title: Protein tyrosine phosphatase-1B dephosphorylation of the insulin receptor occurs in a perinuclear endosome compartment in human embryonic kidney 293 cells.
abstract: Protein tyrosine phosphatase-1B (PTP-1B) is a negative regulator of insulin signaling. It is thought to carry out this role by interacting with and dephosphorylating the activated insulin receptor (IR). However, little is known regarding the nature of the cellular interaction between these proteins, especially because the IR is localized to the plasma membrane and PTP-1B to the endoplasmic reticulum. Using confocal microscopy and fluorescence resonance energy transfer (FRET), the interaction between PTP-1B and the IR was examined in co-transfected human embryonic kidney 293 cells. Biological activities were not significantly affected for either PTP-1B or the IR with the fusion of W1B-green fluorescent protein (GFP) to the N terminus of PTP-1B (W1B-PTP-1B) or the fusion of Topaz-GFP to the C terminus of the IR (Topaz-IR). FRET between W1B and Topaz was monitored in cells transfected with either wild type PTP-1B (W1B-PTP-1B) or the substrate-trapping form PTP-1B(D181A) (W1B-PTP-1B(D181A)) and Topaz-IR. Co-expression of W1B-PTP-1B with Topaz-IR resulted in distribution of Topaz-IR to the plasma membrane, but no FRET was obtained upon insulin treatment. In contrast, co-expression of W1B-PTP-1B(D181A) with Topaz-IR caused an increase in cytosolic Topaz-IR fluorescence and, in some cells, a significant basal FRET signal, suggesting that PTP-1B is interacting with the IR during its synthesis. Stimulation of these cells with insulin resulted in a rapid induction of FRET that increased over time and was localized to a perinuclear spot. Co-expression of Topaz-IR with a GFP-labeled RhoB endosomal marker and treatment of the cells with insulin identified a perinuclear endosome compartment as the site of localization. Furthermore, the insulin-induced FRET could be prevented by the treatment of the cells with a specific PTP-1B inhibitor. These results suggest that PTP-1B appears not only to interact with and dephosphorylate the insulin-stimulated IR in a perinuclear endosome compartment but is also involved in maintaining the IR in a dephosphorylated state during its biosynthesis.
authors.affiliation: Department of Biochemistry & Molecular Biology, Merck Frosst Centre for Therapeutic Research, Pointe-Claire-Dorval, Quebec H9R 4P8, Canada.
authors.forename: Yolanda
authors.initials: Y
authors.lastname: Romsicki
authors.forename: Mark
authors.initials: M
authors.lastname: Reece
authors.forename: Jacques-Yves
authors.initials: JY
authors.lastname: Gauthier
authors.forename: Ernest
authors.initials: E
authors.lastname: Asante-Appiah
authors.forename: Brian P
authors.initials: BP
authors.lastname: Kennedy
mesh_terms: D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004721:Endoplasmic Reticulum / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D031541:Fluorescence Resonance Energy Transfer; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007328:Insulin / Q000378:metabolism; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008956:Models, Chemical; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry / Q000378:metabolism*; D011972:Receptor, Insulin / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection
chemical_list: D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D007328:Insulin; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D011972:Receptor, Insulin; C516607:PTPN1 protein, human; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases