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Detection of novel intracellular agonist responsive pools of phosphatidylinositol 3,4-bisphosphate using the TAPP1 pleckstrin homology domain in immunoelectron microscopy
- Source :
- Biochemical Journal. 377:653-663
- Publication Year :
- 2004
- Publisher :
- Portland Press Ltd., 2004.
-
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.
- Subjects :
- Phosphatidylinositol 3,4-bisphosphate
Endosome
Immunoelectron microscopy
Phosphatase
Intracellular Space
Down-Regulation
Biology
Endoplasmic Reticulum
Biochemistry
Cell Line
Mice
chemistry.chemical_compound
Phosphatidylinositol Phosphates
Cell Line, Tumor
Sequence Homology, Nucleic Acid
Animals
Humans
Tensin
Endomembrane system
Microscopy, Immunoelectron
Molecular Biology
Platelet-Derived Growth Factor
Swiss 3T3 Cells
Staining and Labeling
Tumor Suppressor Proteins
Endoplasmic reticulum
Intracellular Signaling Peptides and Proteins
PTEN Phosphohydrolase
Membrane Proteins
Blood Proteins
Hydrogen Peroxide
Intracellular Membranes
Cell Biology
Phosphoproteins
Phosphoric Monoester Hydrolases
Protein Structure, Tertiary
Cell biology
Pleckstrin homology domain
chemistry
Carrier Proteins
DNA Probes
Peptides
Research Article
Subjects
Details
- ISSN :
- 14708728 and 02646021
- Volume :
- 377
- Database :
- OpenAIRE
- Journal :
- Biochemical Journal
- Accession number :
- edsair.doi.dedup.....1dfcd04ad66e109c6ce362e107a71c13