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. 1994 Jul;14(7):4902–4911. doi: 10.1128/mcb.14.7.4902

Phosphatidylinositol 3-kinase activation is required for insulin stimulation of pp70 S6 kinase, DNA synthesis, and glucose transporter translocation.

B Cheatham 1, C J Vlahos 1, L Cheatham 1, L Wang 1, J Blenis 1, C R Kahn 1
PMCID: PMC358862  PMID: 8007986

Abstract

Phosphatidylinositol 3-kinase (PI 3-kinase) is stimulated by insulin and a variety of growth factors, but its exact role in signal transduction remains unclear. We have used a novel, highly specific inhibitor of PT 3-kinase to dissect the role of this enzyme in insulin action. Treatment of intact 3T3-L1 adipocytes with LY294002 produced a dose-dependent inhibition of insulin-stimulated PI 3-kinase (50% inhibitory concentration, 6 microM) with > 95% reduction in the levels of phosphatidylinositol-3,4,5-trisphosphate without changes in the levels of phosphatidylinositol-4-monophosphate or its derivatives. In parallel, there was a complete inhibition of insulin-stimulated phosphorylation and activation of pp70 S6 kinase. Inhibition of PI 3-kinase also effectively blocked insulin- and serum-stimulated DNA synthesis and insulin-stimulated glucose uptake by inhibiting translocation of GLUT 4 glucose transporters to the plasma membrane. By contrast, LY294002 had no effect on insulin stimulation of mitogen-activated protein kinase or pp90 S6 kinase. Thus, activation of PI 3-kinase plays a critical role in mammalian cells and is required for activation of pp70 S6 kinase and DNA synthesis and certain forms of intracellular vesicular trafficking but not mitogen-activated protein kinase or pp90 S6 kinase activation. These data suggest that PI 3-kinase is not only an important component but also a point of divergence in the insulin signaling network.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Auger K. R., Serunian L. A., Soltoff S. P., Libby P., Cantley L. C. PDGF-dependent tyrosine phosphorylation stimulates production of novel polyphosphoinositides in intact cells. Cell. 1989 Apr 7;57(1):167–175. doi: 10.1016/0092-8674(89)90182-7. [DOI] [PubMed] [Google Scholar]
  2. Backer J. M., Myers M. G., Jr, Shoelson S. E., Chin D. J., Sun X. J., Miralpeix M., Hu P., Margolis B., Skolnik E. Y., Schlessinger J. Phosphatidylinositol 3'-kinase is activated by association with IRS-1 during insulin stimulation. EMBO J. 1992 Sep;11(9):3469–3479. doi: 10.1002/j.1460-2075.1992.tb05426.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Backer J. M., Myers M. G., Jr, Sun X. J., Chin D. J., Shoelson S. E., Miralpeix M., White M. F. Association of IRS-1 with the insulin receptor and the phosphatidylinositol 3'-kinase. Formation of binary and ternary signaling complexes in intact cells. J Biol Chem. 1993 Apr 15;268(11):8204–8212. [PubMed] [Google Scholar]
  4. Backer J. M., Schroeder G. G., Kahn C. R., Myers M. G., Jr, Wilden P. A., Cahill D. A., White M. F. Insulin stimulation of phosphatidylinositol 3-kinase activity maps to insulin receptor regions required for endogenous substrate phosphorylation. J Biol Chem. 1992 Jan 15;267(2):1367–1374. [PubMed] [Google Scholar]
  5. Birnbaum M. J. The insulin-sensitive glucose transporter. Int Rev Cytol. 1992;137:239–297. [PubMed] [Google Scholar]
  6. Blenis J., Chung J., Erikson E., Alcorta D. A., Erikson R. L. Distinct mechanisms for the activation of the RSK kinases/MAP2 kinase/pp90rsk and pp70-S6 kinase signaling systems are indicated by inhibition of protein synthesis. Cell Growth Differ. 1991 Jun;2(6):279–285. [PubMed] [Google Scholar]
  7. Calderhead D. M., Kitagawa K., Tanner L. I., Holman G. D., Lienhard G. E. Insulin regulation of the two glucose transporters in 3T3-L1 adipocytes. J Biol Chem. 1990 Aug 15;265(23):13801–13808. [PubMed] [Google Scholar]
  8. Cheatham B., Kahn C. R. Cysteine 647 in the insulin receptor is required for normal covalent interaction between alpha- and beta-subunits and signal transduction. J Biol Chem. 1992 Apr 5;267(10):7108–7115. [PubMed] [Google Scholar]
  9. Chen R. H., Blenis J. Identification of Xenopus S6 protein kinase homologs (pp90rsk) in somatic cells: phosphorylation and activation during initiation of cell proliferation. Mol Cell Biol. 1990 Jun;10(6):3204–3215. doi: 10.1128/mcb.10.6.3204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chung J., Chen R. H., Blenis J. Coordinate regulation of pp90rsk and a distinct protein-serine/threonine kinase activity that phosphorylates recombinant pp90rsk in vitro. Mol Cell Biol. 1991 Apr;11(4):1868–1874. doi: 10.1128/mcb.11.4.1868. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chung J., Kuo C. J., Crabtree G. R., Blenis J. Rapamycin-FKBP specifically blocks growth-dependent activation of and signaling by the 70 kd S6 protein kinases. Cell. 1992 Jun 26;69(7):1227–1236. doi: 10.1016/0092-8674(92)90643-q. [DOI] [PubMed] [Google Scholar]
  12. Courtneidge S. A., Heber A. An 81 kd protein complexed with middle T antigen and pp60c-src: a possible phosphatidylinositol kinase. Cell. 1987 Sep 25;50(7):1031–1037. doi: 10.1016/0092-8674(87)90169-3. [DOI] [PubMed] [Google Scholar]
  13. Cushman S. W., Wardzala L. J. Potential mechanism of insulin action on glucose transport in the isolated rat adipose cell. Apparent translocation of intracellular transport systems to the plasma membrane. J Biol Chem. 1980 May 25;255(10):4758–4762. [PubMed] [Google Scholar]
  14. Eberle M., Traynor-Kaplan A. E., Sklar L. A., Norgauer J. Is there a relationship between phosphatidylinositol trisphosphate and F-actin polymerization in human neutrophils? J Biol Chem. 1990 Oct 5;265(28):16725–16728. [PubMed] [Google Scholar]
  15. Fingar D. C., Hausdorff S. F., Blenis J., Birnbaum M. J. Dissociation of pp70 ribosomal protein S6 kinase from insulin-stimulated glucose transport in 3T3-L1 adipocytes. J Biol Chem. 1993 Feb 5;268(4):3005–3008. [PubMed] [Google Scholar]
  16. Hiles I. D., Otsu M., Volinia S., Fry M. J., Gout I., Dhand R., Panayotou G., Ruiz-Larrea F., Thompson A., Totty N. F. Phosphatidylinositol 3-kinase: structure and expression of the 110 kd catalytic subunit. Cell. 1992 Aug 7;70(3):419–429. doi: 10.1016/0092-8674(92)90166-a. [DOI] [PubMed] [Google Scholar]
  17. Hu P., Mondino A., Skolnik E. Y., Schlessinger J. Cloning of a novel, ubiquitously expressed human phosphatidylinositol 3-kinase and identification of its binding site on p85. Mol Cell Biol. 1993 Dec;13(12):7677–7688. doi: 10.1128/mcb.13.12.7677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Huang R. S., Sorisky A., Church W. R., Simons E. R., Rittenhouse S. E. "Thrombin" receptor-directed ligand accounts for activation by thrombin of platelet phospholipase C and accumulation of 3-phosphorylated phosphoinositides. J Biol Chem. 1991 Oct 5;266(28):18435–18438. [PubMed] [Google Scholar]
  19. James D. E., Brown R., Navarro J., Pilch P. F. Insulin-regulatable tissues express a unique insulin-sensitive glucose transport protein. Nature. 1988 May 12;333(6169):183–185. doi: 10.1038/333183a0. [DOI] [PubMed] [Google Scholar]
  20. Janmey P. A., Stossel T. P. Gelsolin-polyphosphoinositide interaction. Full expression of gelsolin-inhibiting function by polyphosphoinositides in vesicular form and inactivation by dilution, aggregation, or masking of the inositol head group. J Biol Chem. 1989 Mar 25;264(9):4825–4831. [PubMed] [Google Scholar]
  21. Kahn C. R., White M. F. The insulin receptor and the molecular mechanism of insulin action. J Clin Invest. 1988 Oct;82(4):1151–1156. doi: 10.1172/JCI113711. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kaplan D. R., Whitman M., Schaffhausen B., Pallas D. C., White M., Cantley L., Roberts T. M. Common elements in growth factor stimulation and oncogenic transformation: 85 kd phosphoprotein and phosphatidylinositol kinase activity. Cell. 1987 Sep 25;50(7):1021–1029. doi: 10.1016/0092-8674(87)90168-1. [DOI] [PubMed] [Google Scholar]
  23. Kasuga M., Karlsson F. A., Kahn C. R. Insulin stimulates the phosphorylation of the 95,000-dalton subunit of its own receptor. Science. 1982 Jan 8;215(4529):185–187. doi: 10.1126/science.7031900. [DOI] [PubMed] [Google Scholar]
  24. Kelly K. L., Ruderman N. B. Insulin-stimulated phosphatidylinositol 3-kinase. Association with a 185-kDa tyrosine-phosphorylated protein (IRS-1) and localization in a low density membrane vesicle. J Biol Chem. 1993 Feb 25;268(6):4391–4398. [PubMed] [Google Scholar]
  25. Kunz J., Henriquez R., Schneider U., Deuter-Reinhard M., Movva N. R., Hall M. N. Target of rapamycin in yeast, TOR2, is an essential phosphatidylinositol kinase homolog required for G1 progression. Cell. 1993 May 7;73(3):585–596. doi: 10.1016/0092-8674(93)90144-f. [DOI] [PubMed] [Google Scholar]
  26. Kuo C. J., Chung J., Fiorentino D. F., Flanagan W. M., Blenis J., Crabtree G. R. Rapamycin selectively inhibits interleukin-2 activation of p70 S6 kinase. Nature. 1992 Jul 2;358(6381):70–73. doi: 10.1038/358070a0. [DOI] [PubMed] [Google Scholar]
  27. Lane H. A., Fernandez A., Lamb N. J., Thomas G. p70s6k function is essential for G1 progression. Nature. 1993 May 13;363(6425):170–172. doi: 10.1038/363170a0. [DOI] [PubMed] [Google Scholar]
  28. Ling L. E., Druker B. J., Cantley L. C., Roberts T. M. Transformation-defective mutants of polyomavirus middle T antigen associate with phosphatidylinositol 3-kinase (PI 3-kinase) but are unable to maintain wild-type levels of PI 3-kinase products in intact cells. J Virol. 1992 Mar;66(3):1702–1708. doi: 10.1128/jvi.66.3.1702-1708.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lowenstein E. J., Daly R. J., Batzer A. G., Li W., Margolis B., Lammers R., Ullrich A., Skolnik E. Y., Bar-Sagi D., Schlessinger J. The SH2 and SH3 domain-containing protein GRB2 links receptor tyrosine kinases to ras signaling. Cell. 1992 Aug 7;70(3):431–442. doi: 10.1016/0092-8674(92)90167-b. [DOI] [PubMed] [Google Scholar]
  30. Myers M. G., Jr, Sun X. J., Cheatham B., Jachna B. R., Glasheen E. M., Backer J. M., White M. F. IRS-1 is a common element in insulin and insulin-like growth factor-I signaling to the phosphatidylinositol 3'-kinase. Endocrinology. 1993 Apr;132(4):1421–1430. doi: 10.1210/endo.132.4.8384986. [DOI] [PubMed] [Google Scholar]
  31. Panayotou G., Bax B., Gout I., Federwisch M., Wroblowski B., Dhand R., Fry M. J., Blundell T. L., Wollmer A., Waterfield M. D. Interaction of the p85 subunit of PI 3-kinase and its N-terminal SH2 domain with a PDGF receptor phosphorylation site: structural features and analysis of conformational changes. EMBO J. 1992 Dec;11(12):4261–4272. doi: 10.1002/j.1460-2075.1992.tb05524.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Parker P. J., Waterfield M. D. Phosphatidylinositol 3-kinase: a novel effector. Cell Growth Differ. 1992 Oct;3(10):747–752. [PubMed] [Google Scholar]
  33. Rosen O. M. After insulin binds. Science. 1987 Sep 18;237(4821):1452–1458. doi: 10.1126/science.2442814. [DOI] [PubMed] [Google Scholar]
  34. Rothman J. E., Orci L. Molecular dissection of the secretory pathway. Nature. 1992 Jan 30;355(6359):409–415. doi: 10.1038/355409a0. [DOI] [PubMed] [Google Scholar]
  35. Schu P. V., Takegawa K., Fry M. J., Stack J. H., Waterfield M. D., Emr S. D. Phosphatidylinositol 3-kinase encoded by yeast VPS34 gene essential for protein sorting. Science. 1993 Apr 2;260(5104):88–91. doi: 10.1126/science.8385367. [DOI] [PubMed] [Google Scholar]
  36. Serunian L. A., Auger K. R., Cantley L. C. Identification and quantification of polyphosphoinositides produced in response to platelet-derived growth factor stimulation. Methods Enzymol. 1991;198:78–87. doi: 10.1016/0076-6879(91)98010-4. [DOI] [PubMed] [Google Scholar]
  37. Serunian L. A., Haber M. T., Fukui T., Kim J. W., Rhee S. G., Lowenstein J. M., Cantley L. C. Polyphosphoinositides produced by phosphatidylinositol 3-kinase are poor substrates for phospholipases C from rat liver and bovine brain. J Biol Chem. 1989 Oct 25;264(30):17809–17815. [PubMed] [Google Scholar]
  38. Shibasaki F., Homma Y., Takenawa T. Two types of phosphatidylinositol 3-kinase from bovine thymus. Monomer and heterodimer form. J Biol Chem. 1991 May 5;266(13):8108–8114. [PubMed] [Google Scholar]
  39. Shoelson S. E., Sivaraja M., Williams K. P., Hu P., Schlessinger J., Weiss M. A. Specific phosphopeptide binding regulates a conformational change in the PI 3-kinase SH2 domain associated with enzyme activation. EMBO J. 1993 Feb;12(2):795–802. doi: 10.1002/j.1460-2075.1993.tb05714.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Shurtleff S. A., Downing J. R., Rock C. O., Hawkins S. A., Roussel M. F., Sherr C. J. Structural features of the colony-stimulating factor 1 receptor that affect its association with phosphatidylinositol 3-kinase. EMBO J. 1990 Aug;9(8):2415–2421. doi: 10.1002/j.1460-2075.1990.tb07417.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Skolnik E. Y., Lee C. H., Batzer A., Vicentini L. M., Zhou M., Daly R., Myers M. J., Jr, Backer J. M., Ullrich A., White M. F. The SH2/SH3 domain-containing protein GRB2 interacts with tyrosine-phosphorylated IRS1 and Shc: implications for insulin control of ras signalling. EMBO J. 1993 May;12(5):1929–1936. doi: 10.1002/j.1460-2075.1993.tb05842.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Stack J. H., Herman P. K., Schu P. V., Emr S. D. A membrane-associated complex containing the Vps15 protein kinase and the Vps34 PI 3-kinase is essential for protein sorting to the yeast lysosome-like vacuole. EMBO J. 1993 May;12(5):2195–2204. doi: 10.1002/j.1460-2075.1993.tb05867.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Sturgill T. W., Ray L. B., Erikson E., Maller J. L. Insulin-stimulated MAP-2 kinase phosphorylates and activates ribosomal protein S6 kinase II. Nature. 1988 Aug 25;334(6184):715–718. doi: 10.1038/334715a0. [DOI] [PubMed] [Google Scholar]
  44. Sultan C., Breton M., Mauco G., Grondin P., Plantavid M., Chap H. The novel inositol lipid phosphatidylinositol 3,4-bisphosphate is produced by human blood platelets upon thrombin stimulation. Biochem J. 1990 Aug 1;269(3):831–834. doi: 10.1042/bj2690831. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Sun X. J., Rothenberg P., Kahn C. R., Backer J. M., Araki E., Wilden P. A., Cahill D. A., Goldstein B. J., White M. F. Structure of the insulin receptor substrate IRS-1 defines a unique signal transduction protein. Nature. 1991 Jul 4;352(6330):73–77. doi: 10.1038/352073a0. [DOI] [PubMed] [Google Scholar]
  46. Traynor-Kaplan A. E., Harris A. L., Thompson B. L., Taylor P., Sklar L. A. An inositol tetrakisphosphate-containing phospholipid in activated neutrophils. Nature. 1988 Jul 28;334(6180):353–356. doi: 10.1038/334353a0. [DOI] [PubMed] [Google Scholar]
  47. Valius M., Kazlauskas A. Phospholipase C-gamma 1 and phosphatidylinositol 3 kinase are the downstream mediators of the PDGF receptor's mitogenic signal. Cell. 1993 Apr 23;73(2):321–334. doi: 10.1016/0092-8674(93)90232-f. [DOI] [PubMed] [Google Scholar]
  48. Varticovski L., Daley G. Q., Jackson P., Baltimore D., Cantley L. C. Activation of phosphatidylinositol 3-kinase in cells expressing abl oncogene variants. Mol Cell Biol. 1991 Feb;11(2):1107–1113. doi: 10.1128/mcb.11.2.1107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Varticovski L., Druker B., Morrison D., Cantley L., Roberts T. The colony stimulating factor-1 receptor associates with and activates phosphatidylinositol-3 kinase. Nature. 1989 Dec 7;342(6250):699–702. doi: 10.1038/342699a0. [DOI] [PubMed] [Google Scholar]
  50. Vlahos C. J., Matter W. F., Hui K. Y., Brown R. F. A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J Biol Chem. 1994 Feb 18;269(7):5241–5248. [PubMed] [Google Scholar]
  51. Vlahos C. J., Matter W. F. Signal transduction in neutrophil activation. Phosphatidylinositol 3-kinase is stimulated without tyrosine phosphorylation. FEBS Lett. 1992 Sep 14;309(3):242–248. doi: 10.1016/0014-5793(92)80781-b. [DOI] [PubMed] [Google Scholar]
  52. White M. F., Maron R., Kahn C. R. Insulin rapidly stimulates tyrosine phosphorylation of a Mr-185,000 protein in intact cells. Nature. 1985 Nov 14;318(6042):183–186. doi: 10.1038/318183a0. [DOI] [PubMed] [Google Scholar]
  53. Whitman M., Downes C. P., Keeler M., Keller T., Cantley L. Type I phosphatidylinositol kinase makes a novel inositol phospholipid, phosphatidylinositol-3-phosphate. Nature. 1988 Apr 14;332(6165):644–646. doi: 10.1038/332644a0. [DOI] [PubMed] [Google Scholar]
  54. Whitman M., Kaplan D., Roberts T., Cantley L. Evidence for two distinct phosphatidylinositol kinases in fibroblasts. Implications for cellular regulation. Biochem J. 1987 Oct 1;247(1):165–174. doi: 10.1042/bj2470165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Yano H., Nakanishi S., Kimura K., Hanai N., Saitoh Y., Fukui Y., Nonomura Y., Matsuda Y. Inhibition of histamine secretion by wortmannin through the blockade of phosphatidylinositol 3-kinase in RBL-2H3 cells. J Biol Chem. 1993 Dec 5;268(34):25846–25856. [PubMed] [Google Scholar]
  56. Zorzano A., Wilkinson W., Kotliar N., Thoidis G., Wadzinkski B. E., Ruoho A. E., Pilch P. F. Insulin-regulated glucose uptake in rat adipocytes is mediated by two transporter isoforms present in at least two vesicle populations. J Biol Chem. 1989 Jul 25;264(21):12358–12363. [PubMed] [Google Scholar]

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