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PMID: 14604433 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Detection of novel intracellular agonist responsive pools of phosphatidylinositol 3,4-bisphosphate using the TAPP1 pleckstrin homology domain in immunoelectron microscopy.

The Biochemical journal ·Vol. 377 ·No. Pt 3 ·2004-02-01 ·Pages 653-63

Watt SA, Kimber WA, Fleming IN, Leslie NR, Downes CP, Lucocq JM

Abstract

PtdIns(3,4) P (2), a breakdown product of the lipid second messenger PtdIns(3,4,5) P (3), is a key signalling molecule in pathways controlling various cellular events. Cellular levels of PtdIns(3,4) P (2) 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) P (2) 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) P (2). As expected, we found accumulation of PtdIns(3,4) P (2) 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) P (2) 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) P (2) in endomembrane function.

MeSH Terms
Animals Blood Proteins/genetics Carrier Proteins/genetics Cell Line Cell Line, Tumor DNA Probes/genetics Down-Regulation/drug effects Endoplasmic Reticulum/chemistry,drug effects Humans Hydrogen Peroxide/pharmacology Intracellular Membranes/chemistry,drug effects Intracellular Signaling Peptides and Proteins Intracellular Space/chemistry,metabolism Membrane Proteins/genetics Mice Microscopy, Immunoelectron/methods PTEN Phosphohydrolase Peptides/genetics Phosphatidylinositol Phosphates/metabolism Phosphoproteins/genetics Phosphoric Monoester Hydrolases/biosynthesis Platelet-Derived Growth Factor/pharmacology Protein Structure, Tertiary/genetics Sequence Homology, Nucleic Acid Staining and Labeling/methods Swiss 3T3 Cells/chemistry,drug effects Tumor Suppressor Proteins/biosynthesis
Chemicals
Blood Proteins Carrier Proteins DNA Probes Intracellular Signaling Peptides and Proteins Membrane Proteins PLEKHA1 protein, human Peptides Phosphatidylinositol Phosphates Phosphoproteins Platelet-Derived Growth Factor Tumor Suppressor Proteins platelet protein P47 Hydrogen Peroxide Phosphoric Monoester Hydrolases PTEN Phosphohydrolase PTEN protein, human
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Watt Stephen A
Division of Cell Biology and Immunology, School of Life Sciences, MSI/WTB Complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K.
Kimber Wendy A
Fleming Ian N
Leslie Nick R
Downes C Peter
Lucocq John M
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Article Info
Journal
The Biochemical journal
Abbr.
Biochem J
ISSN
1470-8728
DOI
10.1042/BJ20031397
Published
2004-02-01
Pages
653-63
Language
English
Region
England
NLM ID
2984726R
PMCID
PMC1223916
Subset
IM
External Links
PubMed source DOI: 10.1042/BJ20031397 PMC Full Text View journal details
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