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. 2012 Nov 16;287(47):39653-63.
doi: 10.1074/jbc.M112.388009. Epub 2012 Oct 1.

Tollip, an intracellular trafficking protein, is a novel modulator of the transforming growth factor-β signaling pathway

Lu Zhu  1 , Lingdi Wang, Xiaolin Luo, Yongxian Zhang, Qiurong Ding, Xiaomeng Jiang, Xiao Wang, Yi Pan, Yan Chen
Affiliations

Tollip, an intracellular trafficking protein, is a novel modulator of the transforming growth factor-β signaling pathway

Lu Zhu et al. J Biol Chem. .

Abstract

Upon activation, TGF-β type I receptor (TβRI) undergoes active ubiquitination via recruitment of E3 ligases to the receptor complex by Smad7. However, how ubiquitination of TβRI is coupled to intracellular trafficking, and protein degradation remains unclear. We report here that Tollip, an adaptor protein that contains both ubiquitin-associated domains and endosome-targeting domain, plays an important role in modulating trafficking and degradation of TβRI. Tollip was previously demonstrated to possess a functional role in modulating the signaling of interleukin-1 and Toll-like receptors. We identify here that Tollip interacts with Smad7, a major modulatory protein involved in the negative regulation of TGF-β signaling. Overexpression of Tollip antagonizes TGF-β-stimulated transcriptional response, Smad2 phosphorylation, and epithelial-mesenchymal transition. Tollip also interacts with ubiquitinated TβRI, and such interaction requires ubiquitin-associated domains of Tollip. The interaction and intracellular colocalization of Tollip with TβRI is enhanced by Smad7. Overexpression of Tollip accelerates protein degradation of activated TβRI. In addition, Tollip alters subcellular compartmentalization and endosomal trafficking of activated TβRI. Collectively, our studies reveal that Tollip cooperates with Smad7 to modulate intracellular trafficking and degradation of ubiquitinated TβRI, whereby negatively regulates TGF-β signaling pathway.

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Figures

FIGURE 1.
FIGURE 1.
Interaction of Tollip and Smad7. A, association of overexpressed Tollip with overexpressed Smad7 is shown. HEK293T cells were transfected with the plasmids as indicated. At 48 h post-transfection, the cell lysate was used in immunoprecipitation (IP) and immunoblotting (IB) using the antibodies as indicated. B, the interaction between Tollip and Smad7 was not affected by TGF-β1 treatment. HEK293T cells were transfected with the plasmids as indicated and then treated with TGF-β1 (2 ng/ml) for 2 h before being harvested for IP and IB. C, interaction of endogenous Tollip with endogenous Smad7 is shown. HEK293T cell lysate was subjected to IP with control rabbit IgG or anti-Smad7 antibody followed by IB with an anti-Tollip antibody. D, shown is a diagram depicting the functional domains of Tollip and the mutant Tollip used in the study. E and F, shown is identification of the functional domains involved in the interaction between Tollip and Smad7. Different mutants of Tollip and Smad7 were expressed in HEK293T cells, and coimmunoprecipitation assay were used to identify the region(s) involved in the protein-protein interaction.
FIGURE 2.
FIGURE 2.
Tollip inhibits TGF-β signaling. A and B, Tollip suppresses TGF-β-induced transcriptional response. HEK293T cells were transfected with a luciferase reporter containing four repeats of Smad binding element, Renilla-luciferase, Tollip, and constitutively active TβRI (caTβRI, A) as indicated. Luciferase activity was measured 28 h after transfection (A). The cells were treated with TGF-β1 (2 ng/ml) for 24 h before luciferase assay (B). Each experiment was performed in triplicate. The data are presented as the mean ± S.D. after being normalized to Renilla activity. ** indicates p < 0.01 as comparison between the groups as indicated by Student's t test. C, Tollip attenuates TGF-β-induced Smad2 phosphorylation. HepG2 cells were transfected with the plasmids as indicated and then treated with 2 ng/ml TGF-β1 for various times. The cell lysate was used in immunoblotting using the antibody as indicated. D, Tollip knockdown increases TGF-β-stimulated Smad2 phosphorylation. HepG2 cells were transfected with nonspecific shRNA or Tollip-specific shRNA and then treated with TGF-β1 (2 ng/ml) for 1 h. Total cell lysate was subjected to immunoblotting (IB) with the antibodies as indicated. The knockdown efficiency is shown in supplemental Fig. S3A. E, Smad7 is required for suppressing TGF-β signaling by Tollip. HepG2 cells were transfected with nonspecific shRNA or Smad7-specific shRNA in the presence or absence of Tollip expression. The cells were then treated with TGF-β1 (2 ng/ml) for different length of time before being harvested for immunoblotting. The knockdown efficiency of Smad7 is shown in supplemental Fig. S3B.
FIGURE 3.
FIGURE 3.
Tollip attenuates TGF-β-induced epithelial-mesenchymal transition. HepG2 cells were transfected with FLAG-tagged Tollip as indicated and treated with TGF-β1 (2 ng/ml) for 24 h. The cell lysate was used in immunoblotting (A) and quantitative RT-PCR (B). The data are presented as mean ± S.D. ** indicates p < 0.01 by Student's t test. NS stands for non-significant.
FIGURE 4.
FIGURE 4.
Tollip interacts with TβRI in a Smad7-dependent manner. A, interaction between overexpressed Tollip with TβRI is shown. HEK293T cells were transfected with the plasmids as indicated. At 48 h post-transfection, the cell lysate was used in immunoprecipitation (IP) and immunoblotting (IB) using the antibodies as indicated. B, interaction of endogenous Tollip with endogenous TβRI. HepG2 cell lysate was used in IP with control rabbit IgG or an anti-TβRI antibody followed by IB with an anti-Tollip antibody. C, TGF-β1 treatment enhances Tollip association with TβRI. HEK293T cells were transfected with the plasmids as indicated and then treated with 2 ng/ml TGF-β1 for 24 h. The cell lysate was subjected to IP and IB using the antibodies as indicated. D, Smad7 knockdown abolished the interaction between Tollip and caTβRI. After infection with lentivirus containing nonspecific shRNA or Smad7-specific shRNA, HEK293T cells were transfected with the plasmids as indicated and then used in IP and IB using the antibodies as indicated. E, Smad7 enhances Tollip-caTβRI interaction. HEK293T cells were transfected with the plasmids as indicated followed by IP and IB using the antibodies as indicated. F, Smad7 increases colocalization of Tollip and caTβRI. HeLa cells were transiently transfected with the plasmids as indicated followed by immunofluorescence staining and confocal analysis. The arrows indicate apparent colocalization of Tollip with Smad7 and caTβRI. The quantitative analysis is shown in supplemental Fig. S5A. The nuclei were stained with Hoechst 33342.
FIGURE 5.
FIGURE 5.
Tollip interacts with ubiquitinated TβRI. HEK293T cells were cotransfected with the plasmids as indicated, and the cell lysate was used in immunoprecipitation (IP) and immunoblotting (IB) using the antibodies as indicated. Note that a second IP was used for B after the Tollip/TβRI complex was dissociated from the beads by a free FLAG peptide.
FIGURE 6.
FIGURE 6.
Tollip accelerates degradation of TβRI and alters compartmentalization of TβRI. A, Tollip accelerates turnover of TβRI. HEK293T cells were transfected with constitutively active TβR1 (HA-tagged), Tollip, and mutant Tollip as indicated. At 24 h after transfection, the cells were treated with 50 μg/ml cycloheximide (CHX) for various times and then harvested for immunoblotting with antibodies against HA (to detect TβRI) or actin. The result was quantified by Bandscan software and is shown on the right (mean ± S.D.). *, p < 0.05; **, p < 0.01 between Tollip and control groups; ^, p < 0.05 between TollipΔTBD/CUE and control groups. B, Tollip alters compartmentalization of TβRI. HEK293T cells were transfected with HA-tagged constitutively active TβRI (HA-caTβRI) and FLAG-tagged Tollip as indicated followed by fractionation with iodixanol gradients as described under “Experimental Procedures.” Equal volumes of each fraction were analyzed by immunoblotting with antibodies against HA (to detect HA-caTβRI), EEA1, and GM130. C, Tollip tethers caTβRI to early endosomes. HeLa cells were transiently transfected with the plasmids as indicated followed by immunofluorescence staining and confocal analysis. The arrows indicate colocalization of Tollip with TβRI at early endosomal structures. The quantitative analysis is shown in supplemental Fig. S5B. The early endosomes were stained with EEA1 antibody. The nuclei were stained with Hoechst 33342.
FIGURE 7.
FIGURE 7.
A model to depict Tollip cooperating with Smad7 to mediate degradation of the TGF-β receptor complex. Tollip is recruited to TGF-β receptor complex by its interaction with Smad7 via its C2 domain. Tollip then forms a complex with ubiquitinated TβRI via its two ubiquitin-associated domains: CUE and TBD. The complex is trafficked to the endosomes through the endosomes-targeting domain of Tollip and then undergoes protein degradation.
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