Importin beta mediates nuclear translocation of Smad 3.
Smad proteins are intracellular mediators of transforming growth factor-beta (TGF-beta) and related cytokines. Although ligand-induced nuclear translocation of Smad proteins is clearly established, the pathway mediating this import is yet to be determined. We previously identified a nuclear localization signal (NLS) in the N-terminal region of Smad 3, the major Smad protein involved in TGF-beta signal transduction. This basic motif (Lys(40-)Lys-Leu-Lys-Lys(44)), conserved among all the pathway-specific Smad proteins, is required for Smad 3 nuclear import in response to ligand. Here we studied the nuclear import pathway of Smad 3 mediated by this NLS. We demonstrate that the isolated Smad 3 MH1 domain displays significant specific binding to importin beta, which is diminished or eliminated by mutations in the NLS. Full-size Smad 3 exhibits weak but specific binding to importin beta, which is enhanced after phosphorylation by the type I TGF-beta receptor. In contrast, no interaction was observed between importin alpha and Smad 3 or its MH1 domain, indicating that nuclear translocation of Smad proteins may occur through direct binding to importin beta. We propose that activation of all of the pathway-specific Smad proteins (Smads 1, 2, 3, 5, 8, and 9) exposes the conserved NLS motif, which then binds directly to importin beta and triggers nuclear translocation.
Xiao
Z
Z
Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142, USA.
Liu
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X
Lodish
H F
HF
D030201:Activin Receptors, Type I; D020816:Amino Acid Motifs; D001692:Biological Transport; D002451:Cell Compartmentation; D002467:Cell Nucleus / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D028884:Karyopherins; D008164:Luminescent Proteins / Q000235:genetics / Q000302:isolation & purification; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D000077293:Receptor, Transforming Growth Factor-beta Type I; D018125:Receptors, Transforming Growth Factor beta / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D051900:Smad3 Protein; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D028884:Karyopherins; D008164:Luminescent Proteins; D009687:Nuclear Proteins; D018125:Receptors, Transforming Growth Factor beta; D011993:Recombinant Fusion Proteins; D051900:Smad3 Protein; D015534:Trans-Activators; D049452:Green Fluorescent Proteins; D017346:Protein Serine-Threonine Kinases; D030201:Activin Receptors, Type I; D000077293:Receptor, Transforming Growth Factor-beta Type I