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. 1997 Sep 30;94(20):10669-74.
doi: 10.1073/pnas.94.20.10669.

Transforming growth factor beta-induced phosphorylation of Smad3 is required for growth inhibition and transcriptional induction in epithelial cells

X Liu  1 , Y Sun, S N Constantinescu, E Karam, R A Weinberg, H F Lodish
Affiliations

Transforming growth factor beta-induced phosphorylation of Smad3 is required for growth inhibition and transcriptional induction in epithelial cells

X Liu et al. Proc Natl Acad Sci U S A. .

Abstract

Drosophila Mad proteins are intracellular signal transducers of decapentaplegic (dpp), the Drosophila transforming growth factor beta (TGF-beta)/bone morphogenic protein (BMP) homolog. Studies in which the mammalian Smad homologs were transiently overexpressed in cultured cells have implicated Smad2 in TGF-beta signaling, but the physiological relevance of the Smad3 protein in signaling by TGF-beta receptors has not been established. Here we stably expressed Smad proteins at controlled levels in epithelial cells using a novel approach that combines highly efficient retroviral gene transfer and quantitative cell sorting. We show that upon TGF-beta treatment Smad3 becomes rapidly phosphorylated at the SSVS motif at its very C terminus. Either attachment of an epitope tag to the C terminus or replacement of these three serine residues with alanine abolishes TGF-beta-induced Smad3 phosphorylation; these proteins act in a dominant-negative fashion to block the antiproliferative effect of TGF-beta in mink lung epithelial cells. A Smad3 protein in which the three C-terminal serines have been replaced by aspartic acids is also a dominant inhibitor of TGF-beta signaling, but can activate plasminogen activator inhibitor 1 (PAI-1) transcription in a ligand-independent fashion when its nuclear localization is forced by transient overexpression. Phosphorylation of the three C-terminal serine residues of Smad3 by an activated TGF-beta receptor complex is an essential step in signal transduction by TGF-beta for both inhibition of cell proliferation and activation of the PAI-1 promoter.

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Figures

Figure 1
Figure 1
Stable expression of Smad3 proteins in Mv1Lu L20 cells using the pMX-IRES-GFP retroviral vector. (A) Diagram of the expression vector and the wild-type and mutant Smad3 genes used in this study. (B) Mink lung L20 cells are a derivative of mink lung CCL64 cells that stably express a transfected murine receptor for retroviruses containing the murine ecotropic envelope glycoprotein; L20 cells are highly infectable by the ecotropic retroviruses generated by from BOSC 23 cells. A population of 5 × 105 L20 cells was infected with pMX-IRES-GFP-Smad3 retroviruses encoding the wild-type or mutant Smad3 proteins; shown here is a typical profile of cells infected with pMX-Flag-Smad3-IRES-GFP. After 3 days growth 105 infected GFP-positive cells were sorted by flow cytometry. Indicated by the dotted lines is the population of GFP-positive cells that were selected and used for all subsequent experiments. As expected, cells infected by the same virus but lacking the GFP gene did not exhibit any green fluorescence.
Figure 2
Figure 2
TGF-β-induced phosphorylation of Smad3. (Upper) Ligand-dependent phosphorylation of Smad3 in response to TGF-β treatment. Stable pools of L20 cells expressing various Smad3 mutants were metabolically labeled with [32P]orthophosphate for 2 hr and subsequently incubated in the presence or absence of 500 pM TGF-β1 for 30 min. Cells were lysed in lysis buffer and the cleared supernatants were subjected to immunoprecipitation with the M2 Flag antibody and analyzed by 8% SDS/PAGE. (Lower) Western blot analysis of Smad3 protein levels in the transfected cells demonstrate equivalent expression of the wild-type and mutant Smad3 proteins. Lysates prepared from unlabeled cells were subjected to Western blot analysis using the Flag-M2 antibody (Kodak). The steady-state level of Smad3-C-Flag is higher than that of the N-terminal Flag-tagged Smad3s; this difference could be due to a positional effect of the Flag tag on stability of the Smad3 protein or because higher levels of expression of this form of the protein are well tolerated by these cells.
Figure 3
Figure 3
TGF-β induced phosphorylation of Smad3 occurs at the three C-terminal serine residues, and mutation of the C-terminal serines or addition of a Flag tag at the C terminus blocks TGF-β-induced phosphorylation of Smad3. Cells expressing Flag-N-Smad3, Smad3-C-Flag, or mutants of Flag-N-Smad3 in which the three C-terminal serine residues was changed to alanine (Smad3A) or aspartate (Smad3D) were labeled with [32P]orthophosphate for 2 hr and subsequently incubated in the presence or absence of 500 pM of TGF-β1 for 30 min. Anti-Flag immunoprecipitates were digested overnight with trypsin, oxidized, and resolved in two dimensions by electrophoresis in pH 1.9 buffer followed by chromatography. Spots 1 and 2 in the resultant autoradiogram derive from the C-terminal tryptic peptide. Spot 3 is probably identical to spot 4 and likely corresponds to a peptide from the N terminus of the protein, because the migration pattern of this peptide depends on whether the Flag tag is attached to the N or C terminus of the Smad3 protein.
Figure 4
Figure 4
Expression of Smad3 variants in mink lung epithelial cells affects the antiproliferative effects of TGF-β. Pools of L20 cells stably expressing similar amounts of Flag-N-Smad3 (▴) or Flag-N-Smad3A (▾) or Flag-N-Smad3D (○) or Smad3-C-Flag (⧫) or wild-type Smad3 (▪) or only GFP (vector; •) were incubated with the indicated concentration of TGF-β1 for 20 hr, then labeled with [3H]thymidine for 3 hr, fixed, and counted. Results for each pool of cells are normalized to the amount of thymidine uptake in cells incubated without TGF-β, and each data point represents a average from three independent wells. Variation is less than 10% for each data point.
Figure 5
Figure 5
Effect of Smad3 mutants on transcriptional activation of the TGF-β-responsive PAI-1 promoter (p3TP-Lux) in the absence or presence of 240 pM TGF-β. One million Mv1Lu cells were transiently transfected with 1 μg of vector DNA encoding the indicated Smad3 variants. Open bars, expression of the luciferase reporter gene in the absence of TGF-β; filled bars, in the presence of TGF-β. The data are normalized to β-galactosidase activity to control for transfection efficiency.
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