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. 2003 Dec 1;376(Pt 2):497–503. doi: 10.1042/BJ20031212

Identification and characterization of differentially active pools of type IIalpha phosphatidylinositol 4-kinase activity in unstimulated A431 cells.

Mark G Waugh 1, Shane Minogue 1, Deena Blumenkrantz 1, J Simon Anderson 1, J Justin Hsuan 1
PMCID: PMC1223785  PMID: 12954081

Abstract

The seven known polyphosphoinositides have been implicated in a wide range of regulated and constitutive cell functions, including cell-surface signalling, vesicle trafficking and cytoskeletal reorganization. In order to understand the spatial and temporal control of these diverse cell functions it is necessary to characterize the subcellular distribution of a wide variety of polyphosphoinositide synthesis and signalling events. The predominant phosphatidylinositol kinase activity in many mammalian cell types involves the synthesis of the signalling precursor, phosphatidylinositol 4-phosphate, in a reaction catalysed by the recently cloned PI4KIIalpha (type IIalpha phosphatidylinositol 4-kinase). However the regulation of this enzyme and the cellular distribution of its product in different organelles are very poorly understood. This report identifies the existence, in unstimulated cells, of two major subcellular membrane fractions, which contain PI4KIIalpha possessing different levels of intrinsic activity. Separation of these membranes from each other and from contaminating activities was achieved by density gradient ultracentrifugation at pH 11 in a specific detergent mixture in which both membrane fractions, but not other membranes, were insoluble. Kinetic comparison of the purified membrane fractions revealed a 4-fold difference in K (m) for phosphatidylinositol and a 3.5-fold difference in V (max), thereby indicating a different mechanism of regulation to that described previously for agonist-stimulated cells. These marked differences in basal activity and the occurrence of this isozyme in multiple organelles emphasize the need to investigate cell signalling via PI4KIIalpha at the level of individual organelles rather than whole-cell lysates.

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Selected References

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

  1. Akhtar R. A., Wilmoth T. L. Phorbol esters inhibit ionomycin-induced hydrolysis of phosphoinositides and phosphatidylcholine in bovine corneal epithelial cells. Curr Eye Res. 1992 Feb;11(2):135–145. doi: 10.3109/02713689209000064. [DOI] [PubMed] [Google Scholar]
  2. Andersson K., Hjorth R. Isopycnic centrifugation of rat-liver microsomes in isoosmotic gradients of Percoll and release of microsomal material by low concentrations of sodium deoxycholate. Biochim Biophys Acta. 1984 Feb 29;770(1):97–100. doi: 10.1016/0005-2736(84)90078-6. [DOI] [PubMed] [Google Scholar]
  3. Balla Andras, Tuymetova Galina, Barshishat Michal, Geiszt Miklos, Balla Tamas. Characterization of type II phosphatidylinositol 4-kinase isoforms reveals association of the enzymes with endosomal vesicular compartments. J Biol Chem. 2002 Mar 28;277(22):20041–20050. doi: 10.1074/jbc.M111807200. [DOI] [PubMed] [Google Scholar]
  4. Chatah N. E., Abrams C. S. G-protein-coupled receptor activation induces the membrane translocation and activation of phosphatidylinositol-4-phosphate 5-kinase I alpha by a Rac- and Rho-dependent pathway. J Biol Chem. 2001 Jun 28;276(36):34059–34065. doi: 10.1074/jbc.M104917200. [DOI] [PubMed] [Google Scholar]
  5. Chun M., Liyanage U. K., Lisanti M. P., Lodish H. F. Signal transduction of a G protein-coupled receptor in caveolae: colocalization of endothelin and its receptor with caveolin. Proc Natl Acad Sci U S A. 1994 Nov 22;91(24):11728–11732. doi: 10.1073/pnas.91.24.11728. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Duncan Mara C., Payne Gregory S. ENTH/ANTH domains expand to the Golgi. Trends Cell Biol. 2003 May;13(5):211–215. doi: 10.1016/s0962-8924(03)00076-x. [DOI] [PubMed] [Google Scholar]
  7. Fruman D. A., Meyers R. E., Cantley L. C. Phosphoinositide kinases. Annu Rev Biochem. 1998;67:481–507. doi: 10.1146/annurev.biochem.67.1.481. [DOI] [PubMed] [Google Scholar]
  8. Gehrmann T., Heilmeyer L. M., Jr Phosphatidylinositol 4-kinases. Eur J Biochem. 1998 Apr 15;253(2):357–370. doi: 10.1046/j.1432-1327.1998.2530357.x. [DOI] [PubMed] [Google Scholar]
  9. Gullick W. J., Downward J., Parker P. J., Whittle N., Kris R., Schlessinger J., Ullrich A., Waterfield M. D. The structure and function of the epidermal growth factor receptor studied by using antisynthetic peptide antibodies. Proc R Soc Lond B Biol Sci. 1985 Oct 22;226(1242):127–134. doi: 10.1098/rspb.1985.0087. [DOI] [PubMed] [Google Scholar]
  10. Heilmeyer Ludwig M. G., Jr, Vereb György, Jr, Vereb György, Kakuk Annamária, Szivák Ilona. Mammalian phosphatidylinositol 4-kinases. IUBMB Life. 2003 Feb;55(2):59–65. doi: 10.1002/tbmb.718540873. [DOI] [PubMed] [Google Scholar]
  11. Hsuan J. J., Minogue S., dos Santos M. Phosphoinositide 4- and 5-kinases and the cellular roles of phosphatidylinositol 4,5-bisphosphate. Adv Cancer Res. 1998;74:167–216. doi: 10.1016/s0065-230x(08)60767-8. [DOI] [PubMed] [Google Scholar]
  12. Kauffmann-Zeh A., Klinger R., Endemann G., Waterfield M. D., Wetzker R., Hsuan J. J. Regulation of human type II phosphatidylinositol kinase activity by epidermal growth factor-dependent phosphorylation and receptor association. J Biol Chem. 1994 Dec 9;269(49):31243–31251. [PubMed] [Google Scholar]
  13. Kauffmann-Zeh A., Thomas G. M., Ball A., Prosser S., Cunningham E., Cockcroft S., Hsuan J. J. Requirement for phosphatidylinositol transfer protein in epidermal growth factor signaling. Science. 1995 May 26;268(5214):1188–1190. doi: 10.1126/science.7761838. [DOI] [PubMed] [Google Scholar]
  14. Levine Timothy P., Munro Sean. Targeting of Golgi-specific pleckstrin homology domains involves both PtdIns 4-kinase-dependent and -independent components. Curr Biol. 2002 Apr 30;12(9):695–704. doi: 10.1016/s0960-9822(02)00779-0. [DOI] [PubMed] [Google Scholar]
  15. Mills Ian G., Praefcke Gerrit J. K., Vallis Yvonne, Peter Brian J., Olesen Lene E., Gallop Jennifer L., Butler P. Jonathan G., Evans Philip R., McMahon Harvey T. EpsinR: an AP1/clathrin interacting protein involved in vesicle trafficking. J Cell Biol. 2003 Jan 21;160(2):213–222. doi: 10.1083/jcb.200208023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Olsson H., Martínez-Arias W., Jergil B. Phosphatidylcholine enhances the activity of rat liver type II phosphatidylinositol-kinase. FEBS Lett. 1993 Aug 2;327(3):332–336. doi: 10.1016/0014-5793(93)81015-r. [DOI] [PubMed] [Google Scholar]
  17. Park S. J., Itoh T., Takenawa T. Phosphatidylinositol 4-phosphate 5-kinase type I is regulated through phosphorylation response by extracellular stimuli. J Biol Chem. 2000 Nov 21;276(7):4781–4787. doi: 10.1074/jbc.M010177200. [DOI] [PubMed] [Google Scholar]
  18. Pike L. J., Eakes A. T. Epidermal growth factor stimulates the production of phosphatidylinositol monophosphate and the breakdown of polyphosphoinositides in A431 cells. J Biol Chem. 1987 Feb 5;262(4):1644–1651. [PubMed] [Google Scholar]
  19. Röper K., Corbeil D., Huttner W. B. Retention of prominin in microvilli reveals distinct cholesterol-based lipid micro-domains in the apical plasma membrane. Nat Cell Biol. 2000 Sep;2(9):582–592. doi: 10.1038/35023524. [DOI] [PubMed] [Google Scholar]
  20. Sargiacomo M., Sudol M., Tang Z., Lisanti M. P. Signal transducing molecules and glycosyl-phosphatidylinositol-linked proteins form a caveolin-rich insoluble complex in MDCK cells. J Cell Biol. 1993 Aug;122(4):789–807. doi: 10.1083/jcb.122.4.789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Stephens L., Jackson T. R., Hawkins P. T. Activation of phosphatidylinositol 4,5-bisphosphate supply by agonists and non-hydrolysable GTP analogues. Biochem J. 1993 Dec 1;296(Pt 2):481–488. doi: 10.1042/bj2960481. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Thomas G. M., Cunningham E., Fensome A., Ball A., Totty N. F., Truong O., Hsuan J. J., Cockcroft S. An essential role for phosphatidylinositol transfer protein in phospholipase C-mediated inositol lipid signaling. Cell. 1993 Sep 10;74(5):919–928. doi: 10.1016/0092-8674(93)90471-2. [DOI] [PubMed] [Google Scholar]
  23. Walker D. H., Pike L. J. Phosphatidylinositol kinase is activated in membranes derived from cells treated with epidermal growth factor. Proc Natl Acad Sci U S A. 1987 Nov;84(21):7513–7517. doi: 10.1073/pnas.84.21.7513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Waugh M. G., Lawson D., Hsuan J. J. Epidermal growth factor receptor activation is localized within low-buoyant density, non-caveolar membrane domains. Biochem J. 1999 Feb 1;337(Pt 3):591–597. [PMC free article] [PubMed] [Google Scholar]
  25. Waugh M. G., Lawson D., Tan S. K., Hsuan J. J. Phosphatidylinositol 4-phosphate synthesis in immunoisolated caveolae-like vesicles and low buoyant density non-caveolar membranes. J Biol Chem. 1998 Jul 3;273(27):17115–17121. doi: 10.1074/jbc.273.27.17115. [DOI] [PubMed] [Google Scholar]
  26. Waugh Mark G., Minogue Shane, Anderson J. Simon, Balinger Adam, Blumenkrantz Deena, Calnan Denis P., Cramer Rainer, Hsuan J. Justin. Localization of a highly active pool of type II phosphatidylinositol 4-kinase in a p97/valosin-containing-protein-rich fraction of the endoplasmic reticulum. Biochem J. 2003 Jul 1;373(Pt 1):57–63. doi: 10.1042/BJ20030089. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Wei Yong Jie, Sun Hui Qiao, Yamamoto Masaya, Wlodarski Pawel, Kunii Kaiko, Martinez Manuel, Barylko Barbara, Albanesi Joseph P., Yin Helen L. Type II phosphatidylinositol 4-kinase beta is a cytosolic and peripheral membrane protein that is recruited to the plasma membrane and activated by Rac-GTP. J Biol Chem. 2002 Sep 24;277(48):46586–46593. doi: 10.1074/jbc.M206860200. [DOI] [PubMed] [Google Scholar]

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