Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1997 Feb 1;321(Pt 3):849–856. doi: 10.1042/bj3210849

Molecular cloning and biochemical characterization of a Drosophila phosphatidylinositol-specific phosphoinositide 3-kinase.

C Linassier 1, L K MacDougall 1, J Domin 1, M D Waterfield 1
PMCID: PMC1218144  PMID: 9032475

Abstract

Molecular, biochemical and genetic characterization of phosphoinositide 3-kinases (PI3Ks) have identified distinct classes of enzymes involved in processes mediated by activation of cell-surface receptors and in constitutive intracellular protein trafficking events. The latter process appears to involve a PtdIns-specific PI3K first described in yeast as a mutant, vps34, defective in the sorting of newly synthesized proteins from the Golgi to the vacuole. We have identified a representative member of each class of PI3Ks in Drosophila using a PCR-based approach. In the present paper we describe the molecular cloning of a PI3K from Drosophila, P13K_59F, that shows sequence similarity to Vps34. PI3K_59F encodes a protein of 108 kDa co-linear with Vps34 homologues, and with three regions of sequence similarity to other PI3Ks. Biochemical characterization of the enzyme, by expression of the complete coding sequence as a glutathione S-transferase fusion protein in Sf9 cells, demonstrates that PI3K_59F is a PtdIns-specific PI3K that can utilize either Mg2+ or Mn2+. This activity is sensitive to inhibition both by non-ionic detergent (Nonidet P40) and by wortmannin (IC50 10 nM). PI3K_59F, therefore, conserves both the structural and biochemical properties of the Vps34 class of enzymes.

Full Text

The Full Text of this article is available as a PDF (771.8 KB).

Selected References

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

  1. 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]
  2. Carlberg K., Tapley P., Haystead C., Rohrschneider L. The role of kinase activity and the kinase insert region in ligand-induced internalization and degradation of the c-fms protein. EMBO J. 1991 Apr;10(4):877–883. doi: 10.1002/j.1460-2075.1991.tb08020.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Carpenter C. L., Cantley L. C. Phosphoinositide kinases. Biochemistry. 1990 Dec 25;29(51):11147–11156. doi: 10.1021/bi00503a001. [DOI] [PubMed] [Google Scholar]
  4. Cavener D. R., Ray S. C. Eukaryotic start and stop translation sites. Nucleic Acids Res. 1991 Jun 25;19(12):3185–3192. doi: 10.1093/nar/19.12.3185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chapman R. E. Vacuolar sorting. Tracking down an elusive receptor. Curr Biol. 1994 Nov 1;4(11):1019–1022. doi: 10.1016/s0960-9822(00)00231-1. [DOI] [PubMed] [Google Scholar]
  6. Clarke N. G., Dawson R. M. Alkaline O leads to N-transacylation. A new method for the quantitative deacylation of phospholipids. Biochem J. 1981 Apr 1;195(1):301–306. doi: 10.1042/bj1950301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Davies A. H., Jowett J. B., Jones I. M. Recombinant baculovirus vectors expressing glutathione-S-transferase fusion proteins. Biotechnology (N Y) 1993 Aug;11(8):933–936. doi: 10.1038/nbt0893-933. [DOI] [PubMed] [Google Scholar]
  9. De Camilli P., Emr S. D., McPherson P. S., Novick P. Phosphoinositides as regulators in membrane traffic. Science. 1996 Mar 15;271(5255):1533–1539. doi: 10.1126/science.271.5255.1533. [DOI] [PubMed] [Google Scholar]
  10. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. Downing J. R., Roussel M. F., Sherr C. J. Ligand and protein kinase C downmodulate the colony-stimulating factor 1 receptor by independent mechanisms. Mol Cell Biol. 1989 Jul;9(7):2890–2896. doi: 10.1128/mcb.9.7.2890. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Fry M. J. Structure, regulation and function of phosphoinositide 3-kinases. Biochim Biophys Acta. 1994 Jul 18;1226(3):237–268. doi: 10.1016/0925-4439(94)90036-1. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. 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]
  19. Hong Z., Verma D. P. A phosphatidylinositol 3-kinase is induced during soybean nodule organogenesis and is associated with membrane proliferation. Proc Natl Acad Sci U S A. 1994 Sep 27;91(20):9617–9621. doi: 10.1073/pnas.91.20.9617. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Hunter T. When is a lipid kinase not a lipid kinase? When it is a protein kinase. Cell. 1995 Oct 6;83(1):1–4. doi: 10.1016/0092-8674(95)90225-2. [DOI] [PubMed] [Google Scholar]
  22. Joly M., Kazlauskas A., Fay F. S., Corvera S. Disruption of PDGF receptor trafficking by mutation of its PI-3 kinase binding sites. Science. 1994 Feb 4;263(5147):684–687. doi: 10.1126/science.8303278. [DOI] [PubMed] [Google Scholar]
  23. MacDougall L. K., Domin J., Waterfield M. D. A family of phosphoinositide 3-kinases in Drosophila identifies a new mediator of signal transduction. Curr Biol. 1995 Dec 1;5(12):1404–1415. doi: 10.1016/s0960-9822(95)00278-8. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. Pons S., Asano T., Glasheen E., Miralpeix M., Zhang Y., Fisher T. L., Myers M. G., Jr, Sun X. J., White M. F. The structure and function of p55PIK reveal a new regulatory subunit for phosphatidylinositol 3-kinase. Mol Cell Biol. 1995 Aug;15(8):4453–4465. doi: 10.1128/mcb.15.8.4453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Rodriguez-Viciana P., Warne P. H., Vanhaesebroeck B., Waterfield M. D., Downward J. Activation of phosphoinositide 3-kinase by interaction with Ras and by point mutation. EMBO J. 1996 May 15;15(10):2442–2451. [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. 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]
  30. Stack J. H., Emr S. D. Vps34p required for yeast vacuolar protein sorting is a multiple specificity kinase that exhibits both protein kinase and phosphatidylinositol-specific PI 3-kinase activities. J Biol Chem. 1994 Dec 16;269(50):31552–31562. [PubMed] [Google Scholar]
  31. 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]
  32. 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]
  33. Stephens L., Cooke F. T., Walters R., Jackson T., Volinia S., Gout I., Waterfield M. D., Hawkins P. T. Characterization of a phosphatidylinositol-specific phosphoinositide 3-kinase from mammalian cells. Curr Biol. 1994 Mar 1;4(3):203–214. doi: 10.1016/s0960-9822(00)00049-x. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. Takegawa K., DeWald D. B., Emr S. D. Schizosaccharomyces pombe Vps34p, a phosphatidylinositol-specific PI 3-kinase essential for normal cell growth and vacuole morphology. J Cell Sci. 1995 Dec;108(Pt 12):3745–3756. doi: 10.1242/jcs.108.12.3745. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. Welters P., Takegawa K., Emr S. D., Chrispeels M. J. AtVPS34, a phosphatidylinositol 3-kinase of Arabidopsis thaliana, is an essential protein with homology to a calcium-dependent lipid binding domain. Proc Natl Acad Sci U S A. 1994 Nov 22;91(24):11398–11402. doi: 10.1073/pnas.91.24.11398. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Whitman M., Kaplan D. R., Schaffhausen B., Cantley L., Roberts T. M. Association of phosphatidylinositol kinase activity with polyoma middle-T competent for transformation. Nature. 1985 May 16;315(6016):239–242. doi: 10.1038/315239a0. [DOI] [PubMed] [Google Scholar]
  39. Wymann M. P., Bulgarelli-Leva G., Zvelebil M. J., Pirola L., Vanhaesebroeck B., Waterfield M. D., Panayotou G. Wortmannin inactivates phosphoinositide 3-kinase by covalent modification of Lys-802, a residue involved in the phosphate transfer reaction. Mol Cell Biol. 1996 Apr;16(4):1722–1733. doi: 10.1128/mcb.16.4.1722. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. 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]
  41. Zhou K., Takegawa K., Emr S. D., Firtel R. A. A phosphatidylinositol (PI) kinase gene family in Dictyostelium discoideum: biological roles of putative mammalian p110 and yeast Vps34p PI 3-kinase homologs during growth and development. Mol Cell Biol. 1995 Oct;15(10):5645–5656. doi: 10.1128/mcb.15.10.5645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Zvelebil M. J., MacDougall L., Leevers S., Volinia S., Vanhaesebroeck B., Gout I., Panayotou G., Domin J., Stein R., Pages F. Structural and functional diversity of phosphoinositide 3-kinases. Philos Trans R Soc Lond B Biol Sci. 1996 Feb 29;351(1336):217–223. doi: 10.1098/rstb.1996.0019. [DOI] [PubMed] [Google Scholar]

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

RESOURCES