PtdIns(3,4)P2, a breakdown product of the lipid second messenger PtdIns(3,4,5)P3, is a key signalling molecule in pathways controlling various cellular events. Cellular levels of PtdIns(3,4)P2 are elevated upon agonist stimulation, mediating downstream signalling pathways by recruiting proteins containing specialized lipid-binding modules, such as the pleckstrin homology (PH) domain. A recently identified protein, TAPP1 (tandem-PH-domain-containing protein 1), has been shown to interact in vitro with high affinity and specificity with PtdIns(3,4)P2 through its C-terminal PH domain. In the present study, we have utilized this PH domain tagged with glutathione S-transferase (GST–TAPP1-PH) as a probe in an on-section immunoelectron microscopy labelling procedure, mapping the subcellular distribution of PtdIns(3,4)P2. As expected, we found accumulation of PtdIns(3,4)P2 at the plasma membrane in response to the agonists platelet-derived growth factor and hydrogen peroxide. Importantly, however, we also found agonist stimulated PtdIns(3,4)P2 labelling of intracellular organelles, including the endoplasmic reticulum and multivesicular endosomes. Expression of the 3-phosphatase PTEN (phosphatase and tensin homologue deleted on chromosome 10) in PTEN-null U87MG cells revealed differential sensitivity of these lipid pools to the enzyme. These data suggest a role for PtdIns(3,4)P2 in endomembrane function.
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February 2004
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Research Article|
February 01 2004
Detection of novel intracellular agonist responsive pools of phosphatidylinositol 3,4-bisphosphate using the TAPP1 pleckstrin homology domain in immunoelectron microscopy
Stephen A. WATT;
Stephen A. WATT
*Division of Cell Biology and Immunology, School of Life Sciences, MSI/WTB Complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K.
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Wendy A. KIMBER;
Wendy A. KIMBER
†MRC Protein Phosphorylation Unit, MSI/WTB Complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K.
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Ian N. FLEMING;
Ian N. FLEMING
1
‡Division of Cell Signalling, School of Life Sciences, MSI/WTB Complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K.
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Nick R. LESLIE;
Nick R. LESLIE
‡Division of Cell Signalling, School of Life Sciences, MSI/WTB Complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K.
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C. Peter DOWNES;
C. Peter DOWNES
‡Division of Cell Signalling, School of Life Sciences, MSI/WTB Complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K.
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John M. LUCOCQ
John M. LUCOCQ
2
*Division of Cell Biology and Immunology, School of Life Sciences, MSI/WTB Complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K.
2To whom correspondence should be addressed (e-mail j.m.lucocq@dundee.ac.uk).
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Publisher: Portland Press Ltd
Received:
September 15 2003
Revision Received:
October 31 2003
Accepted:
November 03 2003
Accepted Manuscript online:
November 07 2003
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2004
2004
Biochem J (2004) 377 (3): 653–663.
Article history
Received:
September 15 2003
Revision Received:
October 31 2003
Accepted:
November 03 2003
Accepted Manuscript online:
November 07 2003
Citation
Stephen A. WATT, Wendy A. KIMBER, Ian N. FLEMING, Nick R. LESLIE, C. Peter DOWNES, John M. LUCOCQ; Detection of novel intracellular agonist responsive pools of phosphatidylinositol 3,4-bisphosphate using the TAPP1 pleckstrin homology domain in immunoelectron microscopy. Biochem J 1 February 2004; 377 (3): 653–663. doi: https://doi.org/10.1042/bj20031397
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