Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1996 Dec 1;320(Pt 2):643–649. doi: 10.1042/bj3200643

Cloning and characterization of a 92 kDa soluble phosphatidylinositol 4-kinase.

T Nakagawa 1, K Goto 1, H Kondo 1
PMCID: PMC1217978  PMID: 8973579

Abstract

A phosphatidylinositol (PtdIns) 4-kinase cDNA cloned from a rat brain cDNA library encoded a protein of 816 amino acids with a calculated molecular mass of 91654 Da. This molecule contained a lipid-kinase-unique domain and a presumed lipid/ protein kinase homology domain that are found in other PtdIns 4-kinases and PtdIns 3-kinases. Furthermore, this kinase molecule had 43.3% shared identity with the presumed catalytic domain of yeast PtdIns 4-kinase, PtdInsK1, and the two molecules had a region of similarity that is not conserved in other lipid kinases. By examining PtdIns kinase activity in transfected COS-7 cells using epitope tag immunoprecipitation as well as conventional methods, the product PtdIns phosphate was identified as phosphatidylinositol 4-phosphate (PtdIns4P), but not phosphatidylinositol 3-phosphate (PtdIns3P). The PtdIns 4-kinase activity was recovered predominantly from the soluble fraction and the activity was markedly enhanced in the presence of Triton X-100 and was relatively insensitive to inhibition by adenosine. In addition, the PtdIns 4-kinase activity was completely inhibited in the presence of 10 microM wortmannin. When examined by epitope tag immunocytochemistry, the immunoreactivity for the PtdIns 4-kinase molecule was dominantly aggregated in a cytoplasmic region juxtaposed to the nuclei and was faintly but widely dispersed in the cytoplasm. By in situ hybridization analysis, the mRNA for PtdIns 4-kinase was expressed ubiquitously and was detected in most neurons throughout the grey matter of the brain, with higher expression intensity found in fetal than in adult brain.

Full Text

The Full Text of this article is available as a PDF (752.8 KB).

Selected References

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

  1. Berridge M. J. Inositol trisphosphate and diacylglycerol as second messengers. Biochem J. 1984 Jun 1;220(2):345–360. doi: 10.1042/bj2200345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Berridge M. J., Irvine R. F. Inositol trisphosphate, a novel second messenger in cellular signal transduction. Nature. 1984 Nov 22;312(5992):315–321. doi: 10.1038/312315a0. [DOI] [PubMed] [Google Scholar]
  3. Campbell C. R., Fishman J. B., Fine R. E. Coated vesicles contain a phosphatidylinositol kinase. J Biol Chem. 1985 Sep 15;260(20):10948–10951. [PubMed] [Google Scholar]
  4. Cardenas M. E., Heitman J. FKBP12-rapamycin target TOR2 is a vacuolar protein with an associated phosphatidylinositol-4 kinase activity. EMBO J. 1995 Dec 1;14(23):5892–5907. doi: 10.1002/j.1460-2075.1995.tb00277.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Carpenter C. L., Cantley L. C. Phosphoinositide kinases. Biochemistry. 1990 Dec 25;29(51):11147–11156. doi: 10.1021/bi00503a001. [DOI] [PubMed] [Google Scholar]
  6. Cockcroft S., Taylor J. A., Judah J. D. Subcellular localisation of inositol lipid kinases in rat liver. Biochim Biophys Acta. 1985 May 30;845(2):163–170. doi: 10.1016/0167-4889(85)90173-9. [DOI] [PubMed] [Google Scholar]
  7. Collins C. A., Wells W. W. Identification of phosphatidylinositol kinase in rat liver lysosomal membranes. J Biol Chem. 1983 Feb 25;258(4):2130–2134. [PubMed] [Google Scholar]
  8. 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]
  9. Del Vecchio R. L., Pilch P. F. Phosphatidylinositol 4-kinase is a component of glucose transporter (GLUT 4)-containing vesicles. J Biol Chem. 1991 Jul 15;266(20):13278–13283. [PubMed] [Google Scholar]
  10. Flanagan C. A., Schnieders E. A., Emerick A. W., Kunisawa R., Admon A., Thorner J. Phosphatidylinositol 4-kinase: gene structure and requirement for yeast cell viability. Science. 1993 Nov 26;262(5138):1444–1448. doi: 10.1126/science.8248783. [DOI] [PubMed] [Google Scholar]
  11. Garcia-Bustos J. F., Marini F., Stevenson I., Frei C., Hall M. N. PIK1, an essential phosphatidylinositol 4-kinase associated with the yeast nucleus. EMBO J. 1994 May 15;13(10):2352–2361. doi: 10.1002/j.1460-2075.1994.tb06519.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Goto K., Kondo H. Molecular cloning and expression of a 90-kDa diacylglycerol kinase that predominantly localizes in neurons. Proc Natl Acad Sci U S A. 1993 Aug 15;90(16):7598–7602. doi: 10.1073/pnas.90.16.7598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Goto K., Watanabe M., Kondo H., Yuasa H., Sakane F., Kanoh H. Gene cloning, sequence, expression and in situ localization of 80 kDa diacylglycerol kinase specific to oligodendrocyte of rat brain. Brain Res Mol Brain Res. 1992 Nov;16(1-2):75–87. doi: 10.1016/0169-328x(92)90196-i. [DOI] [PubMed] [Google Scholar]
  14. Hay J. C., Fisette P. L., Jenkins G. H., Fukami K., Takenawa T., Anderson R. A., Martin T. F. ATP-dependent inositide phosphorylation required for Ca(2+)-activated secretion. Nature. 1995 Mar 9;374(6518):173–177. doi: 10.1038/374173a0. [DOI] [PubMed] [Google Scholar]
  15. Hay J. C., Martin T. F. Phosphatidylinositol transfer protein required for ATP-dependent priming of Ca(2+)-activated secretion. Nature. 1993 Dec 9;366(6455):572–575. doi: 10.1038/366572a0. [DOI] [PubMed] [Google Scholar]
  16. Hay J. C., Martin T. F. Resolution of regulated secretion into sequential MgATP-dependent and calcium-dependent stages mediated by distinct cytosolic proteins. J Cell Biol. 1992 Oct;119(1):139–151. doi: 10.1083/jcb.119.1.139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kato H., Uno I., Ishikawa T., Takenawa T. Activation of phosphatidylinositol kinase and phosphatidylinositol-4-phosphate kinase by cAMP in Saccharomyces cerevisiae. J Biol Chem. 1989 Feb 25;264(6):3116–3121. [PubMed] [Google Scholar]
  18. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  19. 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]
  20. Liscovitch M., Chalifa V., Pertile P., Chen C. S., Cantley L. C. Novel function of phosphatidylinositol 4,5-bisphosphate as a cofactor for brain membrane phospholipase D. J Biol Chem. 1994 Aug 26;269(34):21403–21406. [PubMed] [Google Scholar]
  21. Matsuoka K., Bassham D. C., Raikhel N. V., Nakamura K. Different sensitivity to wortmannin of two vacuolar sorting signals indicates the presence of distinct sorting machineries in tobacco cells. J Cell Biol. 1995 Sep;130(6):1307–1318. doi: 10.1083/jcb.130.6.1307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Miller E. S., Ascoli M. Anti-phosphotyrosine immunoprecipitation of phosphatidylinositol 3' kinase activity in different cell types after exposure to epidermal growth factor. Biochem Biophys Res Commun. 1990 Nov 30;173(1):289–295. doi: 10.1016/s0006-291x(05)81055-1. [DOI] [PubMed] [Google Scholar]
  23. Nakagawa T., Goto K., Kondo H. Cloning, expression, and localization of 230-kDa phosphatidylinositol 4-kinase. J Biol Chem. 1996 May 17;271(20):12088–12094. doi: 10.1074/jbc.271.20.12088. [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. Okayama H., Kawaichi M., Brownstein M., Lee F., Yokota T., Arai K. High-efficiency cloning of full-length cDNA; construction and screening of cDNA expression libraries for mammalian cells. Methods Enzymol. 1987;154:3–28. doi: 10.1016/0076-6879(87)54067-8. [DOI] [PubMed] [Google Scholar]
  26. Sabatini D. M., Pierchala B. A., Barrow R. K., Schell M. J., Snyder S. H. The rapamycin and FKBP12 target (RAFT) displays phosphatidylinositol 4-kinase activity. J Biol Chem. 1995 Sep 8;270(36):20875–20878. doi: 10.1074/jbc.270.36.20875. [DOI] [PubMed] [Google Scholar]
  27. Sakane F., Imai S., Yamada K., Kanoh H. The regulatory role of EF-hand motifs of pig 80K diacylglycerol kinase as assessed using truncation and deletion mutants. Biochem Biophys Res Commun. 1991 Dec 31;181(3):1015–1021. doi: 10.1016/0006-291x(91)92038-l. [DOI] [PubMed] [Google Scholar]
  28. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Tooke N. E., Hales C. N., Hutton J. C. Ca2+-sensitive phosphatidylinositol 4-phosphate metabolism in a rat beta-cell tumour. Biochem J. 1984 Apr 15;219(2):471–480. doi: 10.1042/bj2190471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. Walsh J. P., Caldwell K. K., Majerus P. W. Formation of phosphatidylinositol 3-phosphate by isomerization from phosphatidylinositol 4-phosphate. Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):9184–9187. doi: 10.1073/pnas.88.20.9184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. Wong K., Cantley L. C. Cloning and characterization of a human phosphatidylinositol 4-kinase. J Biol Chem. 1994 Nov 18;269(46):28878–28884. [PubMed] [Google Scholar]
  35. Yoshida S., Ohya Y., Goebl M., Nakano A., Anraku Y. A novel gene, STT4, encodes a phosphatidylinositol 4-kinase in the PKC1 protein kinase pathway of Saccharomyces cerevisiae. J Biol Chem. 1994 Jan 14;269(2):1166–1172. [PubMed] [Google Scholar]

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

RESOURCES