Nuclear Targeting of Transforming Growth Factor-β-activated Smad Complexes

Abstract

Upon stimulation by the transforming growth factor ␤ (TGF-␤), Smad2 and Smad3 are phosphorylated at their C termini and assemble into stable heteromeric complexes with Smad4. These complexes are the functional entities that translocate into the nucleus and regulate the expression of TGF-␤ target genes. Here we report that the TGF-␤-activated phospho-Smad3/Smad4 complex utilizes an importin-independent mechanism for nuclear import and engages different nucleoporins for nuclear import compared with the monomeric Smad4. Within the heteromeric complex, phospho-Smad3 appears to dominate over Smad4 in the nuclear import process and guides the complex to its nuclear destination. We also demonstrate that the binding of phosphoSmad3 to Smad4 prevents Smad4 from interacting with the nuclear export receptor chromosome region maintenance 1. In this way, TGF-␤ signaling suppresses nuclear export of Smad4 by chromosome region maintenance 1 and thereby targets Smad4 into the nucleus. Indeed tumorigenic mutations in Smad4 that affect its interaction with Smad2 or Smad3 impair nuclear accumulation of Smad4 in response to TGF-␤.

Nucleocytoplasmic shuttling of Smad proteins

“…There is currently no explanation as to why the motifs are functional in Smad1 but not in Smad2 and 3. Nuclear export of Smad2 and 3 is clearly independent of CRM1, in both the absence and the presence of a TGF-b signal [18,19,24,27]. For Smad2, a karyopherin-independent export mechanism mediated by direct binding to nucleoporins has been suggested [19], similar to what was originally proposed for Smad2/3 import.…”

“…As it is the C-terminal phosphorylated serines in the R-Smads that are responsible for stabilizing the R-Smad-Smad4 complexes [14], these results suggested that R-Smad-Smad4 complexes dissociate in the nucleus as a result of R-Smad dephosphorylation, and that monomeric R-Smads and Smad4 are then exported separately by distinct mechanisms. In agreement with this idea, CRM1-dependent Smad4 export is thought to be inhibited by complex formation of Smad4 with R-Smads, which physically prevents Smad4 from interaction with CRM1 [27]. Moreover, R-Smad C-terminal phosphatases have recently been identified.…”

“…An identical mechanism was proposed to be responsible for Smad4 import, except that in this case the full-length protein was required for import [40]. A further follow-up study, aimed at assessing the import mechanism by which TGF-b-induced homomeric Smad3 and heteromeric Smad3-Smad4 complexes are imported, demonstrated karyopherin-independent import for such complexes in an in vitro import assay [27].…”

“…As Smad4 is dispensable for nuclear accumulation of R-Smads and its own nuclear accumulation is strictly dependent on R-Smad accumulation [8,27,37], the mechanism of R-Smad nuclear accumulation is of most interest. An anchor-release model was originally suggested for Smad2 based on the finding that overexpression of SARA sequesters Smad2 into clusters in the cytoplasm [45,46], and that overexpression of the Smad2-interacting nuclear protein FAST-1/FoxH1 traps Smad2 in the nucleus even in uninduced cells [19].…”

Cytokine-induced nuclear translocation of signaling proteins and their analysis using the inducible translocation trap system

“…The cytoplasmic/nuclear ratio is higher for the latent form because those forms associate with microtubule in the cytoplasm as well as specific anchor proteins such as SARA and dissociate from them upon TGFβ stimulation. Interaction of R-Smads to Smad4 also facilitates the retention of Smad4 in the nucleus [54,63,64]. In stimulated cells phosphorylated R-Smads/Smad4 complex enters the nucleus to regulate gene transcription, becomes dephosphorylated by a phosphatase [65], dissociates from each other, and exits the nucleus separately where R-Smads can be activated again.…”