Phosphorylation of threonine 276 in Smad4 is involved in transforming growth factor-{beta}-induced nuclear accumulation

Bernard A. J. Roelen,1,2,* Ori S. Cohen,1,2,* Malay K. Raychowdhury,2 Deborah N. Chadee,3 Ying Zhang,1,2 John M. Kyriakis,3 Alessandro A. Alessandrini,2 and Herbert Y. Lin1,2

1Program in Membrane Biology and 2Renal Unit, Massachusetts General Hospital, and Department of Medicine, Harvard Medical School, Charlestown 02129; and 3Molecular Cardiology Research Institute, New England Medical Center, Tufts University School of Medicine, Boston, Massachusetts 02111

Submitted 6 February 2003 ; accepted in final form 2 June 2003


    ABSTRACT
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Smad4, the common Smad, is central for transforming growth factor (TGF)-{beta} superfamily ligand signaling. Smad4 has been shown to be constitutively phosphorylated (Nakao A, Imamura T, Souchelnytskyi S, Kawabata M, Ishisaki A, Oeda E, Tamaki K, Hanai J, Heldin C-H, Miyazono K, and ten Dijke P. EMBO J 16: 5353-5362, 1997), but the site(s) of phosphorylation, the kinase(s) that performs this phosphorylation, and the significance of the phosphorylation of Smad4 are currently unknown. This report describes the identification of a consensus ERK phosphorylation site in the linker region of Smad4 at Thr276. Our data show that ERK can phosphorylate Smad4 in vitro but not Smad4 with mutated Thr276. Flag-tagged Smad4-T276A mutant protein accumulates less efficiently in the nucleus after stimulation by TGF-{beta} and is less efficient in generating a transcriptional response than Smad4 wild-type protein. Tryptic phosphopeptide mapping identified a phosphopeptide in Smad4 wild-type protein that was absent in phosphorylated Smad4-T276A mutant protein. Our results suggest that MAP kinase can phosphorylate Thr276 of Smad4 and that phosphorylation can lead to enhanced TGF-{beta}-induced nuclear accumulation and, as a consequence, enhanced transcriptional activity of Smad4.

signal transduction; mitogen-activated protein kinase; phosphopeptide mapping; extracellular signal-regulated kinase phosphorylation site; luciferase reporter


TRANSFORMING GROWTH FACTOR (TGF)-{beta} signaling from the cell surface to the nucleus is exerted via Smad proteins. Smads have been classified into three sub-types: receptor-regulated Smads (R-Smads), common mediator Smads (Co-Smads), and inhibitory Smads (I-Smads). Except for the I-Smads, Smad proteins are composed of conserved NH2-terminal Mad homology (MH)1 and COOH-terminal MH2 domains separated by a more diverse linker region. R-Smads are phosphorylated on a specific SSV/MS motif at the COOH terminus by activated TGF-{beta} type I receptors and then form a heteromeric complex with the Co-Smad Smad4. The resulting R-Smad-Smad4 complex translocates from the cytoplasm to the nucleus, where it interacts with DNA and other proteins to form functional transcriptional complexes (12).

Smad4 is the product of the tumor suppressor gene deleted in pancreatic carcinoma-4 (DPC-4) (10). Its MH1 domain can directly interact with DNA (26) but is also thought to bind to the phosphorylated C-tail of activated R-Smads (11). The MH2 domain is important for transcriptional activation (3, 5, 27) and for direct protein interactions with the MH1 domain of R-Smads (23). Smad4 lacks the COOH-terminal SSV/MS motif found in R-Smads and neither binds nor is phosphorylated by activated type I receptors (15, 17). However, Smad4 has been shown to be constitutively phosphorylated (17), but the site(s) of phosphorylation, the kinase(s) that performs this phosphorylation, and the significance of the phosphorylation of Smad4 have never been elucidated.

A domain in the COOH-terminal part of the linker region between amino acid residues 275 and 322, named the Smad activation domain (SAD), was demonstrated to be important for full transcriptional activity of Smad4 (3). How the SAD regulates Smad4 transcriptional activity is unknown but may involve binding to transcriptional coactivators (5). It was suggested that Smad4 shuttles continuously between the nucleus and the cytoplasm as a consequence of a functional nuclear localization signal (NLS) located in the MH1 domain and a nuclear export signal (NES) located in the NH2-terminal part of the linker region (18). Activation by TGF-{beta} causes Smad4 to accumulate in the nucleus, but little is known about the mechanism of this nuclear accumulation.

An interplay between TGF-{beta}/activin/bone morphogenetic protein (BMP) signaling and Ras-activating mitogens was demonstrated by the identification of functional ERK phosphorylation sites in the linker regions of R-Smads that are distinct from the receptor-mediated phosphorylation sites at the COOH terminus. ERK-mediated phosphorylation of R-Smads could potentially inhibit the nuclear accumulation of these proteins and block TGF-{beta}/BMP-induced signaling (13, 14). In contrast, others demonstrated that phosphorylation of R-Smads by mitogen-activated protein (MAP) kinases leads to enhanced nuclear accumulation of R-Smads (4, 7).

This report describes the identification and characterization of a consensus ERK phosphorylation site in the linker region of the Co-Smad Smad4 at threonine 276 (Thr276). Phosphorylation of Thr276 is shown to be important for TGF-{beta}-induced nuclear accumulation and, as a consequence, transcriptional activity of Smad4.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture and transfection. LLC-PK1 and MDA-MB468 cells were obtained from American Type Culture Collection and cultured in DMEM (Bio-Whittaker, Walkersville, MD) containing 10% fetal bovine serum (FBS; Bio-Whittaker, Walkersville, MD). All plasmid (pcDNA3.1; Invitrogen, Carlsbad, CA) transfections were performed with FuGENE 6 reagent (Roche, Indianapolis, IN). Stably transfected cells were selected and cultured in 1 mg/ml G418 (Life Technologies, Rockville, MD). TGF-{beta}1 (R&D Systems, Minneapolis, MN) was added at 10 ng/ml in DMEM with 2% FBS for the time and concentrations indicated after serum starvation for 8-24 h. U0126 (Promega, Madison, NY) was used at 10-70 µM; leptomycin B (LMB; Sigma, St. Louis, MO) was used at 10 ng/ml.

Immunoprecipitation, SDS-PAGE, and Western blot analysis. Cells were lysed in lysis buffer [50 mM Tris · HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1x protease inhibitor cocktail (Sigma)]. Protein extracts were immunoprecipitated with M2 anti-Flag antibody resin (Sigma) or analyzed directly. SDS-PAGE was performed with precast NuPAGE Novex 4-12% Bis-Tris gels (Invitrogen). Samples were electroblotted to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA), and membranes were probed with anti-human Smad4 rabbit polyclonal IgG (1:400; Santa Cruz, Santa Cruz, CA), monoclonal M2 anti-Flag antibody (1:5,000; Sigma), anti-Smad4 (1:500; Santa Cruz), anti-P-ERK1/2 (1:1,000; Cell Signaling Technology, Beverly, MA), anti-ERK1/2 (1:1,000; Cell Signaling Technology), or monoclonal anti-{beta}-actin antibody (1:5,000; Abcam, Cambridge, UK) followed by anti-rabbit or anti-mouse horseradish peroxidase (HRP) antibody (1:10,000; Santa Cruz). Antibody binding was detected with chemiluminescence reagent (NEN Life Sciences Products, Boston, MA) and exposure to X-Omat film (Kodak, Rochester, NY).

In vitro phosphorylation. The cDNAs encoding Smad4 wild-type and Smad4-T276A were inserted into a pGEX 4T vector (Amersham, Piscataway, NJ) and transformed into BL21 Escherichia coli cells for glutathione S-transferase (GST)-fusion protein expression induced by adding 0.2 mM isopropyl-{beta}-D-thiogalactopyranoside. Purification of the proteins was performed with GST beads according to the manufacturer's protocol (Novagen, Madison, WI). One microgram of each protein was incubated with 100 U of recombinant ERK2 (New England Biolabs, Beverly, MA), MAP kinase buffer, 100 µM ATP, and 5 µCi of [{gamma}-32P]ATP (NEN Life Sciences Products) for 30 min at 30°C. The samples were analyzed by SDS-PAGE, electroblotting, and autoradiography.

Luciferase assay. Cells were transiently transfected with the (CAGA)12Luc reporter construct (6) and with a pRL-TK vector (Promega). Cells were then serum starved and treated with 400 pM TGF-{beta}1 (16 h). Experiments were performed in triplicate wells. Cells were lysed, and luciferase activity was determined with the Dual Reporter Assay (Promega). Relative light units were calculated as ratios of firefly (reporter) and Renilla (transfection control) luciferase values.

Orthophosphate labeling, phosphoamino acid analysis, and tryptic phosphopeptide mapping. Subconfluent cells were washed with phosphate-free medium and labeled with 500 µCi/ml [32P]orthophosphate (NEN Life Science Products) for 4 h in phosphate-free medium containing 1% bovine serum albumin (Sigma) in the presence or absence of TGF-{beta}1. Cells were lysed with (in mM) 50 Tris · HCl pH 7.4, 150 NaCl, 1 EDTA, 1 {beta}-glycerophosphate (Sigma), and 1 phenylmethylsulfonyl fluoride (Sigma) with 1% Triton X-100 and subjected to immunoprecipitation with M2 anti-Flag-antibody (Sigma). Samples separated by SDS-PAGE were electroblotted onto PVDF membrane and subjected to autoradiography. The Smad4 bands were excised and prepared for phosphoamino acid analysis as described previously (2). Samples were electrophoresed in one dimension on TLC plates in pH 3.5 buffer. Unlabeled standards were visualized with ninhydrin spray (Sigma), and the plates were subjected to autoradiography. For tryptic phosphopeptide mapping, Smad4 bands were excised directly from the gel, prepared as described previously (2), and electrophoresed on TLC plates in pH 1.9 buffer. The TLC plates were subsequently subjected to chromatography for 6 h (2). Radioactivity was detected with phosphor-imager screens.

Immunofluorescence. Cells grown on coverslips were serum starved (8 h) before treatment with TGF-{beta}1 for 1 h, fixed in 4% paraformaldehyde, incubated with M5 anti-Flag monoclonal antibody (dilution 1:300; Sigma) followed by incubation with anti-mouse CY-3 (dilution 1:800; Jackson Immunoresearch, West Grove, PA), and mounted with mounting medium (Kirkegaard and Perry Laboratories, Gaithersburg, MD). Epifluorescence analysis was performed on an Eclipse E8000 microscope (Nikon). For quantification of nuclear accumulation, the number of nuclei in which the fluorescence was brighter than in the cytoplasm was counted on photographs by an independent observer. Cells were counter-stained with 4',6-diamidino-2-phenylindole (DAPI) to identify nuclei. At least 70 nuclei were counted for each condition.


    RESULTS
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Presence of consensus ERK phosphorylation site in linker region of Smad4. Examination of the linker region of Smad4 revealed a consensus ERK phosphoacceptor site (PXS/TP) at amino acids 274-277 (Fig. 1A). No other consensus ERK phosphoacceptor sites were identified in the entire Smad4 protein sequence. Smad4 cDNA constructs were generated in which Thr276 was replaced by alanine (T276A), and GST-fusion proteins consisting of GST alone, GST-Smad4 wild type and GST-Smad4-T276A were analyzed with an in vitro ERK2 kinase assay (Fig. 1B). The results show that GST-Smad4 wild type can be phosphorylated by ERK2 in vitro. In contrast, GST-Smad4-T276A was not phosphorylated (Fig. 1B). The stoichiometry of phosphorylation of GST-Smad4 wild type by ERK was estimated at ~22% (data not shown). Phosphoamino acid analysis demonstrated that ERK phosphorylation of GST-Smad4 wild type was indeed on threonine (Fig. 1C). These results suggest that Smad4 can be phosphorylated by ERK2 at Thr276.



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Fig. 1. Phosphorylation of Smad4 by ERK2 in vitro. A: schematic representation of the Smad4 structure. The predicted ERK phosphorylation site is underlined, with Thr276 in bold. Bottom: a comparison with other species. Conserved amino acids are in bold. MH, Mad homology. B: glutathione S-transferase (GST) fusion proteins as indicated were analyzed as substrates for recombinant ERK2 in the presence of [{gamma}-32P]ATP. Protein loading was determined by anti-Smad4 immunoblotting of the same blot (bottom). T276A, Thr276 replaced by alanine. C: the GST-Smad4 phosphoprotein was isolated and subjected to phosphoaminoacid analysis. The relative migration of phosphoserine (P-Ser), phosphothreonine (P-Thr), and phosphotyrosine (PTyr) is indicated.

 

Smad4 wild type and Smad4-T276A are both phosphoproteins. We subsequently investigated whether there is a difference in phosphorylation in vivo between Smad4 wild type and Smad4 in which Thr276 was mutated to Ala (Smad4-T276A). Wild-type LLC-PK1 (porcine kidney epithelial cells) and LLC-PK1 stably transfected with mammalian expression vectors with cDNA constructs coding for Flag-Smad4 wild type and Flag-Smad4-T276A were metabolically labeled with [32P]orthophosphate in the presence or absence of TGF-{beta}1. Whole cell lysates were subsequently subjected to immunoprecipitation with an antibody against Flag. Flag-Smad4 wild type was already phosphorylated in the absence of TGF-{beta}, and that remained unchanged after stimulation with TGF-{beta} (Fig. 2A). Flag-Smad4-T276A was also phosphorylated, irrespective of the presence of TGF-{beta}, and no decrease was observed in gross phosphorylation levels compared with Smad4 wild type (Fig. 2A). These results indicate that there are also other residues in Smad4 that are targets for phosphorylation. Phosphoamino acid analysis revealed phosphorylation predominantly on serine residues and on threonine residues to a lesser extent. No phosphorylation was observed on tyrosine (Fig. 2B). Smad4-T276A still was phosphorylated on threonine residues but to a lesser extent than Smad4 wild type, indicating that threonine residues other than Thr276 are phosphorylated. Tryptic phosphopeptide mapping revealed multiple phosphopeptides, most of these migrating to the cathode. Comparison of the maps of Smad4 wild type and Smad4-T276A with the map of in vitro phosphorylated GST-Smad4 wild type revealed a significant spot that is present in the phosphopeptide maps of in vitro and in vivo phosphorylated Smad4 wild type but absent in the map of Smad4-T276A (Fig. 2C). This spot most likely represents phosphorylation at Thr276.



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Fig. 2. Phosphorylation of Smad4 wild type and Smad4-T276A in cells. Control LLC-PK1 cells and LLC-PK1 cells stably expressing Flag-Smad4 wild type or Flag-Smad4-T276A were labeled with [32P]orthophosphate in the presence or absence of transforming growth factor (TGF)-{beta}. A: cell lysates were subjected to immunoprecipitation with anti-Flag-antibody and analyzed by SDS-PAGE, electroblotting, and autoradiography. B: phosphoamino acid analysis of in vivo labeled Smad4 wild type (wt) and Smad4-T276A (TA). The relative migration of phosphoserine, phosphothreonine, and phosphotyrosine is indicated. C: Tryptic phosphopeptide mapping of in vivo labeled Smad4 wild type and Smad4-T276A and in vitro labeled GST-Smad4 wild type. Indicated are the origins of sample application (black arrowheads), free phosphate (white arrowheads), and phosphopeptide that is present in Smad4 wild type and GST-Smad4 wild type but absent in Smad4-T276A (dotted circles).

 

Thr276 is important for TGF-{beta}-induced nuclear localization of Smad4. To determine the functional significance of Smad4 phosphorylation at amino acid residue Thr276 in cells, the subcellular distribution of Flag-Smad4 in LLC-PK1 cells stably transfected with cDNA constructs coding for Flag-Smad4 wild type and for Flag-Smad4-T276A was followed with indirect immunofluorescence with an anti-Flag antibody. In the absence of exogenous TGF-{beta}, Flag-Smad4 wild-type protein and Flag-Smad4-T276A mutant protein were present throughout the cell (Fig. 3A). On TGF-{beta} addition, Flag-Smad4 wild-type protein accumulated in the nucleus, but, in contrast, the Flag-Smad4-T276A protein was predominantly cytoplasmic in distribution (Fig. 3A). Quantification of the immunofluorescence data showed that in cells expressing Smad4 wild type, a marked increase is observed in the percentage of cells in which Flag-Smad4 is predominantly in the nucleus after stimulation with TGF-{beta}, whereas this percentage remains similar to control values in cells expressing Smad4-T276A (Fig. 3B). Even after 6 h of stimulation with TGF-{beta}, Flag-Smad4-T276A localization was predominantly cytoplasmic (data not shown), indicating that the diminished TGF-{beta}-induced nuclear accumulation is not simply due to slower kinetics. These results demonstrate that Thr276 is an important residue for efficient nuclear accumulation of Smad4 in response to TGF-{beta} and suggest that the phosphorylation of Thr276 can shift the nuclear import vs. export ratio of Smad4.



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Fig. 3. Subcellular localization of Smad4 wild type and Smad4-T276A. A: LLC-PK1 were stably transfected with plasmids expressing Flag-Smad4 wild type or Flag-Smad4-T276A. After stimulation for 1 h with TGF-{beta}1, the subcellular distribution of the Smad4 proteins was visualized by indirect immunofluorescence with an anti-Flag antibody. B: quantification of the percentage of cells with Smad4 predominantly in the nucleus.

 

Thr276 is required for full transcriptional activity of Smad4. We next used the stably transfected LLC-PK1 cells to examine the ability to induce transcription of a (CAGA)12-Luc reporter, which is specific for Smad3/Smad4 signaling by TGF-{beta}/activin (6). Because LLC-PK1 cells express endogenous Smad4, a TGF-{beta} response was observed in untransfected cells after stimulation with TGF-{beta}1 for 16 h (Fig. 4A; compare bars 1 and 2). In stable LLC-PK1 cells expressing Flag-Smad4 wild type, luciferase activation was enhanced compared with untransfected cells after stimulation with TGF-{beta} (Fig. 4A; compare bars 4 and 2). However, in the cell lines expressing the Flag-Smad4-T276A mutant protein, the TGF-{beta}-induced activation of luciferase was not enhanced and was similar to that in control cells (Fig. 4A; compare bars 6 and 2). As a control for protein expression in the stable cells, whole cell extracts were immunoprecipitated with anti-Flag antibody followed by immunoblotting with an anti-Smad4 antibody (Fig. 4B). The results demonstrate that equal amounts of protein are made by the different Smad4 variants in their respective stable cell lines and indicate that the Flag-Smad4-T276A mutant protein is not grossly mis-folded or misaggregated compared with the Flag-Smad4 wild-type protein. Analysis of total cell lysates revealed that the endogenous levels of Smad4 in LLC-PK1 cells are relatively low and that the increase of Smad4 expression in the stable cell lines was approximately sevenfold (Fig. 4B). Equal protein expression was confirmed by immunoblotting of whole cell lysates with an anti-{beta}-actin antibody (Fig. 4B). We next analyzed the transcriptional activity of Flag-Smad4 wild type and Flag-Smad4-T276A in cells that have a homozygous deletion of the Smad4 gene and thus lack endogenous Smad4 expression, the human breast tumor cell line MDA-MB468 (22, 25). Transient transfection of Flag-Smad4 wild type restored TGF-{beta}-dependent reporter gene activation in these cells as measured by (CAGA)12-Luc induction (Fig. 4C). In comparison, transient transfection of Flag-Smad4-T276A in these cells was only partially able to restore TGF-{beta}-specific transcriptional response (Fig. 4C). The levels of Smad4 overexpression in these cells were analyzed by immunoblotting with an anti-Flag antibody, and the results demonstrate that the decrease in luciferase activity in Smad4-T276A-expressing cells was not due to lower levels of expression (Fig. 4D). These results demonstrate that Thr276 is required for full transcriptional activity of Smad4 and suggest that phosphorylation of Smad4 at Thr276 enhances its activity.



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Fig. 4. TGF-{beta}-induced transcriptional activity of Smad4 wild type and Smad4-T276A. A: Control LLC-PK1 cells (bars 1 and 2) and LLC-PK1 cells stably expressing Flag-Smad4 wild type (bars 3 and 4) or Flag-Smad4-T276A (bars 5 and 6) were transiently transfected with the (CAGA)12-Luc expression vector. Cells were cultured with (bars 2, 4, and 6) or without (bars 1, 3, and 5) TGF-{beta}1 and assayed for luciferase activity. Mean + SD values are shown. B: as a control for relative protein expression, whole cell lysates were immunoprecipitated (IP) with anti-Flag antibody, followed by Western blotting (W) with anti-Smad4 antibody (top). Cell lysates were also directly analyzed by SDS-PAGE followed by Western blotting with an anti-pan-Smad4 antibody (middle) or with anti-{beta}-actin antibody (bottom). C: MDA-MB468 cells were transiently transfected with control, Flag-Smad4 wild type, or Flag-Smad4-T276A plasmids together with (CAGA)12-Luc reporter plasmid. Cells were cultured in the presence or absence of TGF-{beta}1 and assayed for luciferase activity. Mean + SD values are shown. D: cell extracts of transfected MDA-MB468 cells were analyzed by SDS-PAGE and subsequent immunoblotting with an anti-Flag antibody (top) or with an anti-{beta}-actin antibody (bottom).

 

To further investigate the effect of phosphorylation by ERK on Smad4 cellular distribution, LLC-PK1 cells stably expressing Flag-Smad4 wild-type and mutant proteins were pretreated with the MEK inhibitor U0126 (8) for 1 h before stimulation with TGF-{beta}1. Analysis of lysates from LLC-PK1 cells confirmed that cells treated for 1 h with U0126 exhibit a dramatic reduction in levels of ERK phosphorylation (Fig. 5B). Treatment for 1 h with TGF-{beta} resulted in a minor increase in levels of phosphorylated ERK (Fig. 5B). In cells expressing Smad4 wild type, Smad4 accumulated in the nucleus after exposure to TGF-{beta}. However, pretreatment of these cells with U0126 inhibited the TGF-{beta}-induced nuclear accumulation of Smad4 (Fig. 5A). We have used concentrations of U0126 ranging from 10 to 70 µM and observed similar results with all concentrations, although the results were more pronounced at the higher concentrations (data not shown). Pretreatment with U0126 did not affect the distribution of Smad4-T276A, which was predominantly cytoplasmic even in the presence of TGF-{beta} (Fig. 5A).



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Fig. 5. Nuclear accumulation and transcriptional activity of Smad4. A: LLC-PK1 cells stably expressing Flag-Smad4 wild type or Flag-Smad4-T276A were incubated for 1 h with TGF-{beta}1 or leptomycin B (LMB) or treated for 1 h with U0126 followed by 1-h incubation with TGF-{beta}1 and U0126. The subcellular distribution of the Smad4 proteins was visualized with an anti-Flag antibody. B: LLC-PK1 cells were incubated in the presence or absence of TGF-{beta} and U0126 for 1 h, after which the cell lysates were subjected to SDS-PAGE and immunoblotting with antibodies that recognize phosphorylated ERK (top) or total ERK (bottom). C and D: LLC-PK1 cells stably expressing Flag-Smad4 wild type and Flag-Smad4-T276A were transiently transfected with (CAGA)12-Luc expression vector. Cells were cultured with or without (control) LMB (C) or with TGF-{beta}1 or TGF-{beta}1 + LMB (D) and assayed for luciferase activity. Mean + SD fold increase in luciferase activity is shown.

 

It was previously demonstrated that Smad4 nuclear export requires the CRM1 protein and that inhibition of CRM1-mediated nuclear export with LMB results in accumulation of Smad4 in the nucleus even in the absence of TGF-{beta} (18). Treatment of cells expressing Flag-Smad4 wild type with LMB for 1 h resulted in nuclear localization of Smad4 (Fig. 5A). In cells expressing Flag-Smad4-T276A, treatment with LMB led to strong nuclear accumulation of Flag-Smad4-T276A (Fig. 5A), suggesting that Smad4-T276A transports to the nucleus and that the diminished TGF-{beta}-induced nuclear accumulation of Smad4-T276A is not due to an absolute inability of Smad4-T276A to enter the nucleus but rather to a decreased nuclear import-to-export ratio. When Smad4 wild type or Smad4-T276A was forced to accumulate in the nucleus with LMB, the transcriptional activity in the absence of TGF-{beta} was enhanced approximately twofold (Fig. 5C). When LLC-PK1 cells were simultaneously treated with LMB and TGF-{beta}1, the fold increase in transcriptional activity of Smad4-T276A reached levels similar to the transcriptional activity of Smad4 wild type with TGF-{beta}1 (Fig. 5D), indicating that the reduced transcriptional activity observed with the Smad4-T276A mutant protein results from a reduced nuclear accumulation.


    DISCUSSION
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We have demonstrated that Smad4 wild-type protein can be phosphorylated by ERK in vitro. Clearly, Thr276 is the likely phosphoacceptor site for ERK, because Smad4-T276A was not phosphorylated by ERK (Fig. 1B). Thr276 is located at the NH2-terminal part of the SAD, and the crystal structure of this region revealed that part of the SAD forms a loop that reinforces a structure of the MH2 domain known as the TOWER structure (20). This TOWER structure functions as a docking site for transcription factors, and it could legitimately be argued that the effect of the T276A mutation is due to constraints on protein confirmation instead of lack of phosphorylation. However, alanine is unlikely to impose constraints on protein formation because of its small side chain. Furthermore, Thr276 is at the NH2 terminus of the SAD, at a position that from the description of the published crystal structure is a loose end, which does not seem to be important for reinforcement of the TOWER structure or for stabilization of the core structure (20).

The consensus ERK site in Smad4 is a viable phosphorylation site in vitro (Fig. 1B) and seems to be conserved in most mammals but is absent in porcine Smad4. Importantly, the LLC-PK1 cells used in this study are porcine epithelial cells, whose endogenous Smad4 was confirmed by RT-PCR to contain a proline and not a threonine at the analogous site (data not shown). Therefore, the endogenous porcine Smad4 will not result in background Thr276 phosphorylation in the metabolic labeling experiments. The significance of the substitution of threonine by proline at the analogous site is unknown. Phosphoamino acid analysis of Flag-tagged mouse Smad4 expressed in LLC-PK1 cells showed phosphorylation on serine and threonine but not on tyrosine residues (Fig. 2B), although our results do not preclude tyrosine phosphorylation of Smad4 under other physiological conditions.

Smad4 subcellular distribution is thought to involve NLS and NES domains that would cause Smad4 to shuttle continuously between the cytoplasm and the nucleus (18). Our data suggest that phosphorylation of Thr276 can regulate Smad4 subcellular distribution, because Smad4-T276A mutants, which cannot be phosphorylated at position 276, do not accumulate efficiently in the nucleus in response to TGF-{beta}1 (Fig. 3A) and pretreatment of LLC-PK1 cells expressing mouse Smad4 wild type with U0126 inhibited the TGF-{beta}-induced nuclear accumulation (Fig. 5A). It is unlikely that phosphorylation of Smad4 at Thr276 is sufficient to cause nuclear accumulation in the absence of TGF-{beta}, because stimulation of ERK activity with EGF alone did not result in enhanced nuclear localization of Smad4 (data not shown).

Interestingly, residue Thr276 is in the COOH-terminal part of the linker region previously identified as important for full Smad4 activation. The significance of amino acids 275-322 in the linker region for TGF-{beta}-induced transcriptional activity was recognized by examining the function of deletion mutants or naturally occurring splice variants of Smad4 in this region (3, 5, 18, 25). These Smad4 deletion mutants were transcriptionally less efficient compared with Smad4 wild type, although, importantly, signaling activity was not completely abolished (3). We demonstrate a consistent finding because the signaling activity of Smad4-T276A is decreased compared with Smad4 wild type but not completely absent (Fig. 3). The decreased activity that we observed is most likely not due to impaired heterooligomerization with the R-Smads, because Smad4 mutants that lacked regions containing Thr276, or in fact the entire linker region, were still able to physically interact with R-Smads (5, 18). In addition, when LMB, which inhibits Smad4 nuclear export (18) and therefore forces Smad4 to accumulate in the nucleus, was added to cells, the TGF-{beta}-induced transcriptional activity was similar for Smad4 wild type and Smad4-T276A (Fig. 5D). Therefore, we hypothesize that phosphorylation of Thr276 only affects the nuclear import vs. export ratio of Smad4, but once Smad4 is in the nucleus Smad4 wild type and Smad4-T276A have equal transcriptional activity. In contrast to previous studies, we observe a lack of nuclear accumulation of Smad-T276A, whereas others have reported no effect on nuclear accumulation of Smad4 mutants lacking the SAD. This could be explained either by the importance of residues in the SAD other than Thr276 or by a difference in assay conditions, because we examined nuclear accumulation within 1 h of addition of TGF-{beta}, whereas others used a constitutively active type I receptor and examined nuclear accumulation after 24 h (5). Several studies have pointed to a significant cross talk between the TGF-{beta} and the MAP kinase pathways, most of them demonstrating activation of MAP kinase pathways by TGF-{beta} (16). Conversely, the MAP kinase pathways have been shown to regulate TGF-{beta} signaling. Phosphorylation of R-Smads by MAP kinases was shown either to enhance (13, 14) or inhibit (7) TGF-{beta}-induced nuclear accumulation of the R-Smads. Our results now also incorporate regulation of Smad4 activity in the MAP kinase pathway, enhancing the complexity of the interactions between the TGF-{beta} and MAP kinase pathways. These effects are likely to be cell type dependent. Previously, it was reported that in RIE cells, stimulation of the ERK pathway by high levels of oncogenic Ras repressed TGF-{beta} signaling as a result of degradation of Smad4 through the ubiquitin-proteasome pathway (21). These results are opposite to our findings, possibly because of differences in cell context.

In conclusion, we propose that Smad4 nuclear localization can be regulated by MAP kinase phosphorylation of residue Thr276 to promote nuclear accumulation of Smad4 and therefore to enhance Smad signaling. This regulation of Smad activity could occur during important physiological and pathophysiological processes such as organogenesis and wound healing, in which TGF-{beta} and the MAP kinase signaling pathways are known to function in a cooperative manner. In vivo examples of such activities would include the synergistic or enhanced effects of TGF-{beta}/BMP and EGF/FGF signaling during induction of chondrogenesis during otic capsule formation (9), paraxial mesoderm myogenesis (24), cardiogenic induction of non-precardiac mesoderm (1), and tubule formation in metanephric mesenchyme (19).


    DISCLOSURES
 
H. Y. Lin was supported by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Grant DK-02716, B. A. J. Roelen was supported by NIDDK Grant DK-19406.


    ACKNOWLEDGMENTS
 
Drs. S. Dennler and P. ten Dijke are thanked for the generous gift of the (CAGA)12MLP-Luc reporter construct. Dr. J. Wrana is thanked for Smad4 constructs. Dr. D. A. Ausiello and Dr. D. Brown of the Program in Membrane Biology at the Massachusetts General Hospital are thanked for their continuing support.

Present address of B. A. J. Roelen: Dept. of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 7, 3584 CL Utrecht, The Netherlands.


    FOOTNOTES
 

Address for reprint requests and other correspondence: H. Y. Lin, Renal Unit, Massachusetts General Hospital, and Dept. of Medicine, Harvard Medical School, 13th St. Bldg. 149, Charlestown, MA 02129 (E-mail: hlin{at}partners.org).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* B. A. J. Roelen and O. S. Cohen contributed equally to this work. Back


    REFERENCES
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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