Interaction of Jab1/CSN5 with Smad7. (A) GST-Jab1/CSN5 was transfected into HepG2 cells with the Flag-tagged Smad7 construct. Cell extracts were subjected to GST pull-down assay with glutathione-Sepharose beads followed by immunoblotting with anti-Flag antibody. Expression of GST, GST-Jab1/CSN5, and Smad7 was monitored as indicated. (B) GST-Jab1/CSN5 was transfected into HepG2 cells with the Myc-tagged Smad4 construct. Cell extracts were subjected to GST pull-down assay with glutathione-Sepharose beads followed by immunoblotting with anti-Myc antibody. Expression of GST, GST-Jab1/CSN5, and Smad4 was monitored as indicated. (C) HA-tagged Jab1/CSN5 constructs were transfected into HepG2 cells. After transfection, the cells were infected with adenoviruses carrying Smad2, Smad3, Smad6, and Smad7 at an MOI of 300. Adenovirus carrying β-galactosidase (MOI, 800) was used as the control (Con). The cells were then treated with lactacyctin (10 μM). Twenty-four h after transfection, the interaction between Smads and Jab1/CSN5 was analyzed by immunoblotting with the anti-Flag antibody after immunoprecipitation (I.P.) with anti-HA antibody. Expression of Smads was confirmed by anti-FLAG immunoblotting. Similar results were achieved in three separate experiments. (D) The interaction between Jab1/CSN5 and Smad7 or Smad3 was examined by GST pull-down assay in vitro. Bacterially expressed GST-Jab1/CSN5 and GST alone were incubated with [³⁵S]methionine-labeled Smad7 protein. Twenty-five percent of [³⁵S]methionine-labeled Smad7 or Smad3 protein used for the assay was applied as the control (Input). (E) Interaction of Jab1/CSN5 with Smad7 in vivo. Flag-tagged Smad7 was stably transfected into HepG2 cells. Transcription of the tagged Smad7 was under the control of doxycycline with an improved tetracycline-inducible system in which the expression of the reverse tetracycline repressor was driven by the EF-1α promoter (39). After the cells were incubated with the indicated amount of doxycycline for 24 h, they were lysed and subjected to immunoblotting with the anti-Jab1 antibody, following immunoprecipitation (IP) with anti-Flag antibody. (F) Interaction between endogenous Jab1/CSN5 and Smad7 in HaCat cells. Cells were incubated in the presence or absence of TGF-β1 (5 ng/ml) for 6 and 24 h, treated with MG-132 (2 μM) for 2 h before cell harvest, and then lysed and subjected to immunoblotting (IB) with the anti-Jab1 antibody following immunoprecipitation with anti-Smad7 antibody.

Jab1/CSN5 controls the subcellular localization of Smad7. (A) COS7 cells were transiently transfected with either HA-tagged Smad7 (0.5 μg/ml) or Jab1/CSN5 (1 μg/ml) alone or in combination with Myc-tagged Jab1/CSN5 (1.5 mg/ml), fixed in 3.5% paraformaldehyde, permeabilized with methanol, stained, and analyzed with laser scanning confocal microscopy. Smad7 was visualized by polyclonal anti-HA antibody and fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (green). Jab1/CSN5 was visualized by monoclonal Myc antibody and a tetramethyl rhodamine isocyanate-conjugated goat anti-mouse IgG (red). DAPI staining (blue) highlights the location of nuclei. (B) Quantitative analysis of Smad7 nuclear staining in the absence or presence of Jab1/CSN5. The Smad7 nuclear signal was plotted as the percentage of cells with prominent nuclear versus cytoplasmic staining ± the standard deviation duplicated from a representative experiment. For each condition, >100 cells were counted in each experiment. (C) HepG2 cells were transiently transfected with HA-tagged Smad7 (0.5 μg/ml) and Myc-tagged Jab1/CSN5 (1.5 μg/ml), fixed in 3.5% paraformaldehyde, permeabilized with methanol, stained, and analyzed with laser scanning confocal microscopy. (D) COS7 cells were transfected as described for panel A and then treated with the proteosomal inhibitor MG132 (2.5 μM) for 6 h. Colocalization of Smad7 and Jab1/CSN5 appears as yellow. Areas marked by a rectangle are enlarged and shown as insets.

Jab1/CSN5 induces degradation of Smad7. (A) Ectopic expression of Jab1/CSN5 down regulates Smad7. HepG2 cells were transfected with increasing amounts of HA-Jab1/CSN5 expression vector or control plasmid together with Flag-Smad7 expression vector. A GFP expression construct was used as the control. After 24 h, cell extracts were analyzed by immunoblotting using antibodies against HA, Flag, and GFP. (B) Jab1/CSN5 specifically degrades Smad7 but not Smad2, Smad3, or Smd6. HepG2 cells were transfected with HA-Jab1 expression construct or control plasmid together with the vectors indicated at the top, and after 24 h, total cell extracts were analyzed by immunoblotting using anti-Flag antibody. (C) The PY motif in Smad7 is important for Jab1-/CSN5-mediated degradation of Smad7. 293T cells were transfected with HA-Jab1/CSN5 or control plasmid together with either wild-type (WT), ΔPY, or mutant Y211A versions of Smad7. Total cell extracts were analyzed by immunoblotting by using anti-HA antibody, and Smad7 and Jab1/CSN5 were identified on the basis of their molecular weights. A GFP expression construct was used as the loading control. (D) Effect of Smurf2 on the Jab1-mediated degradation of Smad7. A Smad7 expression construct was transfected into 293T cells with either HA-Jab1/CSN5 alone or together with increasing amounts of Flag-Smurf2 expression vector. Total cell extracts were analyzed by immunoblotting using antibodies against Smad7, Jab1, and Flag. (E and F) Degradation of Smad7 by Jab1/CSN5 is sensitive to 26S proteosome inhibitors. 293T cells transfected with Flag-Smad7 alone or together with HA-Jab1/CSN5 were incubated with lactacystin (10 μM) or MG132 (2 μM), and expression of Flag-Smad7 was analyzed by immunoblotting.

Jab1/CSN5 induces degradation of Smad7 and enhances TGF-β-induced phosphorylation of Smad2. (A) HepG2 cells were transfected with a HA-Jab1/CSN5 expression construct or control plasmid (pcDNA3) and then infected with adenoviruses carrying Flag-Smad7 at an MOI of 300. After a 30-min treatment with TGF-β1 (5 ng/ml), a band of 58 kDa representing phosphorylated Smad2 (Smad2P) was detected in cell extracts by immunoblotting using rabbit anti-Smad2P antibodies. Adenovirus carrying β-galactosidase (MOI, 800) was used as a control. Expression of Smad7 and HA-Jab1 was confirmed by immunoblotting. Similar results were achieved in three separate experiments with comparable outcomes. (B) Generation of HepG2 cells stably expressing Jab1/CSN5. Expression of endogenous Smad7 protein was decreased in HepG2 cells stably expressing Jab1/CSN5. (C) HepG2 cells stably expressing Jab1/CSN5 and control HepG2 cells were stimulated with 5 ng of TGF-β1/ml for 2 h, and Smad3-binding to CAGA-biotinylated DNA was examined. (D) TGF-β1-induced Smad2 phosphorylation is markedly increased in HepG2 cells stably expressing Jab1/CSN5.

siRNA-mediated inhibition of Jab1/CSN5 induces Smad7 expression. (A) Jab1 siRNA efficiently blocks expression of endogenous Jab1/CSN5. HepG2 cells were transfected with either control (scrambled) siRNA or Jab1 siRNA, and 24 h after transfection, cell lysates were extracted for Western blot analysis. Cell lysates were immunoblotted with antibodies against Jab1/CSN5, Smad7, p27, and β-actin (loading control). (B) Suppression of endogenous Jab1/CSN5 expression by Jab1 siRNA in HepG2 cells results in a decrease of TGF-β1-dependent Smad transcriptional activity. HepG2 cells were cotransfected with a SBE4-Luc reporter vector and either control siRNA (scrambled) or Jab1 siRNA. Cells were lysed and assayed for luciferase activity 24 h after transfection.

Mapping of Smad7 domains which interact with Jab1/CSN5. (A) The PY motif of Smad7 is not required for Smad7-Jab1/CSN5 interaction. 293T cells were transfected with GST or GST-Jab1/CSN5 either alone or together with wild-type or ΔPY versions of Smad7. Cell extracts were subjected to GST pull-down assay with glutathione-Sepharose beads followed by immunoblotting with anti-HA antibody. Expression of GST, GST-Jab1/CSN5, HA-Smad7, and HA-Smad7ΔPY was monitored as indicated. (B) Jab1/CSN5 associates with the C-terminal domain of Smad7. FLAG-tagged Smad7 deletion mutants (7C; amino acids 204 to 407 and the N-terminal domain of Smad7; amino acids 1 to 261) (25) were cotransfected into the 293T cells together with either GST or GST-Jab1/CSN5. Cell extracts were subjected to GST pull-down assay with glutathione-Sepharose beads followed by immunoblotting with anti-FLAG antibody. Expression of Smad7 deletion mutants, GST, and GST-Jab1 was confirmed by immunoblotting aliquots of total cell lysates (bottom panels). The higher band in lanes 2 and 4 is a nonspecific band.

Smad7 is constitutively present in the nucleus, and TGF-β1 treatment increases Smad7 in the cytoplasm. HaCaT cells treated or not treated with 5 ng of TGF-β1/ml for 14 h were incubated in the presence or absence of MG-132, an inhibitor of proteosom-mediated degradation, for 2 h before lysis. Lysates were separated into nuclear and cytoplasmic fractions and immunoblotted with antibodies against Smad7 (top panel) and, to confirm the purity of the fractions, α-Tubulin (middle panel) and Lamin (lower panel). All three blots were prepared from the same lysate and are representative of the results obtained in three separate experiments.

Jab1/CSN5 induces the ubiquitination of Smad7. 293T cells were transfected with HA-tagged ubiquitin together with combinations of Smad7, Smad7ΔPY, Jab1/CSN5, Smurf2, TβRI, or TβRII as indicated. Cell lysates were subjected to immunoprecipitation with Smad7 antiserum and boiled in sodium dodecyl sulfate and were then subjected to immunoblotting with anti-HA antibodies. Expression of the proteins was confirmed by immunoblotting the total cell lysates.

Jab1/CSN5 relieves Smad7-mediated suppression of transcriptional activity induced by TGF-β1. HepG2 cells were cotransfected with either p3TP-Lux (B) or SBE4-Luc (A) together with various combinations of Jab1 and Smad7 expression constructs. Luciferase activity was measured 24 h after TGF-β1 stimulation.

Models for repression of TGF-β signaling by the Smurf-Smad7 complex and reactivation of TGF-β signaling by the Jab1/CSN5-Smad7 complex. (A) Smad7 inhibits TGF-β signaling by preventing access of Smad2 or Smad3 to the TGF-β receptor. (B) Smad7 inhibits TGF-β1 signaling by binding directly to Smurfs, bringing the Smurf/Smad7 complex to TβRI, and inducing the turnover of TβRI. (C) Jab1/CSN5 enhances TGF-β signaling by binding to Smad7 and inducing degradation of Smad7 through the COP9-proteosome pathway.