Regulation of intracellular dynamics of Smad4 by its leucine‐rich nuclear export signal

Abstract

Smad family proteins play a pivotal role in transmitting the transforming growth factor-β (TGF-β) superfamily signals from the cell surface to the nucleus. In response to ligand stimulation, Smad4 forms a complex with respective receptor-specific Smads, and the complex translocates into the nucleus and regulates gene expression. Thus, the nuclear entry of the Smad complex is one of the key steps in signal transduction. However, little is known about regulatory mechanisms for nucleocytoplasmic transport of Smads. Here we report identification of a functional, leucine-rich nuclear export signal (NES) in Smad4, which regulates subcellular distribution of Smad4. We then show evidence suggesting that the NESdependent cytoplasmic localization of Smad4 is important for ensuring optimal TGF-β responsivenesses in transcriptional activation. Moreover, we show that the NES of Smad4 is specifically inactivated by the stimulus-dependent heterooligomerization with receptor-specific Smads during the TGF-β-induced nuclear translocation of Smad4. Taken together, these results suggest an important regulatory role of the NES of Smad4 in TGF-β signaling.

Nucleocytoplasmic shuttling of Smad proteins

“…In the absence of a TGF-b-superfamily ligand, Smad4 is distributed equally between the nucleus and the cytoplasm. Treatment of cells with leptomycin B (LMB), a specific inhibitor of the prototypic export receptor CRM1 or Exportin 1 [16], led to rapid nuclear accumulation of Smad4 [17,18]. This simple experiment showed that CRM1 activity is required for nuclear export of Smad4, and demonstrated that Smad4 must be continuously shuttling between the nucleus and the cytoplasm in the absence of a TGF-b signal.…”

Regulation of cell proliferation by Smad proteins

“…Xenopus Smad4b lacks the NES providing an explanation for its constitutive nuclear localization Watanabe et al, 2000). The nuclear export of Smad4 is mediated by CRM1; inhibition of CRM1-dependent nuclear export by treatment with leptomycin B results in rapid accumulation of Smad4 (but not Smad2 and Smad3) in the nucleus .…”

“…In contrast with Smad2 and Smad3, Smad4 has a leucine-rich nuclear export signal (NES) in its linker region Watanabe et al, 2000). Xenopus Smad4b lacks the NES providing an explanation for its constitutive nuclear localization Watanabe et al, 2000).…”

“…Smad4, which in its basal state is predominantly located in the cytoplasm, is proposed therefore to rapidly and constitutively shuttle between the nucleus and the cytoplasm. Upon heteromeric complex formation between R-Smad and Co-Smad, the nuclear accumulation of the complex may be stimulated by the shielding of the NES and/or unmasking the NLS on R-and/or Co-Smad Watanabe et al, 2000). Recently, Smad1, but not Smad2 and Smad3, was found to contain a functional NES in the MH2 domain ) Thus, Smad1 may also constantly shuttle between nucleus and cytoplasm, and receptor-induced Smad1 phosphorylation shifts the balance towards nuclear accumulation of Smad1.…”

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

“…Functional NES have been identified in several of the Smads including Smad1 and Smad4 and the nuclear export of these particular Smads have been shown to be CRM1-dependent [50,61,62]. However, Smad2 export is CRM1-independent and mediated by direct nucleoporins contact [55].…”