Activation of JNK cascade inhibits Smad2-dependent transcription. (A) Mv1Lu cells were transfected with ARE-Lux alone or with FAST1 in the presence or absence of constitutively activated mutants of MEKK1 (MEKK1.EE) and MKK4 (MKK4.ED). (B) HepG2 cells were transfected with gsc-Lux and FAST2 in the presence or absence of MEKK1.EE and MKK4.ED. (C) Mv1Lu cells were transfected with ARE-Lux alone or with FAST1 in the presence of absence of dominant-negative mutants of MEKK1 (MEKK1.K432A) and MKK4 (MKK4.Ala). In all cases, cells were treated with (filled bars) or without (open bars) TGF-β for 16 h before lysis and then assayed for luciferase activity. Luciferase activity was normalized to β-galactosidase activity and was expressed as mean ± SD of triplicates from a representative experiment performed at least three times.

c-Jun inhibits Smad2 transcriptional activity. (A) COS-7 cells were transfected with Myc-FAST1 and Flag-Smad2 in the presence or absence of MKK4.ED and wild-type (HA-TβRI), or constitutively activated (HA-TβRI.act) TGF-β type I receptor. Cell lysates were subjected to immunoprecipitation with anti-Myc antibody and then immunoblotted with anti-Flag antibody. The expression of transfected DNA was determined by immunoblotting whole cell extracts with anti-Myc or anti-Flag antibodies. (B) HepG2 cells were cotransfected with ARE-Lux, together with FAST1 and HA-c-Jun or HA-c-JunbZip, and cell lysates were assayed for luciferase activity. Cells were treated with (filled bars) or without (open bars) TGF-β for 16 h before lysis and then assayed for luciferase activity (Upper). Expression of HA-c-Jun or HA-c-JunbZip was assessed by Western blotting of cell lysates with anti-HA antibody (Lower).

c-Jun stabilizes the Smad2/TGIF complex. (A) COS-7 cells were transfected with Flag-p300(1892–2441) and Myc-Smad2 in the presence or absence of HA-c-Jun and HA-TβRI or HA-TβRI.act. Association of p300(1892–2441) with Smad2 was analyzed by blotting the Flag immunoprecipitates with the anti-Myc antibody. (B) COS-7 cells were transfected with various combinations of Flag-TGIF, Myc-Smad2, HA-c-Jun, and HA-TβRI or HA-TβRI.act as indicated. Flag immunoprecipitates were subjected to immunoblotting with anti-Myc or anti-HA antibodies.

Association of c-Jun with TGIF. (A) Cell lysates from transiently transfected COS-7 cells were subjected to immunoprecipitation with anti-Flag or anti-Myc antibodies and then immunoblotted by using anti-HA that recognizes HA-c-Jun or HA-c-Jun-ala. (B) In vitro interaction of c-Jun with TGIF or Smad2 was examined by incubating full-length [³⁵S]methionine-labeled c-Jun produced by in vitro transcription/translation with Sepharose-bound bacterially expressed GST-TGIF, GST-Smad2, or GST. Bound material was visualized by SDS and autoradiography. Ponceau staining of the membrane showed that similar amounts of GST, GST-Smad2, and GST-TGIF were used in this assay (data not shown). (C) HepG2 cells were cotransfected with AP1-Lux together with c-Jun and increasing amounts of TGIF. After 48 h, luciferase activity was determined and normalized to β-galactosidase activity. (D) COS-7 cells were transfected with wild-type Flag-TGIF and HA-c-Jun, together with HA-TβRI or HA-TβRI.act, either in the absence or the presence of MKK4.ED. Cell lysates were subjected to anti-Flag immunoprecipitation and then immunoblotted with anti-HA antibody. (E) Proteins were precipitated from Mv1Lu and from Mv1Lu cells treated with TGF-β for 1 h by using an anti-rabbit polyclonal antibody specific for c-Jun (Oncogene; Top) or normal rabbit antiserum (Middle). Precipitated proteins were analyzed by Western blotting with a goat antibody specific for TGIF (Santa Cruz). For comparison, a portion of cell lysates was probed with anti-c-Jun or anti-TGIF (Bottom) antibodies.

Expression of the mutant c-JunbZip inhibits TGIF-mediated repression of Smad2 transcriptional activity. (A) HA-c-Jun or HA-c-JunbZip was cotransfected in COS-7 cells with Flag-TGIF and with HA-TβRI or HA-TβRI.act. Cell lysates were subjected to anti-Flag immunoprecipitation and then immunoblotted with anti-HA antibody. (B) Cell lysates from transiently transfected COS-7 cells were subjected to immunoprecipitation with anti-Myc antibody and then immunoblotted with anti-Flag antibody. (C) HepG2 cells were cotransfected with the indicated combination of ARE-Lux, FAST1, TGIF, HA-c-Jun, and HA-c-JunbZip. Cells were treated with (filled bars) or without (open bars) TGF-β for 16 h before lysis and then assayed for luciferase activity.