The transcription factor Smad2 is released from cytoplasmic retention by TGFbeta receptor-mediated phosphorylation, accumulating in the nucleus where it associates with cofactors to regulate transcription. We uncovered direct interactions of Smad2 with the nucleoporins CAN/Nup214 and Nup153. These interactions mediate constitutive nucleocytoplasmic shuttling of Smad2. CAN/Nup214 and Nup153 compete with the cytoplasmic retention factor SARA and the nuclear Smad2 partner FAST-1 for binding to a hydrophobic corridor on the MH2 surface of Smad2. TGFbeta receptor-mediated phosphorylation stimulates nuclear accumulation of Smad2 by modifying its affinity for SARA and Smad4 but not for CAN/Nup214 or Nup153. Thus, by directly contacting the nuclear pore complex, Smad2 undergoes constant shuttling, providing a dynamic pool that is competitively drawn by cytoplasmic and nuclear signal transduction partners.

Smad proteins undergo rapid nuclear translocation upon stimulation by transforming growth factor-␤ (TGF␤) and in so doing transduce the signal into the nucleus. In this report we unraveled nuclear import mechanisms of Smad3 and Smad4 that are dependent on their interaction with FG-repeat-containing nucleoporins such as CAN/Nup214, without the involvement of importin molecules that are responsible for most of the known nuclear import events. A surface hydrophobic corridor within the MH2 domain of Smad3 is critical for association with CAN/Nup214 and nuclear import, whereas Smad4 interaction with CAN/Nup214, and nuclear import requires structural elements present only in the full-length Smad4. As exemplified by the different susceptibility to inhibition of import by cytoplasmic retention factor SARA (Smad anchor for receptor activation), such utilization of distinct domains for nuclear import of Smad3 and Smad4 suggests that nuclear transport of Smad3 and Smad4 is subject to control by different retention factors.Signal from the transforming growth factor-␤ (TGF␤) 1 cytokines originates at the cell surface upon engagement of TGF␤ ligands with their corresponding Type I/Type II receptor complexes and is transduced into the nucleus by the family of Smad proteins (1). Smad proteins are phosphorylated by the corresponding receptor kinases activated upon ligand binding: Smad2 and Smad 3 by TGF␤ and activin receptors; Smads 1, 5, and 8 by bone morphogenetic protein (BMP) receptors. One key event accompanying ligand binding is the rapid movement of Smads, including the unphosphorylated Smad4, into the nucleus. As transcription factors, these nuclear-bound Smads activate or repress gene expression leading to diverse cellular responses to TGF␤ cytokines (2, 3).Translocation of macromolecules across the nuclear envelope occurs via the nuclear pore complex (NPC), which consists of over 20 nucleoporins and many of them contain multiple phenylalanine-glycine (FG) dipeptide repeats (4 -6). In many cases, movement of cargo molecules through the NPC is mediated by the importin ␤ family of transport receptors, which bind cargo molecules and the FG dipeptide repeats simultaneously through separate domains (7). In the case of classical nuclear localization signal (NLS)-containing proteins, importin ␣ bridges the interaction between the NLS motif and importin ␤ (4 -7). Another common element in importin ␤-mediated nuclear import is the small GTPase Ran in its GTP-bound form (RanGTP), whose binding to importin ␤ disrupts all characterized association between importin ␤ and their cargoes (4 -7). Ran is mostly in its GTP-bound form in the nucleus and a GDP-bound form in the cytoplasm. Such Ran-GTP gradient across the nuclear envelope prompts release of cargo only in the nucleoplasm, ensuring that the cargoes will accumulate in the nucleus (4 -7).Since elucidation of the mechanism of importin ␤-mediated nuclear import, there have been efforts to investigate if similar principles apply to Smads. In Smad3, the N-terminal or MH1 domain cont...