Detection of novel intracellular agonist responsive pools of phosphatidylinositol 3,4-bisphosphate using the TAPP1 pleckstrin homology domain in immunoelectron microscopy.
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.
Watt
Stephen A
SA
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
WA
Fleming
Ian N
IN
Leslie
Nick R
NR
Downes
C Peter
CP
Lucocq
John M
JM
D000818:Animals; D001798:Blood Proteins / Q000235:genetics*; D002352:Carrier Proteins / Q000235:genetics*; D002460:Cell Line; D045744:Cell Line, Tumor; D015342:DNA Probes / Q000235:genetics; D015536:Down-Regulation / Q000187:drug effects; D004721:Endoplasmic Reticulum / Q000737:chemistry / Q000187:drug effects; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007425:Intracellular Membranes / Q000737:chemistry / Q000187:drug effects; D047908:Intracellular Signaling Peptides and Proteins; D042541:Intracellular Space / Q000737:chemistry / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics*; D051379:Mice; D016253:Microscopy, Immunoelectron / Q000379:methods; D051059:PTEN Phosphohydrolase; D010455:Peptides / Q000235:genetics; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics*; D010744:Phosphoric Monoester Hydrolases / Q000096:biosynthesis; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D017434:Protein Structure, Tertiary / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D013194:Staining and Labeling / Q000379:methods; D041701:Swiss 3T3 Cells / Q000737:chemistry / Q000187:drug effects; D025521:Tumor Suppressor Proteins / Q000096:biosynthesis
D001798:Blood Proteins; D002352:Carrier Proteins; D015342:DNA Probes; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; C435927:PLEKHA1 protein, human; D010455:Peptides; D018129:Phosphatidylinositol Phosphates; D010750:Phosphoproteins; D010982:Platelet-Derived Growth Factor; D025521:Tumor Suppressor Proteins; C039077:platelet protein P47; D006861:Hydrogen Peroxide; D010744:Phosphoric Monoester Hydrolases; D051059:PTEN Phosphohydrolase; C494929:PTEN protein, human