Erbin associates with Smad2 and Smad3. (A) Erbin interacts with Smad2/Smad3 but not Smad1/Smad4 in vivo. Flag-Smads and Myc-Erbin were cotransfected into HEK293T cells with ALK5(T202D) (lanes 5, 7, and 9) or ALK3(Q233D) (lane 3). The cell lysates were split and immunoprecipitated (IP) with either anti-Myc (αMyc) or anti-FLAG (αFLAG) antibody. Erbin or Smads were detected from immunoprecipitates by Western blotting with appropriate antibodies as indicated. (B) Erbin interacts with Smad2/Smad3 but not Smad1/Smad4 in vitro. In vitro-synthesized Erbin proteins were allowed to interact with GST-Smad proteins on glutathione beads. The GST-Smad-bound Erbin was resolved by SDS-8% PAGE and detected by autoradiography. As inputs, ³⁵S-labeled in vitro-translated Erbin proteins and control GFP (left gels) represent 1% of the total amount of protein used in each interaction assay (right gels). Equal amounts of GST-Smad1 to GST-Smad4 are indicated by Coomassie brilliant blue staining (top right). (C) Smad3 interacts with Erbin but not Densin-180. In vitro-translated Erbin or Densin-180 proteins were allowed to interact with GST-Smad2/GST-Smad3 proteins on glutathione beads. GST-Smad2- and GST-Smad3-bound proteins were resolved by 8% SDS-PAGE and detected by autoradiography. Equal amounts of GST-Smad2 and GST-Smad3 are indicated by Coomassie brilliant blue staining (left). (D and E) Erbin interacts with Smad2/Smad3 mutants. HEK293T cells were transfected with Myc-Erbin and Smad2/Smad3 (wild type or deletion mutants) as indicated, and Erbin-bound Smad2/Smad3 were immunoprecipitated with anti-Myc antibody and analyzed by Western blotting with anti-Flag or anti-HA antibody. (F) Erbin interacts with endogenous Smad2/Smad3. Cell lysates were prepared from 90% confluent HaCaT cells after 1 h of TGFβ treatment. Endogenous Smad2/Smad3 bound to Erbin were immunoprecipitated with anti-Erbin antibody (αErbin) and detected by Western blotting with anti-Smad2 (αSmad2) and anti-Smad3 (αSmad3) antibodies. WCL, whole-cell lysates; ALK5(T202D), constitutively active type I TGFβ receptor mutant; ALK3(Q233D), constitutively active type I BMP receptor mutant; WT, wild type; 2SA, inactive Smad2/Smad3 mutants with C-terminal Ser-to-Ala mutation; 2SD, active Smad2/Smad3 mutants harboring C-terminal phosphorylation-mimetic Ser-to-Asp mutation; IB, immunoblot; IgG, immunoglobulin G.

Novel region in Erbin mediates its interaction with Smad2/Smad3. (A) Diagram of various Erbin deletion mutants used for in vitro GST pull-down assays depicted in panel B. The start and end amino acid residues for each fragment are indicated. SID is indicated by a filled box. (B) SID mediates Erbin's interaction with Smad3. The left panel shows an autoradiogram of 1% of ³⁵S-labeled in vitro-translated Erbin deletion proteins and GFPs (as inputs). The right panel shows different in vitro-translated Erbin deletion proteins that interacted with GST-Smad3 proteins on glutathione beads. GST-Smad3-bound proteins were resolved by 10% SDS-PAGE followed by autoradiography. (C) SID directly interacts with Smad2/Smad3. In vitro-synthesized Smad2/Smad3 and GFP interacted with GST-SID proteins on glutathione beads. GST-SID-bound proteins were gel resolved and detected by autoradiography. (D) Erbin directly interacts with the MH2 domain of Smad3. The in vitro-synthesized full-length Smad3 domain (wild type [WT]), the N-terminal MH1 domain (N), the C-terminal plus linker region domain (LC), and the C-terminal MH2 domain (C) of Smad3 as well as GFP were incubated with GST-SID proteins on glutathione beads. Deletion constructs of Smad3 are shown with the MH1 and MH2 domains and the linker region as indicated. GST-SID-bound proteins were detected by SDS-PAGE and autoradiography. (E) Smad3 interacts with Erbin but not Erbin-v7. Myc-Erbin or Myc-Erbin-v7 was cotransfected into HEK293T cells with Flag-Smad3, with or without ALK5(T202D). Smad3-bound Erbin or Erbin-v7 was immunoprecipitated by anti-Flag antibody (αFLAG) and detected by Western blotting with anti-Myc (αMyc) antibody. The N-terminal LRR domain, the C-terminal PDZ domain, and SID (black box) are indicated. ΔLRR, Erbin mutant lacking the LRR domain; ΔPDZ, Erbin mutant lacking the PDZ domain; WCL, whole-cell lysate; IP, immunoprecipitate; IB, immunoblot.

Erbin inhibits Smad-mediated transcriptional activation. (A to E) Erbin inhibits TGFβ signaling but not BMP signaling. HaCaT cells were cotransfected with Myc-Erbin with either the SBE-Luc reporter (A), ARE-Luc reporter (together with Fast-1) (B), or p21-Luc reporter (C and D). Similarly, C2C12 cells were transfected with Myc-Erbin and the Id1-Luc reporter (E). Luciferase activity was measured 20 h after stimulation with TGFβ (A, B, and C) or BMP (D and E). (F) Erbin's inhibition of TGFβ-induced transcriptional activity is independent of MEK inhibition. HaCaT cells were transfected either with SBE-Luc alone or together with Myc-Erbin. Luciferase activity was measured 20 h after TGFβ stimulation in the presence of MEK inhibitor U0126 or the control compound U0124. (G) An ERK inhibitor blocks ERK activation. HEK293T cells were treated with inhibitor U0126 or the control compound U0124 (from the same stock used for panel F) for 2 h. Cells then were stimulated with epidermal growth factor (EGF) (10 ng/ml) for 30 min. Phosphorylation of ERK was examined by Western blotting with phosphospecific ERK antibody (αP-Erk1/2). (H) Erbin inhibits Smad3-LC-mediated transcription activity. HaCaT cells were cotransfected with the SBE-Luc reporter and increasing amounts of HA-Smad3-LC (S3-LC) with or without Myc-Erbin. Luciferase activity was measured 20 h after TGFβ stimulation. RLU, relative luciferase units; WCL, whole-cell lysate; IB, immunoblot.

Erbin inhibits TGFβ signaling through SID. (A) SID is necessary for Erbin's inhibitory effect on SBE promoter activity. HaCaT cells were cotransfected with the SBE-Luc reporter and either wild-type Erbin or Erbin deletion mutants as indicated. Luciferase activity was measured 20 h after TGFβ stimulation. (B) Cell lysates also were subjected to Western blot analysis to examine the expression of deletion mutants. The protein band corresponding to each construct is indicated by a solid box. Asterisks indicate apparent nonspecific bands. (C and D) Erbin, but not Erbin-v7, inhibits TGFβ-induced transcriptional activation of SBE-Luc. Myc-Erbin or Myc-Erbin-v7 was cotransfected into HaCaT cells with SBE-Luc (C) or p21-Luc (D). Luciferase activity was measured 20 h after TGFβ stimulation. The expression level of Erbin or Erbin-v7 from cell lysates shown in panel C was determined with Western blot analysis. RLU, relative luciferase activity; WCL, whole-cell lysate; IB, immunoblot; CTRL, control. αMyc, anti-Myc antibody; αβ-actin, anti-β-actin antibody.

Erbin inhibits mesodermal and endodermal marker induction by activin/Smad2, but not BMP4/Smad1, signals in Xenopus ectodermal explants. Mesendodermal gene induction by 1 pg of activin (lane 2) or 0.2 ng of Smad2 (lane 4) RNA was inhibited by the presence of 1 ng of Erbin RNA (lanes 3 and 5), whereas several BMP4 (20 pg)-induced (lane 6) or Smad1 (1 ng)-induced (lane 8) mesendodermal markers in animal caps were not significantly affected by the presence of Erbin (1 ng RNA). In all of these experiments, RNAs were injected into the animal poles of two-cell-stage frog embryos. Animal caps from the injected embryos were dissected at blastula stages and harvested at gastrula stages or tadpole stages for RT-PCR assays. The gene expression in uninjected control animal caps is shown in lane 1, and the expression pattern of whole embryos processed in the absence (Embryo-RT) or presence (Embryo) of reverse transcriptase in RT-PCRs is shown in lanes 10 and 11, respectively. The RT-PCR assay of the gene EF1-α was used as the loading control.

Knockdown of Erbin expression enhances TGFβ signaling. (A) Different shRNAs against Erbin reduce Erbin protein levels in HEK293T cells. Increasing amounts of Erbin shRNA construct shRNA-795 or shRNA-609 or the empty vector pSRG were transiently transfected into HEK293T cells together with Myc-Erbin. Knockdown of Erbin expression was examined by Western blotting. Immunoblotting with anti-β-actin antibody (αβ-actin) served as a loading control. (B) Reducing Erbin expression enhances TGFβ-induced SBE-Luc expression. HaCaT cells were cotransfected with the SBE-Luc reporter and Erbin shRNA construct shRNA-795 (sh795) or shRNA-609 (sh609). Luciferase activity was measured 20 h after TGFβ stimulation. (C) Erbin expression is effectively reduced in Erbin shRNA stable cell lines. Whole-cell lysates (WCL) were prepared from two independent HaCaT cell lines stably expressing Erbin shRNA-795 (KD-1 and KD-2) or an empty pSRG vector (CTL). The endogenous Erbin expression level was detected by Western blotting. Immunoblotting (IB) with anti-β-actin antibody served as a loading control. (D) Reducing Erbin expression enhances TGFβ-induced growth inhibition. KD-1, KD-2, or CTL cells were stimulated with various concentrations of TGFβ as indicated. Cell proliferation was assessed by incorporation of [³H]thymidine. Data are expressed as mean percentages ± standard deviations of thymidine incorporation relative to basal counts of each cell line from duplicate experiments. (E) Knockdown of endogenous Erbin expression enhances ligand-induced expression of TGFβ target genes. Whole-cell extracts from control HaCaT cells or KD-2 cells treated with TGFβ for 24 h were immunoblotted with the indicated antibodies shown on the right. The β-actin immunoblot served as a loading control. (F to H) Reducing Erbin expression enhances Smad2/Smad3-mediated transcriptional activity. HaCaT line KD-2 or CTL cells were transfected with p21-Luc (F), SBE-Luc (G), or ARE-Luc (H). Luciferase activity was measured 20 h after TGFβ stimulation. (I) Reducing Erbin expression did not affect BMP signaling. HaCaT line KD-2 or CTL cells were transfected with Id1-Luc. Luciferase activity was measured 20 h after BMP2 stimulation. RLU, relative luciferase activity; αMyc, anti-Myc antibody; αErbin, anti-Erbin antibody.

Erbin does not affect phosphorylation of Smad2/Smad3. (A and B) Erbin does not affect ALK5(T202D)-mediated phosphorylation of Smad2/Smad3. Cell lysates were prepared from HEK293T cells cotransfected with Flag-Smad2, Flag-Smad3, ALK5(T202D), and Myc-Erbin as indicated. Phosphorylation of Smad2/Smad3 was detected by Western blotting with anti-P-Smad2 (αP-Smad2) and anti-P-Smad3 (αP-Smad3) antibodies. Expression levels of Myc-Erbin or Flag-Smad2 and Flag-Smad3 were detected by anti-Myc (αMyc) or anti-Flag (αFlag) antibodies. (C and D) Erbin does not affect TGFβ-induced phosphorylation of Smad2/Smad3. Cell lysates were prepared from HEK293T cells that were cotransfected with Flag-Smad2 and Flag-Smad3 with or without Myc-Erbin and treated with or without TGFβ (1 h). Flag-Smad proteins first were immunoprecipitated by anti-Flag antibody and then were subjected to Western blot analysis. WCL, whole-cell lysate; IB, immunoblot; IP, immunoprecipitate.

Erbin physically sequesters R-Smad2/R-Smad3 from binding to Smad4. (A) Erbin disrupts Smad3-Smad4 interaction in vitro. The left blot shows an autoradiogram of 1% input of ³⁵S-labeled in vitro-translated proteins as indicated. The right blot shows in vitro-translated Smad3 in the absence or presence of in vitro-translated Erbin; SID was allowed to interact with GST-Smad4 proteins on glutathione beads. GST-Smad4-bound Smad3 proteins were gel resolved and detected by autoradiography. (B and C) Erbin disrupts Smad3-Smad4 interaction in vivo. HEK293T cells were transfected with expression plasmids for Flag-Smad2/Smad3, HA-Smad4, ALK5(T202D), and Myc-Erbin, as indicated. Smad4-bound Smad2/Smad3 was immunoprecipitated with anti-HA (αHA) antibody and detected by anti-Flag (αFlag) Western blotting. (D) Overexpression of Erbin reduces association of Smad4 with Smad3. HEK293T cells were transfected with Flag-Smad3, HA-Smad4, ALK5(T202D), and increasing amounts of Myc-Erbin. Smad4-bound Smad3 was immunoprecipitated with anti-HA antibody and detected by anti-Flag Western blotting. (E) Overexpression of Smad4 reduces association of Erbin with Smad3. HEK293T cells were transfected with Myc-Erbin, Flag-Smad3, ALK5(T202D), and increasing amounts of HA-Smad4. Erbin-bound Smad3 was immunoprecipitated with anti-Myc antibody (αMyc) and detected by anti-Flag Western blotting. (F) Erbin blocks Smad2 nuclear translocation in response to TGFβ. HaCaT cells stably expressing GFP-Smad2 were transiently transfected with Myc-Erbin (top) or Myc-Erbin-LRR (bottom). Twenty-four hours after transfection, cells were treated with TGFβ or were left untreated for 1 h and fixed. Erbin or the Erbin LRR domain was detected by indirect immunofluorescence staining using anti-Myc antibodies. 4′,6′-Diamidino-2-phenylindole (DAPI)-stained DNA in the nucleus is shown. IP, immunoprecipitate; WCL, whole-cell lysate; IB, immunoblot; IgG, immunoglobulin G.