CHIP Controls the Sensitivity of Transforming Growth Factor-β Signaling by Modulating the Basal Level of Smad3 through Ubiquitin-mediated Degradation

Hong Xin,Xialian Xu,Linyu Li,Hongxiu Ning,Yu Rong,Yu Shang,Yinyin Wang,Xin-Yuan Fu,Zhijie Chang

doi:10.1074/jbc.m412275200

Abstract

Transforming growth factor-beta (TGF-beta) signaling is critical in a variety of biological processes such as cell proliferation, differentiation, and apoptosis. TGF-beta signaling is mediated by a group of proteins including TGF-beta receptors and Smads. It is known that different cells can exhibit different sensitivities to TGF-beta. Several molecular mechanisms, such as the differential expression of the receptor levels, have been suggested as contributing to these differences. Here, we report evidence for a novel mechanism of regulating TGF-beta sensitivity that depends on the role of CHIP (carboxyl terminus of Hsc70-interacting protein) in regulating the basal level of Smad3 via the ubiquitin-dependent degradation pathway. First, using a luciferase assay we found that overexpression of CHIP inhibited TGF-beta signaling, whereas silencing CHIP expression by small interfering RNAs led to increased TGF-beta signaling sensitivity. Second, based on the results of cell proliferation assays and JunB expression, we found that TGF-beta signaling could be abolished by stably overexpressing CHIP. Third, in those cell lines with stably expressed CHIP, we observed that the Smad3 protein level was dramatically decreased. Finally, we demonstrated that CHIP served as a U-box dependent E3 ligase that can directly mediate ubiquitination and degradation of Smad3 and that this action of CHIP was independent of TGF-beta signaling. Taken together, these findings suggest that CHIP can modulate the sensitivity of the TGF-beta signaling by controlling the basal level of Smad3 through ubiquitin-mediated degradation.

Highlights

Transforming growth factor-␤ (TGF-␤) signaling is critical in a variety of biological processes such as cell proliferation, differentiation, and apoptosis
We demonstrated that CHIP served as a U-box dependent E3 ligase that can directly mediate ubiquitination and degradation of Smad3 and that this action of CHIP was independent of TGF-␤ signaling
These findings suggest that CHIP can modulate the sensitivity of the TGF-␤ signaling by controlling the basal level of Smad3 through ubiquitin-mediated degradation

Summary

Introduction

Transforming growth factor-␤ (TGF-␤) signaling is critical in a variety of biological processes such as cell proliferation, differentiation, and apoptosis. It has been shown that TGF-␤-induced Smad2-Smurf association with the transcriptional co-repressor SnoN leads to Smurf targeting SnoN for ubiquitin-mediated degradation by proteasome [29] These findings provided a mechanism for the down-regulation of TGF-␤ signaling via degradation of activated R-Smads [15]. CHIP was identified as mediating the degradation of the cystic fibrosis transmembrane conductance regulator [36], Parkin-associated endothelin-receptorlike receptor (Pael-R) [37], the androgen receptor [38], and ErbB2 [39] In these previous studies, CHIP was considered as a novel E3 ligase to change the function of Hsp70/Hsp chaperons from a protein-folding machine into a degradation factor (35, 36, 39 – 42). We present evidence that CHIP can be an important regulator in controlling the sensitivity of the TGF-␤ signaling by reducing the basal level of Smad through ubiquitination

Methods

Results

Conclusion