Distinct domain utilization by Smad3 and Smad4 for nucleoporin interaction and nuclear import

Smad proteins undergo rapid nuclear translocation upon stimulation by transforming growth factor-beta (TGFbeta) 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.     

Inhibition of TGFbeta1-mediated PAI-1 induction by oltipraz through selective interruption of Smad 3 activation

Transforming growth factor-beta1 (TGFbeta1) induces plasminogen activator inhibitor-1 (PAI-1) as a major target protein. PAI-1 is associated with fibrosis, thrombosis, and metabolic disorders. TGFbeta1 induces PAI-1 via phosphorylation and nuclear translocation of Smads. Oltipraz inhibits TGFbeta1 expression and also regenerates cirrhotic liver. Nevertheless, whether oltipraz modulates TGFbeta1-mediated cell signaling is unclear. First, this study examined the effect of oltipraz on PAI-1 expression in cirrhotic rat liver. The cells immunochemically stained with anti-PAI-1 antibody accumulated around and within fibrous nodules in cirrhotic liver, which was notably decreased by oltipraz treatment. Next, whether oltipraz inhibits TGFbeta1-mediated Smads activation or Smad-mediated PAI-1 induction was determined in L929 fibroblasts. Oltipraz inhibited the ability of TGFbeta1 to induce PAI-1, as indicated by repression of TGFbeta1-mediated luciferase induction from the plasmid comprising the human PAI-1 promoter and of TGFbeta1-induced Smad-DNA-binding activity. TGFbeta1 induced nuclear transport of receptor-regulated Smad 2 and Smad 3, of which oltipraz selectively inhibited the transport and phosphorylation of Smad 3, thereby reducing formation of Smad 3/4 complex in the nucleus. In summary, oltipraz inhibits PAI-1 induction via a decrease in the formation of Smad 3/4 complex due to selective interruption of Smad 3 activation, indicating that oltipraz regulates the cellular responses downstream of ligand-activated TGFbeta1 receptor.     

Identification of three novel Smad binding proteins involved in cell polarity

A yeast two-hybrid screen was utilized to identify novel Smad 3 binding proteins expressed in developing mouse orofacial tissue. Three proteins (Erbin, Par-3, and Dishevelled) were identified that share several similar structural and functional characteristics. Each contains at least one PDZ domain and all have been demonstrated to play a role in the establishment and maintenance of cell polarity. In GST (glutathione S-transferase) pull-down assays, Erbin, Par-3, and Dishevelled bound strongly to the isolated MH2 domain of Smad 3, with weaker binding to a full-length Smad 3 protein. Failure of Erbin, Par-3, and Dishevelled to bind to a Smad 3 mutant protein that was missing the MH2 domain confirms that the binding site resides within the MH2 domain. Erbin, Par-3, and Dishevelled also interacted with the MH2 domains of other Smads, suggesting broad Smad binding specificity. Dishevelled and Erbin mutant proteins, in which the PDZ domain was removed, still retained their ability to bind Smad 3, albeit with lower affinity. While transforming growth factor beta (TGFbeta) has been suggested to alter cell polarity through a Smad-independent mechanism involving activation of members of the RhoA family of GTP binding proteins, the observation that Smads can directly interact with proteins involved in cell polarity, as shown in the present report, suggests an additional means by which TGFbeta could alter cell polarity via a Smad-dependent signaling mechanism.     

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.