Different subcellular localization and phosphoinositides binding of insulin receptor substrate protein pleckstrin homology domains.
Insulin evokes diverse biological effects through receptor-mediated tyrosine phosphorylation of the insulin receptor substrate (IRS) proteins. Here, we show that, in vitro, the IRS-1, -2 and -3 pleckstrin homology (PH) domains bind with different specificities to the 3-phosphorylated phosphoinositides. In fact, the IRS-1 PH domain binds preferentially to phosphatidylinositol 3,4,5-trisphosphate (PtdIns-3,4,5-P3), the IRS-2 PH domain to phosphatidylinositol 3,4-bisphosphate (PtdIns-3,4-P2), and the IRS-3 PH domain to phosphatidylinositol 3-phosphate. When expressed in NIH-IR fibroblasts and L6 myocytes, the IRS-1 and -2 PH domains tagged with green fluorescent protein (GFP) are localized exclusively in the cytoplasm. Stimulation with insulin causes a translocation of the GFP-IRS-1 and -2 PH domains to the plasma membrane within 3-5 min. This translocation is blocked by the phosphatidylinositol 3-kinase (PI 3-K) inhibitors, wortmannin and LY294002, suggesting that this event is PI 3-K dependent. Interestingly, platelet-derived growth factor (PDGF) did not induce translocation of the IRS-1 and -2 PH domains to the plasma membrane, indicating the existence of specificity for insulin. In contrast, the GFP-IRS-3 PH domain is constitutively localized to the plasma membrane. These results reveal a differential regulation of the IRS PH domains and a novel positive feedback loop in which PI 3-K functions as both an upstream regulator and a downstream effector of IRS-1 and -2 signaling.
Razzini
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Department of Cell Biology & Oncology, Istituto di Ricerche Farmacologiche Mario Negri, Consorzio Mario Negri Sud, Santa Maria Imbaro, Italy.
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D000818:Animals; D001798:Blood Proteins / Q000737:chemistry*; D002462:Cell Membrane / Q000737:chemistry; D003593:Cytoplasm / Q000737:chemistry; D005347:Fibroblasts / Q000648:ultrastructure; D049452:Green Fluorescent Proteins; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D055504:Insulin Receptor Substrate Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins; D051379:Mice; D009132:Muscles / Q000648:ultrastructure; D016297:Mutagenesis, Site-Directed; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism*; D010750:Phosphoproteins / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D017385:Sequence Homology; D013347:Subcellular Fractions / Q000737:chemistry*
D001798:Blood Proteins; C526292:IRS1 protein, human; C526297:IRS2 protein, human; C491585:IRS3P protein, human; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D047908:Intracellular Signaling Peptides and Proteins; C526295:Irs1 protein, mouse; C526298:Irs2 protein, mouse; C108872:Irs3 protein, mouse; D008164:Luminescent Proteins; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols; D010750:Phosphoproteins; C060974:phosphatidylinositol 3,4,5-triphosphate; C060980:phosphatidylinositol 3,4-diphosphate; C055525:phosphatidylinositol 3-phosphate; C039077:platelet protein P47; D049452:Green Fluorescent Proteins