Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1996 Apr;16(4):1722–1733. doi: 10.1128/mcb.16.4.1722

Wortmannin inactivates phosphoinositide 3-kinase by covalent modification of Lys-802, a residue involved in the phosphate transfer reaction.

M P Wymann 1, G Bulgarelli-Leva 1, M J Zvelebil 1, L Pirola 1, B Vanhaesebroeck 1, M D Waterfield 1, G Panayotou 1
PMCID: PMC231159  PMID: 8657148

Abstract

Wortmannin at nanomolar concentrations is a potent and specific inhibitor of phosphoinositide (PI) 3-kinase and has been used extensively to demonstrate the role of this enzyme in diverse signal transduction processes. At higher concentrations, wortmannin inhibits the ataxia telangiectasia gene (ATM)-related DNA-dependent protein kinase (DNA-PKcs). We report here the identification of the site of interaction of wortmannin on the catalytic subunit of PI 3-kinase, p110alpha. At physiological pH (6.5 to 8) wortmannin reacted specifically with p110alpha. Phosphatidylinositol-4,5-diphosphate, ATP, and ATP analogs [adenine and 5'-(4-fluorosulfonylbenzoyl)adenine] competed effectively with wortmannin, while substances containing nucleophilic amino acid side chain functions had no effect at the same concentrations. This suggests that the wortmannin target site is localized in proximity to the substrate-binding site and that residues involved in wortmannin binding have an increased nucleophilicity because of their protein environment. Proteolytic fragments of wortmannin-treated, recombinant p110alpha were mapped with anti-wortmannin and anti-p110alpha peptide antibodies, thus limiting the target site within a 10-kDa fragment, colocalizing with the ATP-binding site. Site-directed mutagenesis of all candidate residues within this region showed that only the conservative Lys-802-to-Arg mutation abolished wortmannin binding. Inhibition of PI 3-kinase occurs, therefore, by the formation of an enamine following the attack of Lys-802 on the furan ring (at C-20) of wortmannin. The Lys-802-to-Arg mutant was also unable to bind FSBA and was catalytically inactive in lipid and protein kinase assays, indicating a crucial role for Lys-802 in the phosphotransfer reaction. In contrast, an Arg-916-to-Pro mutation abolished the catalytic activity whereas covalent wortmannin binding remained intact. Our results provide the basis for the design of novel and specific inhibitors of an enzyme family, including PI kinases and ATM-related genes, that play a central role in many physiological processes.

Full Text

The Full Text of this article is available as a PDF (748.8 KB).

Selected References

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

  1. Arcaro A., Wymann M. P. Wortmannin is a potent phosphatidylinositol 3-kinase inhibitor: the role of phosphatidylinositol 3,4,5-trisphosphate in neutrophil responses. Biochem J. 1993 Dec 1;296(Pt 2):297–301. doi: 10.1042/bj2960297. [DOI] [PMC free article] [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. Baggiolini M., Dewald B., Schnyder J., Ruch W., Cooper P. H., Payne T. G. Inhibition of the phagocytosis-induced respiratory burst by the fungal metabolite wortmannin and some analogues. Exp Cell Res. 1987 Apr;169(2):408–418. doi: 10.1016/0014-4827(87)90201-1. [DOI] [PubMed] [Google Scholar]
  4. Barton G. J., Sternberg M. J. A strategy for the rapid multiple alignment of protein sequences. Confidence levels from tertiary structure comparisons. J Mol Biol. 1987 Nov 20;198(2):327–337. doi: 10.1016/0022-2836(87)90316-0. [DOI] [PubMed] [Google Scholar]
  5. Bonser R. W., Thompson N. T., Randall R. W., Tateson J. E., Spacey G. D., Hodson H. F., Garland L. G. Demethoxyviridin and wortmannin block phospholipase C and D activation in the human neutrophil. Br J Pharmacol. 1991 May;103(1):1237–1241. doi: 10.1111/j.1476-5381.1991.tb12330.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brown E. J., Albers M. W., Shin T. B., Ichikawa K., Keith C. T., Lane W. S., Schreiber S. L. A mammalian protein targeted by G1-arresting rapamycin-receptor complex. Nature. 1994 Jun 30;369(6483):756–758. doi: 10.1038/369756a0. [DOI] [PubMed] [Google Scholar]
  7. Brown W. J., DeWald D. B., Emr S. D., Plutner H., Balch W. E. Role for phosphatidylinositol 3-kinase in the sorting and transport of newly synthesized lysosomal enzymes in mammalian cells. J Cell Biol. 1995 Aug;130(4):781–796. doi: 10.1083/jcb.130.4.781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Chuang T. H., Bohl B. P., Bokoch G. M. Biologically active lipids are regulators of Rac.GDI complexation. J Biol Chem. 1993 Dec 15;268(35):26206–26211. [PubMed] [Google Scholar]
  10. Chung J., Grammer T. C., Lemon K. P., Kazlauskas A., Blenis J. PDGF- and insulin-dependent pp70S6k activation mediated by phosphatidylinositol-3-OH kinase. Nature. 1994 Jul 7;370(6484):71–75. doi: 10.1038/370071a0. [DOI] [PubMed] [Google Scholar]
  11. Claesson-Welsh L. Platelet-derived growth factor receptor signals. J Biol Chem. 1994 Dec 23;269(51):32023–32026. [PubMed] [Google Scholar]
  12. Cross D. A., Alessi D. R., Vandenheede J. R., McDowell H. E., Hundal H. S., Cohen P. The inhibition of glycogen synthase kinase-3 by insulin or insulin-like growth factor 1 in the rat skeletal muscle cell line L6 is blocked by wortmannin, but not by rapamycin: evidence that wortmannin blocks activation of the mitogen-activated protein kinase pathway in L6 cells between Ras and Raf. Biochem J. 1994 Oct 1;303(Pt 1):21–26. doi: 10.1042/bj3030021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Davidson H. W. Wortmannin causes mistargeting of procathepsin D. evidence for the involvement of a phosphatidylinositol 3-kinase in vesicular transport to lysosomes. J Cell Biol. 1995 Aug;130(4):797–805. doi: 10.1083/jcb.130.4.797. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dhand R., Hara K., Hiles I., Bax B., Gout I., Panayotou G., Fry M. J., Yonezawa K., Kasuga M., Waterfield M. D. PI 3-kinase: structural and functional analysis of intersubunit interactions. EMBO J. 1994 Feb 1;13(3):511–521. doi: 10.1002/j.1460-2075.1994.tb06289.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Dhand R., Hiles I., Panayotou G., Roche S., Fry M. J., Gout I., Totty N. F., Truong O., Vicendo P., Yonezawa K. PI 3-kinase is a dual specificity enzyme: autoregulation by an intrinsic protein-serine kinase activity. EMBO J. 1994 Feb 1;13(3):522–533. doi: 10.1002/j.1460-2075.1994.tb06290.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. End P., Gout I., Fry M. J., Panayotou G., Dhand R., Yonezawa K., Kasuga M., Waterfield M. D. A biosensor approach to probe the structure and function of the p85 alpha subunit of the phosphatidylinositol 3-kinase complex. J Biol Chem. 1993 May 15;268(14):10066–10075. [PubMed] [Google Scholar]
  17. Escobedo J. A., Navankasattusas S., Kavanaugh W. M., Milfay D., Fried V. A., Williams L. T. cDNA cloning of a novel 85 kd protein that has SH2 domains and regulates binding of PI3-kinase to the PDGF beta-receptor. Cell. 1991 Apr 5;65(1):75–82. doi: 10.1016/0092-8674(91)90409-r. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Fry M. J., Panayotou G., Dhand R., Ruiz-Larrea F., Gout I., Nguyen O., Courtneidge S. A., Waterfield M. D. Purification and characterization of a phosphatidylinositol 3-kinase complex from bovine brain by using phosphopeptide affinity columns. Biochem J. 1992 Dec 1;288(Pt 2):383–393. doi: 10.1042/bj2880383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Gélas P., Von Tscharner V., Record M., Baggiolini M., Chap H. Human neutrophil phospholipase D activation by N-formylmethionyl-leucylphenylalanine reveals a two-step process for the control of phosphatidylcholine breakdown and oxidative burst. Biochem J. 1992 Oct 1;287(Pt 1):67–72. doi: 10.1042/bj2870067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hartley K. O., Gell D., Smith G. C., Zhang H., Divecha N., Connelly M. A., Admon A., Lees-Miller S. P., Anderson C. W., Jackson S. P. DNA-dependent protein kinase catalytic subunit: a relative of phosphatidylinositol 3-kinase and the ataxia telangiectasia gene product. Cell. 1995 Sep 8;82(5):849–856. doi: 10.1016/0092-8674(95)90482-4. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Hawkins P. T., Jackson T. R., Stephens L. R. Platelet-derived growth factor stimulates synthesis of PtdIns(3,4,5)P3 by activating a PtdIns(4,5)P2 3-OH kinase. Nature. 1992 Jul 9;358(6382):157–159. doi: 10.1038/358157a0. [DOI] [PubMed] [Google Scholar]
  24. Herman P. K., Emr S. D. Characterization of VPS34, a gene required for vacuolar protein sorting and vacuole segregation in Saccharomyces cerevisiae. Mol Cell Biol. 1990 Dec;10(12):6742–6754. doi: 10.1128/mcb.10.12.6742. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. 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]
  27. Janmey P. A., Iida K., Yin H. L., Stossel T. P. Polyphosphoinositide micelles and polyphosphoinositide-containing vesicles dissociate endogenous gelsolin-actin complexes and promote actin assembly from the fast-growing end of actin filaments blocked by gelsolin. J Biol Chem. 1987 Sep 5;262(25):12228–12236. [PubMed] [Google Scholar]
  28. Kanai F., Ito K., Todaka M., Hayashi H., Kamohara S., Ishii K., Okada T., Hazeki O., Ui M., Ebina Y. Insulin-stimulated GLUT4 translocation is relevant to the phosphorylation of IRS-1 and the activity of PI3-kinase. Biochem Biophys Res Commun. 1993 Sep 15;195(2):762–768. doi: 10.1006/bbrc.1993.2111. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Kaufman R. J., Davies M. V., Pathak V. K., Hershey J. W. The phosphorylation state of eucaryotic initiation factor 2 alters translational efficiency of specific mRNAs. Mol Cell Biol. 1989 Mar;9(3):946–958. doi: 10.1128/mcb.9.3.946. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Kazlauskas A., Cooper J. A. Phosphorylation of the PDGF receptor beta subunit creates a tight binding site for phosphatidylinositol 3 kinase. EMBO J. 1990 Oct;9(10):3279–3286. doi: 10.1002/j.1460-2075.1990.tb07527.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kimura K., Hattori S., Kabuyama Y., Shizawa Y., Takayanagi J., Nakamura S., Toki S., Matsuda Y., Onodera K., Fukui Y. Neurite outgrowth of PC12 cells is suppressed by wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase. J Biol Chem. 1994 Jul 22;269(29):18961–18967. [PubMed] [Google Scholar]
  33. Komatsu H., Ikebe M. Affinity labelling of smooth-muscle myosin light-chain kinase with 5'-[p-(fluorosulphonyl)benzoyl]adenosine. Biochem J. 1993 Nov 15;296(Pt 1):53–58. doi: 10.1042/bj2960053. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Kovacsovics T. J., Bachelot C., Toker A., Vlahos C. J., Duckworth B., Cantley L. C., Hartwig J. H. Phosphoinositide 3-kinase inhibition spares actin assembly in activating platelets but reverses platelet aggregation. J Biol Chem. 1995 May 12;270(19):11358–11366. doi: 10.1074/jbc.270.19.11358. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. 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]
  37. 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]
  38. Madhusudan, Trafny E. A., Xuong N. H., Adams J. A., Ten Eyck L. F., Taylor S. S., Sowadski J. M. cAMP-dependent protein kinase: crystallographic insights into substrate recognition and phosphotransfer. Protein Sci. 1994 Feb;3(2):176–187. doi: 10.1002/pro.5560030203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Nakanishi H., Brewer K. A., Exton J. H. Activation of the zeta isozyme of protein kinase C by phosphatidylinositol 3,4,5-trisphosphate. J Biol Chem. 1993 Jan 5;268(1):13–16. [PubMed] [Google Scholar]
  40. 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]
  41. Nakanishi S., Kakita S., Takahashi I., Kawahara K., Tsukuda E., Sano T., Yamada K., Yoshida M., Kase H., Matsuda Y. Wortmannin, a microbial product inhibitor of myosin light chain kinase. J Biol Chem. 1992 Feb 5;267(4):2157–2163. [PubMed] [Google Scholar]
  42. 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]
  43. Okada T., Sakuma L., Fukui Y., Hazeki O., Ui M. Blockage of chemotactic peptide-induced stimulation of neutrophils by wortmannin as a result of selective inhibition of phosphatidylinositol 3-kinase. J Biol Chem. 1994 Feb 4;269(5):3563–3567. [PubMed] [Google Scholar]
  44. Otsu M., Hiles I., Gout I., Fry M. J., Ruiz-Larrea F., Panayotou G., Thompson A., Dhand R., Hsuan J., Totty N. Characterization of two 85 kd proteins that associate with receptor tyrosine kinases, middle-T/pp60c-src complexes, and PI3-kinase. Cell. 1991 Apr 5;65(1):91–104. doi: 10.1016/0092-8674(91)90411-q. [DOI] [PubMed] [Google Scholar]
  45. Page M. J. p36C: an improved baculovirus expression vector for producing high levels of mature recombinant proteins. Nucleic Acids Res. 1989 Jan 11;17(1):454–454. doi: 10.1093/nar/17.1.454. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. 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]
  47. Reinhold S. L., Prescott S. M., Zimmerman G. A., McIntyre T. M. Activation of human neutrophil phospholipase D by three separable mechanisms. FASEB J. 1990 Feb 1;4(2):208–214. doi: 10.1096/fasebj.4.2.2105252. [DOI] [PubMed] [Google Scholar]
  48. Rusconi S., Severne Y., Georgiev O., Galli I., Wieland S. A novel expression assay to study transcriptional activators. Gene. 1990 May 14;89(2):211–221. doi: 10.1016/0378-1119(90)90008-f. [DOI] [PubMed] [Google Scholar]
  49. Sabatini D. M., Erdjument-Bromage H., Lui M., Tempst P., Snyder S. H. RAFT1: a mammalian protein that binds to FKBP12 in a rapamycin-dependent fashion and is homologous to yeast TORs. Cell. 1994 Jul 15;78(1):35–43. doi: 10.1016/0092-8674(94)90570-3. [DOI] [PubMed] [Google Scholar]
  50. Sabers C. J., Martin M. M., Brunn G. J., Williams J. M., Dumont F. J., Wiederrecht G., Abraham R. T. Isolation of a protein target of the FKBP12-rapamycin complex in mammalian cells. J Biol Chem. 1995 Jan 13;270(2):815–822. doi: 10.1074/jbc.270.2.815. [DOI] [PubMed] [Google Scholar]
  51. Saraste M., Sibbald P. R., Wittinghofer A. The P-loop--a common motif in ATP- and GTP-binding proteins. Trends Biochem Sci. 1990 Nov;15(11):430–434. doi: 10.1016/0968-0004(90)90281-f. [DOI] [PubMed] [Google Scholar]
  52. Scholz G., Barritt G. J., Kwok F. Affinity labelling of the active site of brain phosphatidylinositol 4-kinase with 5'-fluorosulphonylbenzoyl-adenosine. Eur J Biochem. 1992 Dec 1;210(2):461–466. doi: 10.1111/j.1432-1033.1992.tb17443.x. [DOI] [PubMed] [Google Scholar]
  53. 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]
  54. Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
  55. Shibasaki F., Fukui Y., Takenawa T. Different properties of monomer and heterodimer forms of phosphatidylinositol 3-kinases. Biochem J. 1993 Jan 1;289(Pt 1):227–231. doi: 10.1042/bj2890227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Skolnik E. Y., Margolis B., Mohammadi M., Lowenstein E., Fischer R., Drepps A., Ullrich A., Schlessinger J. Cloning of PI3 kinase-associated p85 utilizing a novel method for expression/cloning of target proteins for receptor tyrosine kinases. Cell. 1991 Apr 5;65(1):83–90. doi: 10.1016/0092-8674(91)90410-z. [DOI] [PubMed] [Google Scholar]
  57. Stack J. H., DeWald D. B., Takegawa K., Emr S. D. Vesicle-mediated protein transport: regulatory interactions between the Vps15 protein kinase and the Vps34 PtdIns 3-kinase essential for protein sorting to the vacuole in yeast. J Cell Biol. 1995 Apr;129(2):321–334. doi: 10.1083/jcb.129.2.321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Stephens L. R., Hughes K. T., Irvine R. F. Pathway of phosphatidylinositol(3,4,5)-trisphosphate synthesis in activated neutrophils. Nature. 1991 May 2;351(6321):33–39. doi: 10.1038/351033a0. [DOI] [PubMed] [Google Scholar]
  59. Stephens L., Smrcka A., Cooke F. T., Jackson T. R., Sternweis P. C., Hawkins P. T. A novel phosphoinositide 3 kinase activity in myeloid-derived cells is activated by G protein beta gamma subunits. Cell. 1994 Apr 8;77(1):83–93. doi: 10.1016/0092-8674(94)90237-2. [DOI] [PubMed] [Google Scholar]
  60. Sternberg M. J., Zvelebil M. J. Prediction of protein structure from sequence. Eur J Cancer. 1990;26(11-12):1163–1166. doi: 10.1016/0277-5379(90)90277-z. [DOI] [PubMed] [Google Scholar]
  61. 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]
  62. Summers N. L., Karplus M. Construction of side-chains in homology modelling. Application to the C-terminal lobe of rhizopuspepsin. J Mol Biol. 1989 Dec 20;210(4):785–811. doi: 10.1016/0022-2836(89)90109-5. [DOI] [PubMed] [Google Scholar]
  63. Taylor S. S., Knighton D. R., Zheng J., Ten Eyck L. F., Sowadski J. M. Structural framework for the protein kinase family. Annu Rev Cell Biol. 1992;8:429–462. doi: 10.1146/annurev.cb.08.110192.002241. [DOI] [PubMed] [Google Scholar]
  64. Thelen M., Wymann M. P., Langen H. Wortmannin binds specifically to 1-phosphatidylinositol 3-kinase while inhibiting guanine nucleotide-binding protein-coupled receptor signaling in neutrophil leukocytes. Proc Natl Acad Sci U S A. 1994 May 24;91(11):4960–4964. doi: 10.1073/pnas.91.11.4960. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Toker A., Meyer M., Reddy K. K., Falck J. R., Aneja R., Aneja S., Parra A., Burns D. J., Ballas L. M., Cantley L. C. Activation of protein kinase C family members by the novel polyphosphoinositides PtdIns-3,4-P2 and PtdIns-3,4,5-P3. J Biol Chem. 1994 Dec 23;269(51):32358–32367. [PubMed] [Google Scholar]
  66. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. 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]
  68. 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]
  69. 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]
  70. 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]
  71. Volinia S., Hiles I., Ormondroyd E., Nizetic D., Antonacci R., Rocchi M., Waterfield M. D. Molecular cloning, cDNA sequence, and chromosomal localization of the human phosphatidylinositol 3-kinase p110 alpha (PIK3CA) gene. Genomics. 1994 Dec;24(3):472–477. doi: 10.1006/geno.1994.1655. [DOI] [PubMed] [Google Scholar]
  72. Welsh G. I., Foulstone E. J., Young S. W., Tavaré J. M., Proud C. G. Wortmannin inhibits the effects of insulin and serum on the activities of glycogen synthase kinase-3 and mitogen-activated protein kinase. Biochem J. 1994 Oct 1;303(Pt 1):15–20. doi: 10.1042/bj3030015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  73. 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]
  74. Wennström S., Siegbahn A., Yokote K., Arvidsson A. K., Heldin C. H., Mori S., Claesson-Welsh L. Membrane ruffling and chemotaxis transduced by the PDGF beta-receptor require the binding site for phosphatidylinositol 3' kinase. Oncogene. 1994 Feb;9(2):651–660. [PubMed] [Google Scholar]
  75. Wessel D., Flügge U. I. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal Biochem. 1984 Apr;138(1):141–143. doi: 10.1016/0003-2697(84)90782-6. [DOI] [PubMed] [Google Scholar]
  76. White M. F., Kahn C. R. The insulin signaling system. J Biol Chem. 1994 Jan 7;269(1):1–4. [PubMed] [Google Scholar]
  77. Woscholski R., Kodaki T., McKinnon M., Waterfield M. D., Parker P. J. A comparison of demethoxyviridin and wortmannin as inhibitors of phosphatidylinositol 3-kinase. FEBS Lett. 1994 Apr 4;342(2):109–114. doi: 10.1016/0014-5793(94)80482-6. [DOI] [PubMed] [Google Scholar]
  78. Wymann M., Arcaro A. Platelet-derived growth factor-induced phosphatidylinositol 3-kinase activation mediates actin rearrangements in fibroblasts. Biochem J. 1994 Mar 15;298(Pt 3):517–520. doi: 10.1042/bj2980517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  79. 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]
  80. Yao R., Cooper G. M. Requirement for phosphatidylinositol-3 kinase in the prevention of apoptosis by nerve growth factor. Science. 1995 Mar 31;267(5206):2003–2006. doi: 10.1126/science.7701324. [DOI] [PubMed] [Google Scholar]
  81. Yeh J. I., Gulve E. A., Rameh L., Birnbaum M. J. The effects of wortmannin on rat skeletal muscle. Dissociation of signaling pathways for insulin- and contraction-activated hexose transport. J Biol Chem. 1995 Feb 3;270(5):2107–2111. doi: 10.1074/jbc.270.5.2107. [DOI] [PubMed] [Google Scholar]
  82. Yu F. X., Sun H. Q., Janmey P. A., Yin H. L. Identification of a polyphosphoinositide-binding sequence in an actin monomer-binding domain of gelsolin. J Biol Chem. 1992 Jul 25;267(21):14616–14621. [PubMed] [Google Scholar]
  83. Zakian V. A. ATM-related genes: what do they tell us about functions of the human gene? Cell. 1995 Sep 8;82(5):685–687. doi: 10.1016/0092-8674(95)90463-8. [DOI] [PubMed] [Google Scholar]
  84. Zoller M. J., Nelson N. C., Taylor S. S. Affinity labeling of cAMP-dependent protein kinase with p-fluorosulfonylbenzoyl adenosine. Covalent modification of lysine 71. J Biol Chem. 1981 Nov 10;256(21):10837–10842. [PubMed] [Google Scholar]
  85. Zvelebil M. J., Barton G. J., Taylor W. R., Sternberg M. J. Prediction of protein secondary structure and active sites using the alignment of homologous sequences. J Mol Biol. 1987 Jun 20;195(4):957–961. doi: 10.1016/0022-2836(87)90501-8. [DOI] [PubMed] [Google Scholar]

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

RESOURCES