Fig. 6. TβRImL45 is inactive in TGF-β- and Smad-mediated transcription response. Luciferase reporter assays of wild-type or mutant TβRImL45 in RIBL17 cells. White bars, without TGF-β treatment; black bars, with TGF-β treatment. Values are relative to control vector-transfected cells in the absence of TGF-β. RI, TβRI.

Fig. 2. p38 is required for TGF-β-induced EMT. NMuMG cells were incubated with the p38 kinase inhibitor SB203580 (E–H) or MEK1/2 inhibitor U0126 (I–L) in the absence (A, B, E, F, I and J) or presence of TGF-β (C, D, G, H, K and L) for 36 h, followed by staining for the actin cytoskeleton and E-cadherin. Control cells in the absence of protein kinase inhibitor (A–D).

Fig. 8. TβRImL45(TD) is sufficient to promote apoptosis but not sufficient for EMT. (A) Generation of pools of NMuMG cells that express constitutively active wild-type TβRI(TD) or mutant TβRImL45(TD). Upper panel: expression level of FLAG-tagged TβRI(TD) and TβRImL45(TD); lower panel: TβRI(TD), but not TβRImL45(TD), induced phosphorylation of Smad2. Control: cell lysates of COS-1 cells transfected with FLAG-tagged TβRI. (B) Both constitutively active wild-type receptor and constitutively active mutant type I receptor-promoted apoptosis. Apoptosis was assayed as in Figure 1A. (C) TβRI(TD), but not TβRImL45(TD), promoted EMT. One microgram of each plasmid DNA was used in (A) and (B). For (C), 1 µg of TβRI(TD) or 4 µg of TβRImL45(TD) were used.

Fig. 3. TGF-β-induced p38 activation. (A) Time course of TGF-β-induced activation of p38 (left panel). SB203580 inhibited TGF-β-induced p38 activation (right panel). Cell lysates were harvested after incubation with or without TGF-β for the indicated times in the presence or absence of SB203580. (B) SB203580 did not inhibit TGF-β-induced Smad2 phosphorylation. Cell lysates were harvested after incubation with or without TGF-β for 1 h. (C) TGF-β did not activate JNK kinase, although JNK can be readily activated by anisomycin (Aniso) in NMuMG cells. Cell lysates were subjected to in vitro kinase assay for JNK activities using purified glutathione S-transferase–c-Jun as substrate following immunoprecipitation of JNK1. (D) TGF-β-induced activation of MKK3 and MKK6, the MKKs upstream of p38.

Fig. 4. Dominant-negative TGF-β receptor but not dominant-negative Smad3 or inhibitory Smad, Smad7, blocks TGF-β-induced p38 activation and apoptosis. (A) TβRI(KR), a kinase-deficient mutant type I receptor, but not Smad3ΔSSVS, or inhibitory Smad, Smad7, inhibited TGF-β-mediated p38 activation. NMuMG cells, transfected with HA-p38 and the indicated expression plasmids, were incubated with or without TGF-β for 2 h, then TGF-β-mediated p38 activation was assayed by phospho-p38 blotting (top panel) following anti-HA immunoprecipitation of transfected p38. The levels of HA-p38 in the immunoprecipitates (middle panel) and FLAG-tagged Smads or receptor in total cell lysates (bottom panel) were shown as indicated. (B) TβRI(KR), Smad3ΔSSVS and Smad7 all inhibited the antiproliferative effect of TGF-β as measured by DNA synthesis. (C) TβRI(KR), but not Smad3ΔSSVS or Smad7, blocks TGF-β-induced apoptosis. Apoptosis was assayed as in Figure 1A. White bars, without TGF-β treatment; black bars, with TGF-β treatment. RI, TβRI.

Fig. 7. TGF-β type I receptor activates p38 independently of Smad activation. (A) TβRImL45 activated p38 in response to TGF-β. RIBL17 cells expressing HA-p38 and the wild-type or mutant receptor were stimulated with TGF-β for 30 min, and activities of p38 were examined by in vitro kinase assay using purified ATF-2 as substrate. (B) Transcriptional assays of constitutively active TβRI(TD) or TβRImL45(TD) in NMuMG cells using 3TP-Lux (open bar) or 4×SBE-Luc (striped bar) reporters. (C) Constitutively active TβRImL45(TD), but not kinase-deficient TβRImL45(KR), activated p38. NMuMG cells were transfected with HA-p38 and the indicated expression plasmids. Activities of transfected p38 were measured by phospho-p38 or phospho-ATF-2 blotting after an in vitro kinase reaction using ATF-2 as substrate following immunoprecipitation of transfected HA-p38. (D) TβRImL45(TD) activated p38 in a dose-dependent manner. Experiments were performed as in (C). The levels of HA-p38 in the immunoprecipitates and FLAG-tagged receptors in total cell lysates were shown as indicated. RI, TβRI.

Fig. 1. p38 is required for TGF-β-induced apoptotic but not antiproliferative responses. (A) Time course of TGF-β-induced apoptosis in NMuMG cells. The percentage of apoptotic cells was measured by Trypan Blue staining (left panel) and DNA fragmentation was quantified by the Cell Death Detection ELISA assay (right panel). (B) Inhibition of TGF-β-induced caspase-3 activation by the p38 kinase inhibitor SB203580. Cell lysates were harvested after incubation with or without TGF-β in the presence or absence of the indicated protein kinase inhibitor and blotted for full-length caspase-3 (inactive), cleaved caspase-3 (active), phospho-Akt and total Akt. (C) Inhibition of TGF-β-induced apoptosis by SB203580, a p38 kinase inhibitor. Cells were treated as in (B), then trypsinized and collected for apoptotic assay as in (A). (D) p38 kinase activity was not required for TGF-β-induced growth arrest.

Fig. 5. Generation of a mutant TGF-β type I receptor that no longer activates Smad. (A) Schematic representation of TβRI and amino acid sequence alignment of L45 loops of the wild-type and mutated type I receptor. The three residues that differ between the TGF-β and BMP type I receptors are underlined. Mutations in TβRImL45 are shown in lower case. (B) L45 loop-mutated type I receptors can no longer associate with Smad2 or Smad3. COS-1 cells were transfected with expression plasmids for FLAG-tagged RII and RI, and for C-terminal Myc-tagged Smad2 or Smad3. [¹²⁵I]TGF-β binding and cross-linking allowed the detection of receptor complex at the cell surface (top panel). Immunoprecipitation using Myc antibody showed co-precipitation of Smad2 or Smad3 with ligand-bound RII and wild-type RI, but not with mutated RI (middle panel). Smad2 and Smad3 levels were assessed by anti-Myc blotting (lower panel). (C) In vitro kinase activity of type I receptors. Transfected type I receptors in COS-1 cells were immunoprecipitated using anti-FLAG antibodies and subjected to in vitro kinase assay using [γ-³³P]ATP and SDS–PAGE. (D) RIBL17 cells expressing the TβRI or TβRImL45 were stimulated with TGF-β for 1 h and C-terminal serine phosphorylation of Smad2 was examined by blotting of cell lysates using the anti-phospho-Smad2 antibody (upper panel). The level of Smad2 protein was analyzed by anti-Smad2 blotting (lower panel). RI, TβRI; RII, TβRII.