin (TOR), mediates TOR action. Cell. 2002 Jul 26;110(2):177–189. [PubMed]
  • Kim Do-Hyung, Sarbassov DD, Ali Siraj M, King Jessie E, Latek Robert R, Erdjument-Bromage Hediye, Tempst Paul, Sabatini David M. mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery. Cell. 2002 Jul 26;110(2):163–175. [PubMed]
  • Jacinto Estela, Hall Michael N. Tor signalling in bugs, brain and brawn. Nat Rev Mol Cell Biol. 2003 Feb;4(2):117–126. [PubMed]
  • Biondi RM, Kieloch A, Currie RA, Deak M, Alessi DR. The PIF-binding pocket in PDK1 is essential for activation of S6K and SGK, but not PKB. EMBO J. 2001 Aug 15;20(16):4380–4390. [PMC free article] [PubMed]
  • Biondi Ricardo M, Komander David, Thomas Christine C, Lizcano Jose M, Deak Maria, Alessi Dario R, van Aalten Daan M F. High resolution crystal structure of the human PDK1 catalytic domain defines the regulatory phosphopeptide docking site. EMBO J. 2002 Aug 15;21(16):4219–4228. [PMC free article] [PubMed]
  • Kozma Sara C, Thomas George. Regulation of cell size in growth, development and human disease: PI3K, PKB and S6K. Bioessays. 2002 Jan;24(1):65–71. [PubMed]
  • Leevers SJ. Growth control: invertebrate insulin surprises! Curr Biol. 2001 Mar 20;11(6):R209–R212. [PubMed]
  • Potter Christopher J, Pedraza Laura G, Xu Tian. Akt regulates growth by directly phosphorylating Tsc2. Nat Cell Biol. 2002 Sep;4(9):658–665. [PubMed]
  • Gao Xinsheng, Zhang Yong, Arrazola Peter, Hino Okio, Kobayashi Toshiyuki, Yeung Raymond S, Ru Binggeng, Pan Duojia. Tsc tumour suppressor proteins antagonize amino-acid-TOR signalling. Nat Cell Biol. 2002 Sep;4(9):699–704. [PubMed]
  • Radimerski Thomas, Montagne Jacques, Rintelen Felix, Stocker Hugo, van der Kaay Jeroen, Downes C Peter, Hafen Ernst, Thomas George. dS6K-regulated cell growth is dPKB/dPI(3)K-independent, but requires dPDK1. Nat Cell Biol. 2002 Mar;4(3):251–255. [PubMed]
  • Oldham S, Montagne J, Radimerski T, Thomas G, Hafen E. Genetic and biochemical characterization of dTOR, the Drosophila homolog of the target of rapamycin. Genes Dev. 2000 Nov 1;14(21):2689–2694. [PMC free article] [PubMed]
  • Andjelković M, Jones PF, Grossniklaus U, Cron P, Schier AF, Dick M, Bilbe G, Hemmings BA. Developmental regulation of expression and activity of multiple forms of the Drosophila RAC protein kinase. J Biol Chem. 1995 Feb 24;270(8):4066–4075. [PubMed]
  • Lizcano Jose M, Deak Maria, Morrice Nick, Kieloch Agnieszka, Hastie C James, Dong Liying, Schutkowski Mike, Reimer Ulf, Alessi Dario R. Molecular basis for the substrate specificity of NIMA-related kinase-6 (NEK6). Evidence that NEK6 does not phosphorylate the hydrophobic motif of ribosomal S6 protein kinase and serum- and glucocorticoid-induced protein kinase in vivo. J Biol Chem. 2002 Aug 2;277(31):27839–27849. [PubMed]
  • Alessi DR, Cohen P, Ashworth A, Cowley S, Leevers SJ, Marshall CJ. Assay and expression of mitogen-activated protein kinase, MAP kinase kinase, and Raf. Methods Enzymol. 1995;255:279–290. [PubMed]
  • Radimerski Thomas, Montagne Jacques, Hemmings-Mieszczak Maja, Thomas George. Lethality of Drosophila lacking TSC tumor suppressor function rescued by reducing dS6K signaling. Genes Dev. 2002 Oct 15;16(20):2627–2632. [PMC free article] [PubMed]
  • Verdu J, Buratovich MA, Wilder EL, Birnbaum MJ. Cell-autonomous regulation of cell and organ growth in Drosophila by Akt/PKB. Nat Cell Biol. 1999 Dec;1(8):500–506. [PubMed]
  • Clemens JC, Worby CA, Simonson-Leff N, Muda M, Maehama T, Hemmings BA, Dixon JE. Use of double-stranded RNA interference in Drosophila cell lines to dissect signal transduction pathways. Proc Natl Acad Sci U S A. 2000 Jun 6;97(12):6499–6503. [PMC free article] [PubMed]
  • Gingras AC, Raught B, Sonenberg N. Regulation of translation initiation by FRAP/mTOR. Genes Dev. 2001 Apr 1;15(7):807–826. [PubMed]
  • Podsypanina K, Lee RT, Politis C, Hennessy I, Crane A, Puc J, Neshat M, Wang H, Yang L, Gibbons J, et al. An inhibitor of mTOR reduces neoplasia and normalizes p70/S6 kinase activity in Pten+/- mice. Proc Natl Acad Sci U S A. 2001 Aug 28;98(18):10320–10325. [PMC free article] [PubMed]
  • Neshat MS, Mellinghoff IK, Tran C, Stiles B, Thomas G, Petersen R, Frost P, Gibbons JJ, Wu H, Sawyers CL. Enhanced sensitivity of PTEN-deficient tumors to inhibition of FRAP/mTOR. Proc Natl Acad Sci U S A. 2001 Aug 28;98(18):10314–10319. [PMC free article] [PubMed]
  • Zhang H, Stallock JP, Ng JC, Reinhard C, Neufeld TP. Regulation of cellular growth by the Drosophila target of rapamycin dTOR. Genes Dev. 2000 Nov 1;14(21):2712–2724. [PMC free article] [PubMed]
  • Stocker Hugo, Radimerski Thomas, Schindelholz Benno, Wittwer Franz, Belawat Priyanka, Daram Pierre, Breuer Sebastian, Thomas George, Hafen Ernst. Rheb is an essential regulator of S6K in controlling cell growth in Drosophila. Nat Cell Biol. 2003 Jun;5(6):559–565. [PubMed]
  • Saucedo Leslie J, Gao Xinsheng, Chiarelli Dominic A, Li Ling, Pan Duoija, Edgar Bruce A. Rheb promotes cell growth as a component of the insulin/TOR signalling network. Nat Cell Biol. 2003 Jun;5(6):566–571. [PubMed]
  • Zhang Yong, Gao Xinsheng, Saucedo Leslie J, Ru Binggen, Edgar Bruce A, Pan Duojia. Rheb is a direct target of the tuberous sclerosis tumour suppressor proteins. Nat Cell Biol. 2003 Jun;5(6):578–581. [PubMed]
  • Brunn GJ, Williams J, Sabers C, Wiederrecht G, Lawrence JC, Jr, Abraham RT. Direct inhibition of the signaling functions of the mammalian target of rapamycin by the phosphoinositide 3-kinase inhibitors, wortmannin and LY294002. EMBO J. 1996 Oct 1;15(19):5256–5267. [PMC free article] [PubMed]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...