title: The mechanism of nuclear export of Smad3 involves exportin 4 and Ran.
abstract: Transforming growth factor beta (TGF-beta) receptors phosphorylate Smad3 and induce its nuclear import so it can regulate gene transcription. Smad3 can return to the cytoplasm to propagate further cycles of signal transduction or to be degraded. We demonstrate that Smad3 is exported by a constitutive mechanism that is insensitive to leptomycin B. The Mad homology 2 (MH2) domain is responsible for Smad3 export, which requires the GTPase Ran. Inactive, GDP-locked RanT24N or nuclear microinjection of Ran GTPase activating protein 1 blocked Smad3 export. Inactivation of the Ran guanine nucleotide exchange factor RCC1 inhibited Smad3 export and led to nuclear accumulation of phosphorylated Smad3. A screen for importin/exportin family members that associate with Smad3 identified exportin 4, which binds a conserved peptide sequence in the MH2 domain of Smad3 in a Ran-dependent manner. Exportin 4 is sufficient for carrying the in vitro nuclear export of Smad3 in cooperation with Ran. Knockdown of endogenous exportin 4 completely abrogates the export of endogenous Smad3. A short peptide representing the minimal interaction domain in Smad3 effectively competes with Smad3 association to exportin 4 and blocks nuclear export of Smad3 in vivo. We thus delineate a novel nuclear export pathway for Smad3.
authors.affiliation: Ludwig Institute for Cancer Research, Box 595 Biomedical Center, SE-751 24 Uppsala, Sweden.
authors.forename: Akira
authors.initials: A
authors.lastname: Kurisaki
authors.forename: Keiko
authors.initials: K
authors.lastname: Kurisaki
authors.forename: Marcin
authors.initials: M
authors.lastname: Kowanetz
authors.forename: Hiromu
authors.initials: H
authors.lastname: Sugino
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Yoneda
authors.forename: Carl-Henrik
authors.initials: CH
authors.lastname: Heldin
authors.forename: Aristidis
authors.initials: A
authors.lastname: Moustakas
mesh_terms: D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D028884:Karyopherins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D051900:Smad3 Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D020931:ran GTP-Binding Protein / Q000378:metabolism*
chemical_list: D028884:Karyopherins; D009687:Nuclear Proteins; C120587:RAN protein, human; D011993:Recombinant Fusion Proteins; C497790:SMAD3 protein, human; D051900:Smad3 Protein; C414440:Xpo4 protein, human; D020931:ran GTP-Binding Protein