Oncogenic H-Ras inhibits Smad-dependent TGFβ transcriptional responses. (A) Activation of the A3–Luciferase reporter construct by the indicated concentrations of TGFβ was analyzed in EpH4 and EpRas mammary epithelial cells. (█) EpH4; (▴) EpRas. (B) TGFβ- and FAST2-dependent activation of the A3–Luc reporter in Mv1Lu mink lung epithelial cells transfected with or without H-Ras^V12. (█) −Ras^V12 + FAST-2; (▴) +Ras^V12 + FAST-2; (♦) −Ras^V12; (●) +Ras^V12. (C) TGFβ antiproliferative response in Mv1Lu cells transfected with or without H-Ras^V12. (█) −Ras^V12; (▴) +Ras^V12. G₁ progression activity was determined with an E2F reporter construct driving luciferase expression. In all cases, data are the average of triplicates ±s.d.

TGFβ-induced nuclear accumulation of Smad2 and Smad3 in EpH4 and EpRas cells. (A) COS cells transfected with Flag-tagged versions of Smad1, Smad2, Smad3, or Smad4 were subjected to Western immunoblotting with anti-Flag antibodies as a positive control, or antibodies raised against the linker region of Smad2. (B) Untransfected EpH4, EpRas, and Mv1Lu cell extracts were subjected to Western immunoblotting with affinity-purified anti-Smad2/Smad3 (top) or nonimmune serum (bottom). (Asterisks) Nonspecific bands. (C) EpRas and EpH4 cells were stimulated with TGFβ for 30 min. Cell lysates were subjected to Western immunoblotting with either antibodies against receptor-phosphorylated Smad2 (top) or anti-Smad2/Smad3 antibodies (bottom). (D) In a similar experiment, endogenous Smad2 and Smad3 were visualized by anti-Smad2/Smad3 immunofluorescence. (E) EpH4 (█) and EpRas (▴) cells were treated with TGFβ for different time periods. Following immunofluorescence staining, the percentage of cells with Smad2/Smad3 staining predominantly or exclusively in the nucleus was determined.

Nuclear accumulation of Smads in response to TGFβ is inhibited by oncogenic H-Ras^V12 or constitutively active Mek1. Mv1Lu cells were transiently transfected with empty vector, H-Ras^V12 or constitutively active Mek1 (ca Mek1), and then treated with TGFβ for 30 min before fixation. Transfected cells were marked by cotransfection with GFP. Endogenous proteins were visualized by anti-Smad2/Smad3 immunofluorescence by a rhodamine-coupled system. (Arrowheads) GFP-positive cells. (A) Representative micrographs of the control and H-Ras^V12 transfectants visualizing the GFP and anti-Smad2/Smad3 signals. (B) Percentage of GFP-positive cells with Smad2/Smad3 predominantly in the nucleus under the three experimental conditions.

Elevated Erk activity and Smad2/Smad3 phosphorylation levels in EpRas cells. (A) The kinase activity associated with anti-Erk immunoprecipitates from EpH4 and EpRas cells was determined with myelin basic protein (MBP) as a substrate. The relative levels of Erk1 and Erk2 were determined by anti-Erk Western immunoblotting. (B) The phosphorylation level of Smad2 and Smad3 in EpH4 and EpRas cells was determined by immunoprecipitation from ³²P-labeled cells. The relative levels of Smads were determined by anti-Smad2/Smad3 Western immunoblotting of unlabeled cell extracts prepared in parallel.

Oncogenic H-Ras^V12 causes phosphorylation of Smad2 and Smad3 through Mek1 at Ser/Thr–Pro sites in the linker region. (A) Schematic representation of phosphorylation sites in the region that links the DNA binding domain (MH1 domain) and the receptor-interaction/transcriptional domain (MH2 domain) in Smad1, Smad2, and Smad3. (●) Erk consensus sites (PXS/TP); (□) serine/proline motifs, which may serve as phosphorylation sites for proline-directed kinases; (⋄) receptor phosphorylation sites. (B,C) Vectors encoding Flag-tagged wild-type (WT) Smads or mutant Smads lacking all four potential phosphorylation sites in the linker region (EPSM constructs), were used. These constructs were cotransfected with vectors encoding H-Ras^V12 or constitutively active Mek1 into Mv1Lu/R1B-L17 cells as indicated. The phosphorylation level of transfected Smads was determined by anti-Flag immunoprecipitation from ³²P-labeled cells. PD98058 (a Mek1 inhibitor) or wortmannin (a phosphatidyl inositol 3′-kinase inhibitor) were added 1.5 hr prior to cell lysis, as indicated. Smad expression was monitored by anti-Flag immunoblotting of cell lysates. (D) Purified, recombinant Smad proteins (wild-type or EPSM mutants) were tested as substrates for recombinant activated Erk2 in in vitro kinase assays. Identical amounts of recombinant Smad protein was added to all assays. (E) Effects of H-Ras^V12 cotransfection or EGF addition (18 nm, 30 min) on the phosphorylation of Flag-tagged Smad constructs containing Ser → Ala mutation in the carboxy-terminal SSXS receptor phosphorylation sites. Other assay conditions and controls were as described above.

EGF induces phosphorylation and inhibits TGFβ-dependent nuclear accumulation of Smad2 and Smad3. (A) Mv1Lu/R1B-L17 cells transfected with vectors encoding the indicated Flag-tagged Smad constructs were labeled with ³²P, and incubated with EGF for 30 min. The phosphorylation level of the transfected Smads was determined by anti-Flag immunoprecipitation. Smad expression was controlled by anti-Flag immunoblotting of cell lysates. (B) Mv1Lu cells were incubated with EGF for 60 min before fixation (bottom) and/or TGFβ for 30 min before fixation (right), and endogenous Smad2/3 visualized by immunofluorescence. Note the lack of cytoplasmic staining in cells treated with TGFβ alone, and the cytoplasmic staining remaining in cells treated with TGFβ plus EGF.

Mek1 inhibition of Smad3 nuclear accumulation requires phosphorylation in the linker domain. (A) Mv1Lu cells were cotransfected with Flag-tagged Smad3 [wild-type (WT) or mutant (EPSM)] and empty vector or constitutively active Mek1 as indicated. TGFβ was added 30 min prior to fixation. Flag–Smad3 was visualized by anti-Flag immunofluorescence. (B) Nuclear accumulation of Flag–Smad3 in the TGFβ-treated cells of A was quantitated by determining the proportion of cells with Smad2/Smad3 staining exclusively in the nucleus (e.g., as in A, far right; solid area) and cells with predominant but not exclusive nuclear staining (e.g., as in A, second from right; stippled area).

Ras-resistant Smad3 rescues TGFβ antimitogenic and transcriptional responses in EpRas cells. EpH4 and EpRas cells were cotransfected with wild-type Smad3, Ras-resistant (EPSM) Smad3 mutant, or the corresponding empty vector. These vectors were cotransfected with the E2F–luciferase reporter construct (6×E2F–Luc) in A, or the Smad-responsive ARE–luciferase reporter construct (A3–Luc) and FAST2 in B. Cells were incubated with or without TGFβ and luciferase activity was then measured. Data are the average of triplicates ±s.d.