Transforming growth factor-beta induces nuclear import of Smad3 in an importin-beta1 and Ran-dependent manner

Abstract

Smad proteins are cytoplasmic signaling effectors of transforming growth factor-beta (TGF-beta) family cytokines and regulate gene transcription in the nucleus. Receptor-activated Smads (R-Smads) become phosphorylated by the TGF-beta type I receptor. Rapid and precise transport of R-Smads to the nucleus is of crucial importance for signal transduction. By focusing on the R-Smad Smad3 we demonstrate that 1) only activated Smad3 efficiently enters the nucleus of permeabilized cells in an energy- and cytosol-dependent manner. 2) Smad3, via its N-terminal domain, interacts specifically with importin-beta1 and only after activation by receptor. In contrast, the unique insert of exon3 in the N-terminal domain of Smad2 prevents its association with importin-beta1. 3) Nuclear import of Smad3 in vivo requires the action of the Ran GTPase, which mediates release of Smad3 from the complex with importin-beta1. 4) Importin-beta1, Ran, and p10/NTF2 are sufficient to mediate import of activated Smad3. The data describe a pathway whereby Smad3 phosphorylation by the TGF-beta receptor leads to enhanced interaction with importin-beta1 and Ran-dependent import and release into the nucleus. The import mechanism of Smad3 shows distinct features from that of the related Smad2 and the structural basis for this difference maps to the divergent sequences of their N-terminal domains.

Figure 1

In vitro import assay for Smad3. (A) Profile of bacterially purified Smad3 (lane 1) and Smad3D proteins (lane 2) after SDS-PAGE and Coomassie Brilliant Blue staining. (B) Microinjection of bacterial Smad3 together with bacterial GSTNLSGFP protein in the cytoplasm of human colon carcinoma SW480.7 cells. The cells were previously transiently infected with adenoviruses that encode for LacZ (control) or caALK-5. The caALK-5–infected cells were also treated with TGF-β1 (+TGF-β) immediately after microinjection and for 1 h. (C) In vitro import of Smad3, Smad3D, and GSTNLSGFP in the absence (−) and presence (+) of cytosol in digitonin-permeabilized HeLa cells. In this assay, each protein was added at 0.5 μM. Smad proteins were detected by anti-Flag/Cy3 immunofluorescence and GSTNLSGFP by green autofluorescence. Nuclear morphology was assessed by phase contrast microscopy.

Figure 2

Smad3 interacts with importin-β1 in an activation/phosphorylation-dependent manner. (A-C) SW480.7 and CCL64 cells were used giving essentially identical results. Representative immunoblots are shown in this figure. (A) In vitro interaction assay between GST (lane 1), GST-importin-β1 (GST-β1, lane 2), GST-importin-β2 (GST-β2, lane 3) and Smad3. Detergent extracts from transiently coinfected cells with adenoviruses encoding LacZ and Smad3 or caALK-5 and Smad3 and treated (+TGF-β) or not (LacZ) with TGF-β1 were isolated. Extracts were incubated with the three GST protein affinity columns and the bound Smad3 was analyzed by immunoblotting (IB) with anti-Flag antibody. In lane 4 an aliquot of the total cell lysate was analyzed by immunoblotting directly. (B) In vitro interaction assay between GST (lane 1), GST-importin-α1 (GST-α1, lane 2), GST-importin-α2 (GST-α2, lane 3), GST-importin-α3 (GST-α3, lane 4) and GST-importin-β1 (GST-β1, lane 5) and Smad3. Cell extracts were prepared and analyzed as in A. The total lysate control is in lane 6. (C) Specificity of the Smad3–importin-β1 interaction. In vitro interaction assay between GST-importin-β1 (GST-β1, all lanes) and Smad3. Cell extracts treated as indicated on top of the panel were prepared and analyzed as in A by using anti-Flag (first panel) or anti-phospho-Smad (second panel) immunoblotting. Analysis of total lysate aliquots (Total) with the same two antibodies is shown in the third and fourth panels, respectively. Smad7 was also detected by anti-Flag immunoblotting (fifth panel). Asterisks on the right of the second and fourth panels indicate nonspecific bands resulting from the rabbit anti-phospho-Smad serum. (D) C-Terminal phosphorylation-dependent interaction between Smad3 and importin-β1. In vitro interaction assay between GST (lanes 1 and 2), GST-importin-β1 (GST-β1, lanes 3 and 4) and histidine-tagged baculoviral Smad3 (S3, lanes 1 and 3), or C terminally phosphorylated Smad3 (S3P, lanes 2 and 4). Bound Smad3 proteins were analyzed by immunoblotting by using anti-histidine (His) antibody. Aliquots of the baculoviral proteins were separately analyzed by sequential anti-histidine (lanes 5 and 6) and anti-phospho-serine (P-Ser, lanes 7 and 8) immunoblotting. (A–D) Position of the relevant protein bands is indicated on the sides of the panels.

Figure 2

Smad3 interacts with importin-β1 in an activation/phosphorylation-dependent manner. (A-C) SW480.7 and CCL64 cells were used giving essentially identical results. Representative immunoblots are shown in this figure. (A) In vitro interaction assay between GST (lane 1), GST-importin-β1 (GST-β1, lane 2), GST-importin-β2 (GST-β2, lane 3) and Smad3. Detergent extracts from transiently coinfected cells with adenoviruses encoding LacZ and Smad3 or caALK-5 and Smad3 and treated (+TGF-β) or not (LacZ) with TGF-β1 were isolated. Extracts were incubated with the three GST protein affinity columns and the bound Smad3 was analyzed by immunoblotting (IB) with anti-Flag antibody. In lane 4 an aliquot of the total cell lysate was analyzed by immunoblotting directly. (B) In vitro interaction assay between GST (lane 1), GST-importin-α1 (GST-α1, lane 2), GST-importin-α2 (GST-α2, lane 3), GST-importin-α3 (GST-α3, lane 4) and GST-importin-β1 (GST-β1, lane 5) and Smad3. Cell extracts were prepared and analyzed as in A. The total lysate control is in lane 6. (C) Specificity of the Smad3–importin-β1 interaction. In vitro interaction assay between GST-importin-β1 (GST-β1, all lanes) and Smad3. Cell extracts treated as indicated on top of the panel were prepared and analyzed as in A by using anti-Flag (first panel) or anti-phospho-Smad (second panel) immunoblotting. Analysis of total lysate aliquots (Total) with the same two antibodies is shown in the third and fourth panels, respectively. Smad7 was also detected by anti-Flag immunoblotting (fifth panel). Asterisks on the right of the second and fourth panels indicate nonspecific bands resulting from the rabbit anti-phospho-Smad serum. (D) C-Terminal phosphorylation-dependent interaction between Smad3 and importin-β1. In vitro interaction assay between GST (lanes 1 and 2), GST-importin-β1 (GST-β1, lanes 3 and 4) and histidine-tagged baculoviral Smad3 (S3, lanes 1 and 3), or C terminally phosphorylated Smad3 (S3P, lanes 2 and 4). Bound Smad3 proteins were analyzed by immunoblotting by using anti-histidine (His) antibody. Aliquots of the baculoviral proteins were separately analyzed by sequential anti-histidine (lanes 5 and 6) and anti-phospho-serine (P-Ser, lanes 7 and 8) immunoblotting. (A–D) Position of the relevant protein bands is indicated on the sides of the panels.

Figure 2

Smad3 interacts with importin-β1 in an activation/phosphorylation-dependent manner. (A-C) SW480.7 and CCL64 cells were used giving essentially identical results. Representative immunoblots are shown in this figure. (A) In vitro interaction assay between GST (lane 1), GST-importin-β1 (GST-β1, lane 2), GST-importin-β2 (GST-β2, lane 3) and Smad3. Detergent extracts from transiently coinfected cells with adenoviruses encoding LacZ and Smad3 or caALK-5 and Smad3 and treated (+TGF-β) or not (LacZ) with TGF-β1 were isolated. Extracts were incubated with the three GST protein affinity columns and the bound Smad3 was analyzed by immunoblotting (IB) with anti-Flag antibody. In lane 4 an aliquot of the total cell lysate was analyzed by immunoblotting directly. (B) In vitro interaction assay between GST (lane 1), GST-importin-α1 (GST-α1, lane 2), GST-importin-α2 (GST-α2, lane 3), GST-importin-α3 (GST-α3, lane 4) and GST-importin-β1 (GST-β1, lane 5) and Smad3. Cell extracts were prepared and analyzed as in A. The total lysate control is in lane 6. (C) Specificity of the Smad3–importin-β1 interaction. In vitro interaction assay between GST-importin-β1 (GST-β1, all lanes) and Smad3. Cell extracts treated as indicated on top of the panel were prepared and analyzed as in A by using anti-Flag (first panel) or anti-phospho-Smad (second panel) immunoblotting. Analysis of total lysate aliquots (Total) with the same two antibodies is shown in the third and fourth panels, respectively. Smad7 was also detected by anti-Flag immunoblotting (fifth panel). Asterisks on the right of the second and fourth panels indicate nonspecific bands resulting from the rabbit anti-phospho-Smad serum. (D) C-Terminal phosphorylation-dependent interaction between Smad3 and importin-β1. In vitro interaction assay between GST (lanes 1 and 2), GST-importin-β1 (GST-β1, lanes 3 and 4) and histidine-tagged baculoviral Smad3 (S3, lanes 1 and 3), or C terminally phosphorylated Smad3 (S3P, lanes 2 and 4). Bound Smad3 proteins were analyzed by immunoblotting by using anti-histidine (His) antibody. Aliquots of the baculoviral proteins were separately analyzed by sequential anti-histidine (lanes 5 and 6) and anti-phospho-serine (P-Ser, lanes 7 and 8) immunoblotting. (A–D) Position of the relevant protein bands is indicated on the sides of the panels.

Figure 3

Ran GTPase mediates the nuclear import of Smad3. (A) Dominant negative RanQ69L-GTP inhibits the ligand-dependent nuclear import of Smad3. SW480.7 cells transiently infected with an adenovirus expressing Smad3 were left intact (−), microinjected with GSTNLSGFP plus buffer (Buffer), or with GSTNLSGFP plus RanQ69L-GTP (RanQ69L-GTP). Immediately after microinjection cells were treated with vehicle (−TGF-β) or treated with TGF-β1 (+TGF-β) for 1 h. The proteins were detected by GFP autofluorescence (NLS), anti-Flag/tetramethylrhodamine isothiocyanate immunofluorescence (Smad3), and nuclei were scored by 4′,6-diamidino-2-phenylindole fluorescence. Each three-panel row of photographs represents the same microscopic field. (B) Ran-GTP dissociates Smad3 from the complex with importin-β1. In vitro dissociation assay of purified Smad3D bound to GST–importin-β1 (GST-β1) affinity columns (lanes 1–4). A similar assay was performed between purified MBP-IBB fusion protein and GST–importin-β1 (GST-β1) affinity columns (lanes 5–8). Purified proteins were bound to the affinity columns and eluted with buffer containing Ran-GDP (lanes 1, 2, 5, and 6) or Ran-GTP (lanes 3, 4, 7, and 8). The proteins remaining bound (lanes 1, 3, 5, and 7) to the affinity columns and those eluted (lanes 2, 4, 6, and 8) off the columns were analyzed by SDS-PAGE and Coomassie Brilliant Blue staining. The position of the relevant protein bands is indicated on the sides of the panel. Densitometric quantification of the eluted Smad3D and MBP-IBB protein bands of the gel is presented as a bar graph. The eluted protein band intensity is graphed as percentage of the total band intensity (bound plus eluted) in each of the four binding reactions. Numbers corresponding to the eluted sample lanes of the gel described above are indicated below the bar graph.

Figure 4

Importin-β1 interacts specifically with the Smad3 MH1 domain but not with Smad2. (A) A diagram of Smad3 with its three domains (MH1, linker [L] and MH2) is shown. N and C denote the amino and carboxyl termini. The conserved lysine-rich motif in MH1 is indicated as a striped box. (B) 293T cells were transiently transfected with empty vector (Mock) or the indicated 6-myc-tagged Smad3 (S3) domain mutants together with ca-ALK-5 to stimulate the pathway. Cell extracts were incubated with GST-importin-β1 (GST-β1) and the bound Smad3 proteins were analyzed by immunoblotting (IB) with anti-myc antibody. (C) Aliquots of the same extracts were incubated with GST alone as negative control and analyzed as in B. (D) Aliquots of the same extracts (Total) were analyzed by direct immunoblotting with anti-myc antibody to monitor the expression levels of the different Smad3 mutants in the transfected cell extracts. (E) Diagrams of wild-type Smad2 and the three Smad2-Smad3 chimeras are shown with their three domains (MH1, L, and MH2). N and C denote the amino and carboxyl termini. The conserved lysine-rich motif in MH1 is indicated as a striped box. The two unique inserts of Smad2 are shown as highlighted boxes labeled GAG and TID. Smad2 sequences are shown as black and Smad3 sequences as white boxes. The positions in Smad3 MH1 sequences that mark the absence of the Smad2 unique inserts are also depicted with straight vertical lines and are interconnected with the two inserts between constructs. (F) 293T cells were transiently transfected with the indicated nontagged wild-type Smad2 (lanes 1 and 5) and three Smad2-Smad3 chimeras (lanes 2–4 and 6–8) and then left unstimulated (−, lanes 1–4) or were stimulated (+, lanes 5–8) with TGF-β1. Cell extracts were incubated with GST-importin-β1 (pull-down) and the bound Smad2 proteins were analyzed by IB with the anti-Smad2/3 antibody. Aliquots of the same extracts (total) were analyzed by direct immunoblotting with the anti-Smad2/3 antibody to monitor the expression levels of the different Smad2 variants in the transfected cell extracts. The closely migrating positions of the four Smad2 variants are shown by bracket on the right side of the panels.

Figure 5

In vitro nuclear import assay of various Smad3 domains. In vitro import of N-terminal GFP fusions of Smad3D (GFPS3D, A–E), Smad3 (S3) domain mutants MH1 (GFPS3MH1, F–J), linker (GFPS3L, panels K–O), N-terminal GSTGFP fusion of Smad3 MH2 (GSTGFPS3MH2, P–T), and control GSTNLSGFP (U–Y) proteins in digitonin-permeabilized HeLa cells. Each protein cargo was added at 0.5 μM. The in vitro import conditions are indicated to the left of the panels and included: the absence (−) and presence (+) of cytosol, ATP depletion by hexokinase plus glucose treatment (−ATP), wheat germ agglutinin blocking of the nuclear pores (+WGA), and incubation on ice in the presence of cytosol. Protein detection was obtained by green autofluorescence. Nuclear staining was assessed with phase contrast microscopy.

Figure 6

In vitro reconstitution of Smad3 nuclear import. In vitro import of Smad3D, N-terminal GFP fusion of Smad3 MH1 domain (GFPS3MH1) and control GSTNLSGFP proteins in the presence or absence of cytosol in digitonin-permeabilized HeLa cells. For Smad3D and GFPS3MH1, additional import reactions are shown with purified protein components (no cytosol): importin-β1 alone (+imp β1), Ran-GDP plus p10, importin-β1 plus Ran-GDP plus p10, or importin-β1 plus RanQ69L-GTP plus p10. Smad3D was detected via anti-Flag/Cy3 immunofluorescence and GFPS3MH1 and GSTNLSGFP via their green autofluorescence.