Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1996 Aug;16(8):4117–4127. doi: 10.1128/mcb.16.8.4117

Membrane localization of phosphatidylinositol 3-kinase is sufficient to activate multiple signal-transducing kinase pathways.

A Klippel 1, C Reinhard 1, W M Kavanaugh 1, G Apell 1, M A Escobedo 1, L T Williams 1
PMCID: PMC231408  PMID: 8754810

Abstract

Phosphatidylinositol (PI) 3-kinase is a cytoplasmic signaling molecule recruited to the membrane by activated growth factor receptors. The p85 subunit of PI 3-kinase links the catalytic p110 subunit to activated growth factor receptors and is required for enzymatic activity of p110. In this report, we describe the effects of expressing novel forms of p110 that are targeted to the membrane by either N-terminal myristoylation or C-terminal farnesylation. The expression of membrane-localized p110 is sufficient to trigger downstream responses characteristic of growth factor action, including the stimulation of pp70 S6 kinase, Akt/Rac, and Jun N-terminal kinase (JNK). These responses can also be triggered by expression of a form of p110 (p110*) that is cytosolic but exhibits a high specific activity. Finally, targeting of pl10* to the membrane results in maximal activation of downstream responses. Our data demonstrate that either membrane-targeted forms of p110 or a form of p110 with high specific activity can act as constitutively active PI 3-kinases and induce PI 3-kinase-dependent responses in the absence of growth factor stimulation. The results also show that PI 3-kinase activation is sufficient to stimulate several kinases that appear to function in different signaling pathways.

Full Text

The Full Text of this article is available as a PDF (999.4 KB).

Selected References

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

  1. Aronheim A., Engelberg D., Li N., al-Alawi N., Schlessinger J., Karin M. Membrane targeting of the nucleotide exchange factor Sos is sufficient for activating the Ras signaling pathway. Cell. 1994 Sep 23;78(6):949–961. doi: 10.1016/0092-8674(94)90271-2. [DOI] [PubMed] [Google Scholar]
  2. Burgering B. M., Coffer P. J. Protein kinase B (c-Akt) in phosphatidylinositol-3-OH kinase signal transduction. Nature. 1995 Aug 17;376(6541):599–602. doi: 10.1038/376599a0. [DOI] [PubMed] [Google Scholar]
  3. Cadwallader K. A., Paterson H., Macdonald S. G., Hancock J. F. N-terminally myristoylated Ras proteins require palmitoylation or a polybasic domain for plasma membrane localization. Mol Cell Biol. 1994 Jul;14(7):4722–4730. doi: 10.1128/mcb.14.7.4722. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cantley L. C., Auger K. R., Carpenter C., Duckworth B., Graziani A., Kapeller R., Soltoff S. Oncogenes and signal transduction. Cell. 1991 Jan 25;64(2):281–302. doi: 10.1016/0092-8674(91)90639-g. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Coso O. A., Chiariello M., Yu J. C., Teramoto H., Crespo P., Xu N., Miki T., Gutkind J. S. The small GTP-binding proteins Rac1 and Cdc42 regulate the activity of the JNK/SAPK signaling pathway. Cell. 1995 Jun 30;81(7):1137–1146. doi: 10.1016/s0092-8674(05)80018-2. [DOI] [PubMed] [Google Scholar]
  7. Datta K., Franke T. F., Chan T. O., Makris A., Yang S. I., Kaplan D. R., Morrison D. K., Golemis E. A., Tsichlis P. N. AH/PH domain-mediated interaction between Akt molecules and its potential role in Akt regulation. Mol Cell Biol. 1995 Apr;15(4):2304–2310. doi: 10.1128/mcb.15.4.2304. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Deichaite I., Casson L. P., Ling H. P., Resh M. D. In vitro synthesis of pp60v-src: myristylation in a cell-free system. Mol Cell Biol. 1988 Oct;8(10):4295–4301. doi: 10.1128/mcb.8.10.4295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dérijard B., Hibi M., Wu I. H., Barrett T., Su B., Deng T., Karin M., Davis R. J. JNK1: a protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-Jun activation domain. Cell. 1994 Mar 25;76(6):1025–1037. doi: 10.1016/0092-8674(94)90380-8. [DOI] [PubMed] [Google Scholar]
  10. Evan G. I., Lewis G. K., Ramsay G., Bishop J. M. Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol Cell Biol. 1985 Dec;5(12):3610–3616. doi: 10.1128/mcb.5.12.3610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fabian J. R., Morrison D. K., Daar I. O. Requirement for Raf and MAP kinase function during the meiotic maturation of Xenopus oocytes. J Cell Biol. 1993 Aug;122(3):645–652. doi: 10.1083/jcb.122.3.645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ferguson K. M., Lemmon M. A., Schlessinger J., Sigler P. B. Structure of the high affinity complex of inositol trisphosphate with a phospholipase C pleckstrin homology domain. Cell. 1995 Dec 15;83(6):1037–1046. doi: 10.1016/0092-8674(95)90219-8. [DOI] [PubMed] [Google Scholar]
  13. Franke T. F., Yang S. I., Chan T. O., Datta K., Kazlauskas A., Morrison D. K., Kaplan D. R., Tsichlis P. N. The protein kinase encoded by the Akt proto-oncogene is a target of the PDGF-activated phosphatidylinositol 3-kinase. Cell. 1995 Jun 2;81(5):727–736. doi: 10.1016/0092-8674(95)90534-0. [DOI] [PubMed] [Google Scholar]
  14. Gille H., Strahl T., Shaw P. E. Activation of ternary complex factor Elk-1 by stress-activated protein kinases. Curr Biol. 1995 Oct 1;5(10):1191–1200. doi: 10.1016/s0960-9822(95)00235-1. [DOI] [PubMed] [Google Scholar]
  15. Guan K. L., Dixon J. E. Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase. Anal Biochem. 1991 Feb 1;192(2):262–267. doi: 10.1016/0003-2697(91)90534-z. [DOI] [PubMed] [Google Scholar]
  16. Hancock J. F., Paterson H., Marshall C. J. A polybasic domain or palmitoylation is required in addition to the CAAX motif to localize p21ras to the plasma membrane. Cell. 1990 Oct 5;63(1):133–139. doi: 10.1016/0092-8674(90)90294-o. [DOI] [PubMed] [Google Scholar]
  17. Hawkins P. T., Eguinoa A., Qiu R. G., Stokoe D., Cooke F. T., Walters R., Wennström S., Claesson-Welsh L., Evans T., Symons M. PDGF stimulates an increase in GTP-Rac via activation of phosphoinositide 3-kinase. Curr Biol. 1995 Apr 1;5(4):393–403. doi: 10.1016/s0960-9822(95)00080-7. [DOI] [PubMed] [Google Scholar]
  18. Hu Q., Klippel A., Muslin A. J., Fantl W. J., Williams L. T. Ras-dependent induction of cellular responses by constitutively active phosphatidylinositol-3 kinase. Science. 1995 Apr 7;268(5207):100–102. doi: 10.1126/science.7701328. [DOI] [PubMed] [Google Scholar]
  19. Hunter T. Protein kinases and phosphatases: the yin and yang of protein phosphorylation and signaling. Cell. 1995 Jan 27;80(2):225–236. doi: 10.1016/0092-8674(95)90405-0. [DOI] [PubMed] [Google Scholar]
  20. Joly M., Kazlauskas A., Corvera S. Phosphatidylinositol 3-kinase activity is required at a postendocytic step in platelet-derived growth factor receptor trafficking. J Biol Chem. 1995 Jun 2;270(22):13225–13230. doi: 10.1074/jbc.270.22.13225. [DOI] [PubMed] [Google Scholar]
  21. Jones P. F., Jakubowicz T., Pitossi F. J., Maurer F., Hemmings B. A. Molecular cloning and identification of a serine/threonine protein kinase of the second-messenger subfamily. Proc Natl Acad Sci U S A. 1991 May 15;88(10):4171–4175. doi: 10.1073/pnas.88.10.4171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kapeller R., Cantley L. C. Phosphatidylinositol 3-kinase. Bioessays. 1994 Aug;16(8):565–576. doi: 10.1002/bies.950160810. [DOI] [PubMed] [Google Scholar]
  23. Kapeller R., Chakrabarti R., Cantley L., Fay F., Corvera S. Internalization of activated platelet-derived growth factor receptor-phosphatidylinositol-3' kinase complexes: potential interactions with the microtubule cytoskeleton. Mol Cell Biol. 1993 Oct;13(10):6052–6063. doi: 10.1128/mcb.13.10.6052. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kaplan D. R., Whitman M., Schaffhausen B., Raptis L., Garcea R. L., Pallas D., Roberts T. M., Cantley L. Phosphatidylinositol metabolism and polyoma-mediated transformation. Proc Natl Acad Sci U S A. 1986 Jun;83(11):3624–3628. doi: 10.1073/pnas.83.11.3624. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kaplan J. M., Varmus H. E., Bishop J. M. The src protein contains multiple domains for specific attachment to membranes. Mol Cell Biol. 1990 Mar;10(3):1000–1009. doi: 10.1128/mcb.10.3.1000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Keith C. T., Schreiber S. L. PIK-related kinases: DNA repair, recombination, and cell cycle checkpoints. Science. 1995 Oct 6;270(5233):50–51. doi: 10.1126/science.270.5233.50. [DOI] [PubMed] [Google Scholar]
  27. Klippel A., Escobedo J. A., Hirano M., Williams L. T. The interaction of small domains between the subunits of phosphatidylinositol 3-kinase determines enzyme activity. Mol Cell Biol. 1994 Apr;14(4):2675–2685. doi: 10.1128/mcb.14.4.2675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kodaki T., Woscholski R., Hallberg B., Rodriguez-Viciana P., Downward J., Parker P. J. The activation of phosphatidylinositol 3-kinase by Ras. Curr Biol. 1994 Sep 1;4(9):798–806. doi: 10.1016/s0960-9822(00)00177-9. [DOI] [PubMed] [Google Scholar]
  29. Kotani K., Yonezawa K., Hara K., Ueda H., Kitamura Y., Sakaue H., Ando A., Chavanieu A., Calas B., Grigorescu F. Involvement of phosphoinositide 3-kinase in insulin- or IGF-1-induced membrane ruffling. EMBO J. 1994 May 15;13(10):2313–2321. doi: 10.1002/j.1460-2075.1994.tb06515.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kundra V., Escobedo J. A., Kazlauskas A., Kim H. K., Rhee S. G., Williams L. T., Zetter B. R. Regulation of chemotaxis by the platelet-derived growth factor receptor-beta. Nature. 1994 Feb 3;367(6462):474–476. doi: 10.1038/367474a0. [DOI] [PubMed] [Google Scholar]
  31. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  32. Li G., D'Souza-Schorey C., Barbieri M. A., Roberts R. L., Klippel A., Williams L. T., Stahl P. D. Evidence for phosphatidylinositol 3-kinase as a regulator of endocytosis via activation of Rab5. Proc Natl Acad Sci U S A. 1995 Oct 24;92(22):10207–10211. doi: 10.1073/pnas.92.22.10207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Matthias P., Müller M. M., Schreiber E., Rusconi S., Schaffner W. Eukaryotic expression vectors for the analysis of mutant proteins. Nucleic Acids Res. 1989 Aug 11;17(15):6418–6418. doi: 10.1093/nar/17.15.6418. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Minden A., Lin A., Claret F. X., Abo A., Karin M. Selective activation of the JNK signaling cascade and c-Jun transcriptional activity by the small GTPases Rac and Cdc42Hs. Cell. 1995 Jun 30;81(7):1147–1157. doi: 10.1016/s0092-8674(05)80019-4. [DOI] [PubMed] [Google Scholar]
  35. Ming X. F., Burgering B. M., Wennström S., Claesson-Welsh L., Heldin C. H., Bos J. L., Kozma S. C., Thomas G. Activation of p70/p85 S6 kinase by a pathway independent of p21ras. Nature. 1994 Sep 29;371(6496):426–429. doi: 10.1038/371426a0. [DOI] [PubMed] [Google Scholar]
  36. Nakanishi S., Catt K. J., Balla T. A wortmannin-sensitive phosphatidylinositol 4-kinase that regulates hormone-sensitive pools of inositolphospholipids. Proc Natl Acad Sci U S A. 1995 Jun 6;92(12):5317–5321. doi: 10.1073/pnas.92.12.5317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Nebreda A. R., Hill C., Gomez N., Cohen P., Hunt T. The protein kinase mos activates MAP kinase kinase in vitro and stimulates the MAP kinase pathway in mammalian somatic cells in vivo. FEBS Lett. 1993 Oct 25;333(1-2):183–187. doi: 10.1016/0014-5793(93)80401-f. [DOI] [PubMed] [Google Scholar]
  38. Nobes C. D., Hall A. Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell. 1995 Apr 7;81(1):53–62. doi: 10.1016/0092-8674(95)90370-4. [DOI] [PubMed] [Google Scholar]
  39. Nobes C. D., Hawkins P., Stephens L., Hall A. Activation of the small GTP-binding proteins rho and rac by growth factor receptors. J Cell Sci. 1995 Jan;108(Pt 1):225–233. doi: 10.1242/jcs.108.1.225. [DOI] [PubMed] [Google Scholar]
  40. Okada T., Kawano Y., Sakakibara T., Hazeki O., Ui M. Essential role of phosphatidylinositol 3-kinase in insulin-induced glucose transport and antilipolysis in rat adipocytes. Studies with a selective inhibitor wortmannin. J Biol Chem. 1994 Feb 4;269(5):3568–3573. [PubMed] [Google Scholar]
  41. Paules R. S., Resnick J., Kasenally A. B., Ernst M. K., Donovan P., Vande Woude G. F. Characterization of activated and normal mouse Mos gene in murine 3T3 cells. Oncogene. 1992 Dec;7(12):2489–2498. [PubMed] [Google Scholar]
  42. Posada J., Yew N., Ahn N. G., Vande Woude G. F., Cooper J. A. Mos stimulates MAP kinase in Xenopus oocytes and activates a MAP kinase kinase in vitro. Mol Cell Biol. 1993 Apr;13(4):2546–2553. doi: 10.1128/mcb.13.4.2546. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Ray L. B., Sturgill T. W. Insulin-stimulated microtubule-associated protein kinase is phosphorylated on tyrosine and threonine in vivo. Proc Natl Acad Sci U S A. 1988 Jun;85(11):3753–3757. doi: 10.1073/pnas.85.11.3753. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Rodriguez-Viciana P., Warne P. H., Dhand R., Vanhaesebroeck B., Gout I., Fry M. J., Waterfield M. D., Downward J. Phosphatidylinositol-3-OH kinase as a direct target of Ras. Nature. 1994 Aug 18;370(6490):527–532. doi: 10.1038/370527a0. [DOI] [PubMed] [Google Scholar]
  45. Schultz A. M., Henderson L. E., Oroszlan S., Garber E. A., Hanafusa H. Amino terminal myristylation of the protein kinase p60src, a retroviral transforming protein. Science. 1985 Jan 25;227(4685):427–429. doi: 10.1126/science.3917576. [DOI] [PubMed] [Google Scholar]
  46. 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]
  47. 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]
  48. Stanssens P., Opsomer C., McKeown Y. M., Kramer W., Zabeau M., Fritz H. J. Efficient oligonucleotide-directed construction of mutations in expression vectors by the gapped duplex DNA method using alternating selectable markers. Nucleic Acids Res. 1989 Jun 26;17(12):4441–4454. doi: 10.1093/nar/17.12.4441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Stephens L. R., Jackson T. R., Hawkins P. T. Agonist-stimulated synthesis of phosphatidylinositol(3,4,5)-trisphosphate: a new intracellular signalling system? Biochim Biophys Acta. 1993 Oct 7;1179(1):27–75. doi: 10.1016/0167-4889(93)90072-w. [DOI] [PubMed] [Google Scholar]
  50. Stoyanov B., Volinia S., Hanck T., Rubio I., Loubtchenkov M., Malek D., Stoyanova S., Vanhaesebroeck B., Dhand R., Nürnberg B. Cloning and characterization of a G protein-activated human phosphoinositide-3 kinase. Science. 1995 Aug 4;269(5224):690–693. doi: 10.1126/science.7624799. [DOI] [PubMed] [Google Scholar]
  51. Takebe Y., Seiki M., Fujisawa J., Hoy P., Yokota K., Arai K., Yoshida M., Arai N. SR alpha promoter: an efficient and versatile mammalian cDNA expression system composed of the simian virus 40 early promoter and the R-U5 segment of human T-cell leukemia virus type 1 long terminal repeat. Mol Cell Biol. 1988 Jan;8(1):466–472. doi: 10.1128/mcb.8.1.466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Terada N., Franklin R. A., Lucas J. J., Blenis J., Gelfand E. W. Failure of rapamycin to block proliferation once resting cells have entered the cell cycle despite inactivation of p70 S6 kinase. J Biol Chem. 1993 Jun 5;268(16):12062–12068. [PubMed] [Google Scholar]
  53. Tolias K. F., Cantley L. C., Carpenter C. L. Rho family GTPases bind to phosphoinositide kinases. J Biol Chem. 1995 Jul 28;270(30):17656–17659. doi: 10.1074/jbc.270.30.17656. [DOI] [PubMed] [Google Scholar]
  54. Ui M., Okada T., Hazeki K., Hazeki O. Wortmannin as a unique probe for an intracellular signalling protein, phosphoinositide 3-kinase. Trends Biochem Sci. 1995 Aug;20(8):303–307. doi: 10.1016/s0968-0004(00)89056-8. [DOI] [PubMed] [Google Scholar]
  55. 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]
  56. Volinia S., Dhand R., Vanhaesebroeck B., MacDougall L. K., Stein R., Zvelebil M. J., Domin J., Panaretou C., Waterfield M. D. A human phosphatidylinositol 3-kinase complex related to the yeast Vps34p-Vps15p protein sorting system. EMBO J. 1995 Jul 17;14(14):3339–3348. doi: 10.1002/j.1460-2075.1995.tb07340.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Weng Q. P., Andrabi K., Klippel A., Kozlowski M. T., Williams L. T., 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. doi: 10.1073/pnas.92.12.5744. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Wennström S., Hawkins P., Cooke F., Hara K., Yonezawa K., Kasuga M., Jackson T., Claesson-Welsh L., Stephens L. Activation of phosphoinositide 3-kinase is required for PDGF-stimulated membrane ruffling. Curr Biol. 1994 May 1;4(5):385–393. doi: 10.1016/s0960-9822(00)00087-7. [DOI] [PubMed] [Google Scholar]
  59. 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]
  60. Whitmarsh A. J., Shore P., Sharrocks A. D., Davis R. J. Integration of MAP kinase signal transduction pathways at the serum response element. Science. 1995 Jul 21;269(5222):403–407. doi: 10.1126/science.7618106. [DOI] [PubMed] [Google Scholar]
  61. Wilson I. A., Niman H. L., Houghten R. A., Cherenson A. R., Connolly M. L., Lerner R. A. The structure of an antigenic determinant in a protein. Cell. 1984 Jul;37(3):767–778. doi: 10.1016/0092-8674(84)90412-4. [DOI] [PubMed] [Google Scholar]
  62. 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]
  63. Zhang J., Fry M. J., Waterfield M. D., Jaken S., Liao L., Fox J. E., Rittenhouse S. E. Activated phosphoinositide 3-kinase associates with membrane skeleton in thrombin-exposed platelets. J Biol Chem. 1992 Mar 5;267(7):4686–4692. [PubMed] [Google Scholar]
  64. Zheng Y., Bagrodia S., Cerione R. A. Activation of phosphoinositide 3-kinase activity by Cdc42Hs binding to p85. J Biol Chem. 1994 Jul 22;269(29):18727–18730. [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES