Importin α-importin β complex mediated nuclear translocation of insulin-like growth factor binding protein-5
Copyright policy )Importin α-importin β complex mediated nuclear translocation of insulin-like growth factor binding protein-5
Insulin-like growth factor-binding protein (IGFBP)-5 is a secreted protein that binds to IGFs and modulates IGF actions, as well as regulates cell proliferation, migration, and apoptosis independent of IGF. Proper cellular localization is critical for the effective function of most signaling molecules. In previous studies, we have shown that the nuclear IGFBP-5 comes from ER-cytosol retro-translocation. In this study, we further investigated the pathway mediating IGFBP-5 nuclear import after it retro-translocation. Importin-α5 was identified as an IGFBP-5-interacting protein with a yeast two-hybrid system, and its interaction with IGFBP-5 was further confirmed by GST pull down and co-immunoprecipitation. Binding affinity of IGFBP-5 and importins were determined by surface plasmon resonance (IGFBP-5/importin-β: KD=2.44e-7, IGFBP-5/importin-α5: KD=3.4e-7). Blocking the importin-α5/importin-β nuclear import pathway using SiRNA or dominant negative impotin-β dramatically inhibited IGFBP-5-EGFP nuclear import, though importin-α5 overexpress does not affect IGFBP-5 nuclear import. Furthermore, nuclear IGFBP-5 was quantified using luciferase report assay. When deleted the IGFBP-5 nuclear localization sequence (NLS), IGFBP-5ΔNLS loss the ability to translocate into the nucleus and accumulation of IGFBP-5ΔNLS was visualized in the cytosol. Altogether, our findings provide a substantially evidence showed that the IGFBP-5 nuclear import is mediated by importin-α/importin-β complex, and NLS is critical domain in IGFBP-5 nuclear translocation.
- University of Michigan–Ann Arbor United States
- Central South University China (People's Republic of)
- University of Michigan–Flint United States
Microscopy, Confocal, Recombinant Fusion Proteins, Green Fluorescent Proteins, Nuclear Localization Signals, Active Transport, Cell Nucleus, Surface Plasmon Resonance, Peptide Fragments, Kinetics, HEK293 Cells, Microscopy, Fluorescence, Genes, Reporter, Two-Hybrid System Techniques, Mutation, Humans, Immunoprecipitation, RNA Interference, Protein Multimerization, Insulin-Like Growth Factor Binding Protein 5, Gene Deletion, HeLa Cells
Microscopy, Confocal, Recombinant Fusion Proteins, Green Fluorescent Proteins, Nuclear Localization Signals, Active Transport, Cell Nucleus, Surface Plasmon Resonance, Peptide Fragments, Kinetics, HEK293 Cells, Microscopy, Fluorescence, Genes, Reporter, Two-Hybrid System Techniques, Mutation, Humans, Immunoprecipitation, RNA Interference, Protein Multimerization, Insulin-Like Growth Factor Binding Protein 5, Gene Deletion, HeLa Cells
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