An extended bipartite nuclear localization signal in Smad4 is required for its nuclear import and transcriptional activity

Abstract

Smad proteins are a class of tumor suppressors that play critical roles in inhibiting the proliferation of a variety of cell types by modulating the transcriptions of target genes. Despite recent advances, the mechanism of their nuclear import is not completely understood. Smad proteins contain a conserved basic motif in their N-terminal MH1 domains that resembles a nuclear localization signal (NLS). Previous studies indicate that in receptor-regulated Smads such as Smad1 and Smad3 this motif determines their interactions with nuclear import receptors and mediates their ligand-induced nuclear translocation. Common-Smads such as Smad4 display constant nucleocytoplasmic shuttling and are capable of autonomous nuclear import and export. Mutations of the basic motif in Smad4 disrupted its nuclear accumulation. However, this motif is not sufficient to confer nuclear translocation to a fused heterologous protein, suggesting that it is only part of the bona fide Smad4 NLS. We mapped the Smad4 NLS by fusing various segments of Smad4 sequence covering the basic motif to GFP and tested the localization of the fusion proteins. We identified an extended NLS, starting from the basic motif and extending into the DNA-binding region , that is sufficient to confer nuclear localization to GFP. Among the 14 basic residues in the NLS, only four (K45, K46, K48 and R81) are critical for import. This NLS is critical not only for autonomous nuclear import of Smad4, but also for its nuclear translocation in the presence of activated R-Smads, further confirming the functional relevance of the Smad4 NLS in TGF-b signal transduction. Structural modeling demonstrated that the four critical basic residues are all solvent exposed and map to a single localized segment on one surface of the Smad4 MH1 domain. Their distribution and spacing resemble a classical bipartite NLS. Smad4 displays specific binding to importin a through its MH1 domain, which was abrogated by loss-of-function mutations in Smad4 NLS. Finally, the Smad4 NLS is essential for its transcriptional activity since loss-of-function NLS mutants are also transcriptionally inactive.

αB-Crystallin Regulates Subretinal Fibrosis by Modulation of Epithelial-Mesenchymal Transition

“…In canonical TGF-b signaling, nuclear translocation of R-SMAD2/3 facilitates binding to Smad binding elements via masking the SMAD4 nuclear export signal. 37,38 However, we did not find evidence that nuclear SMAD2/3 accumulation was altered after aB-crystallin knockdown or overexpression. Instead, we considered the possibility that SMAD4 stabilization in the nucleus involved other R-SMADs.…”

“…Because SMAD4 is the common mediator or co-SMAD that displays continuous shuttling between the nucleus and the cytoplasm, 37,38 we determined whether its accumulation in the nucleus after aB-crystallin overexpression was associated with nuclear accumulation of receptor-regulated R-SMADs. We first evaluated SMAD2/3 because these are activated in the canonical TGF-b/SMAD signaling pathway.…”

Nucleocytoplasmic shuttling of Smad proteins

“…Very recently, further work has investigated the possible mechanism of Imp7/8-mediated Smad import. Nuclear import of Smad4, both in the presence and absence of signal has been shown to require Imp7 and 8; and a KKLK motif in the MH1 domain, which is part of the originally identified NLS in Smad4 [39], was shown to be required for Smad4 import, although not directly for binding to Imp8.…”

“…An extended, bipartite NLS has been identified in Smad4 (amino acids 45-110), which overlaps with the corresponding sequence motif responsible for Smad1 and 3 import, but additionally extends into the DNA-binding region of the Smad4 MH1 domain. The isolated Smad4 MH1 domain interacts with importin a1 through this motif [39].…”

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

“…On the other hand, Smad4 uses an extended bipartite NLS that binds to importin α. This NLS is important not only for autonomous nuclear translocation of Smad4 but also its translocation in the presence of R-Smads [53]. The fact that R-Smads directly bind to importin β and Smad4 to importin α makes it an interesting issue to investigate whether importin β bound to R-Smad can still interact with IBB domain of importin α and whether such interaction contributes to stabilization of R-Smad/Smad4 complex during nuclear translocation.…”