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.
Original language | English |
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Pages (from-to) | 653-663 |
Number of pages | 11 |
Journal | Biochemical Journal |
Volume | 377 |
Issue number | 3 |
DOIs | |
Publication status | Published - 1 Feb 2004 |
Keywords
- animals
- blood proteins
- carrier proteins
- cell line
- tumor cell line
- DNA probes
- down-regulation
- endoplasmic reticulum
- humans
- hydrogen peroxide
- intracellular membranes
- intracellular signaling peptides and proteins
- intracellular space
- membrane proteins
- mice
- immunoelectron microscopy
- PTEN phosphohydrolase
- peptides
- phosphatidylinositol phosphates
- phosphoproteins
- phosphoric monoester hydrolases
- platelet-derived growth factor
- tertiary protein structure
- sequence homology, amino acid
- staining and labeling
- swiss 3T3 cells
- tumor suppressor proteins