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 PI4KIIα (type IIα 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 PI4KIIα 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 Km for phosphatidylinositol and a 3.5-fold difference in Vmax, 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 PI4KIIα at the level of individual organelles rather than whole-cell lysates.
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December 2003
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Research Article|
December 01 2003
Identification and characterization of differentially active pools of type IIα phosphatidylinositol 4-kinase activity in unstimulated A431 cells
Mark G. WAUGH;
Mark G. WAUGH
Centre for Molecular Cell Biology, Department of Medicine, Royal Free and University College Medical School, University College London, Rowland Hill Street, London NW3 2PF, U.K.
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Shane MINOGUE;
Shane MINOGUE
Centre for Molecular Cell Biology, Department of Medicine, Royal Free and University College Medical School, University College London, Rowland Hill Street, London NW3 2PF, U.K.
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Deena BLUMENKRANTZ;
Deena BLUMENKRANTZ
Centre for Molecular Cell Biology, Department of Medicine, Royal Free and University College Medical School, University College London, Rowland Hill Street, London NW3 2PF, U.K.
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J. Simon ANDERSON;
J. Simon ANDERSON
Centre for Molecular Cell Biology, Department of Medicine, Royal Free and University College Medical School, University College London, Rowland Hill Street, London NW3 2PF, U.K.
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J. Justin HSUAN
J. Justin HSUAN
1
Centre for Molecular Cell Biology, Department of Medicine, Royal Free and University College Medical School, University College London, Rowland Hill Street, London NW3 2PF, U.K.
1To whom correspondence should be addressed (e-mail j.hsuan@rfc.ucl.ac.uk).
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Publisher: Portland Press Ltd
Received:
August 11 2003
Revision Received:
September 02 2003
Accepted:
September 03 2003
Accepted Manuscript online:
September 03 2003
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2003
2003
Biochem J (2003) 376 (2): 497–503.
Article history
Received:
August 11 2003
Revision Received:
September 02 2003
Accepted:
September 03 2003
Accepted Manuscript online:
September 03 2003
Citation
Mark G. WAUGH, Shane MINOGUE, Deena BLUMENKRANTZ, J. Simon ANDERSON, J. Justin HSUAN; Identification and characterization of differentially active pools of type IIα phosphatidylinositol 4-kinase activity in unstimulated A431 cells. Biochem J 1 December 2003; 376 (2): 497–503. doi: https://doi.org/10.1042/bj20031212
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