Abstract
Smad ubiquitin regulatory factor 1 (Smurf1), a HECT type E3 ubiquitin ligase, interacts with inhibitory Smad7 and induces translocation of Smad7 to the cytoplasm. Smurf1 then associates with the transforming growth factor (TGF)-beta type I receptor, TbetaR-I, enhancing turnover. However, the mechanism of nuclear export of Smad7 by Smurf1 has not been elucidated. Here we identified a functional nuclear export signal (NES) in a C-terminal region of Smurf1. In transfected cells, the Smurf1-Smad7 complex was accumulated in the cytoplasm by the nuclear export receptor, CRM1; this action was prevented by treatment with leptomycin B, a specific inactivator of CRM1 function. A green fluorescence protein fusion protein containing the C-terminal NES motif of Smurf1, located in the cytoplasm, accumulated in the nucleus following treatment with leptomycin B. Moreover, Smurf1 was shown to bind physically to CRM1 through NES, and nuclear export of the Smurf1-Smad7 complex was prevented by mutations of Smurf1 within the NES. Finally, the Smurf1 NES mutant reduced inhibition by Smad7 of the transcriptional activation induced by TGF-beta. These results thus suggest that CRM1-dependent nuclear export of Smurf1 is essential for the negative regulation of TGF-beta signaling by Smad7.
Highlights
In contrast to regulated Smads (R-Smads) and Co-Smad, inhibitory Smads (I-Smads), including Smad6 and Smad7, bind to type I receptors and compete with R-Smads for activation by the receptors, resulting in inhibition of transforming growth factor (TGF)- superfamily signaling (6 – 8)
Because Smad7 is located predominantly in the nucleus in many transfected mammalian cells [9, 12], nuclear export of Smad7 may play an important role in the negative feedback regulation of TGF- signaling
Because endogenous expression of Smad ubiquitin regulatory factor 1 (Smurf1) was negligible in the transfection experiments, Smad7 was exported to the cytoplasm only in cells co-transfected with Smurf1 in the present study
Summary
In contrast to R-Smads and Co-Smad, I-Smads, including Smad and Smad, bind to type I receptors and compete with R-Smads for activation by the receptors, resulting in inhibition of TGF- superfamily signaling (6 – 8). HECT type E3 ubiquitin ligases, Smad ubiquitin regulatory factor 1 (Smurf1) and Smurf, have been reported to interact with Smad in the nucleus and to induce translocation of Smad to the cytoplasm. The transport of many large proteins from the nucleus to the cytoplasm is mediated by a short leucine-rich motif, known as the nuclear export signal (NES) sequence. CRM1, which belongs to the family of importin -related nuclear transport receptors, directly and associates with NES and mediates nuclear export of the protein containing NES [14, 15]. In Smad, phosphorylation of the C-terminal SSXS motif results in conformational changes that expose the nuclear localization signal, so that importin-1 can Nuclear Export of Smad7-Smurf by CRM1
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