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.

Background: CRM1, an evolutionarily conserved protein, was shown to be a receptor for leucinerich nuclear export signal (NES)-dependent protein transport. In lower eukaryotes CRM1 is reported to be required for the export of mRNA, however, involvement of the NES-dependent transport pathway in mRNA export in higher eukaryotes has not been established.