Article |
Address correspondence to Xu Cao, 1670 University Blvd., VH G002, Birmingham, AL 35294-0019. Tel.: (205) 934-0162. Fax: (205) 934-1775. email: cao{at}path.uab.edu
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Abstract |
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Key Words: TGFß; Smad7; GADD34; phosphatase; SARA
Abbreviations used in this paper: GADD, growth arrest and DNA damage; I-1, inhibitor 1; OA, okadaic acid; PP1, protein phosphatase 1; PP1c, catalytic subunit of protein phosphatase 1; PTP, protein tyrosine phosphatase; RNAi, RNA interference; R-Smads, receptor-regulated Smads; SARA, Smad anchor for receptor activation; siRNA, small interfering RNA; TßRI, transforming growth factor ß type I receptor.
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Introduction |
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With no exception, the phosphorylation state of cellular proteins is controlled by the opposing actions of protein kinases and phosphatases. There are two kinds of kinase/phosphatase in the mammalian system: protein tyrosine kinase/protein tyrosine phosphatase (PTP) and serine threonine kinase/protein phosphatase (PP). Receptor protein tyrosine kinases are all type I transmembrane proteins with a cytoplasmic domain that has an intrinsic catalytic activity activated upon ligand binding. These phosphorylated substrates can hence be dephosphorylated by certain PTPs (Egloff et al., 1997; Bollen, 2001; Attisano and Wrana, 2002; Cohen, 2002). Mammalian members of the receptor serine threonine kinase family are receptors for ligands of TGFß family. The counterpart of PTP here is protein phosphatase (PP), but no protein phosphatase was found directly involved in the dephosphorylation of major components in the TGFß signaling pathway.
Here we show that Smad7, an inhibitory Smad whose expression is induced by TGFß (Hayashi et al., 1997; Nakao et al., 1997), interacts with growth arrest and DNA damage protein (GADD34; Hollander et al., 1997; Liebermann and Hoffman, 2002), a regulatory/targeting subunit of the protein phosphatase 1 (PP1) holoenzyme (Egloff et al., 1997; Aggen et al., 2000; Bollen, 2001; Cohen, 2002). The catalytic subunit of PP1, PP1c, is recruited to TFGß type I receptor (TßRI)Smad7GADD34 complex through this regulatory subunit, GADD34, to dephosphorylate TßRI. Furthermore, GADD34 is induced by UV light irradiation along with Smad7 resulting UV lightinduced TGFß resistance in Mv1Lu cells. Blockage of GADD34 and Smad7 by RNA interference (RNAi) restores the resistance to TGFß. Together, these results indicate that the formation of PP1 holoenzyme mediated by TGFß-induced Smad7 functions as a negative feedback in TGFß signaling pathway by dephosphorylating TßRI. This implies an important mechanism by which TGFß regulates the development, maintenance, and tumorigenesis of different tissues.
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Results |
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TGFß regulates the formation of TßRISmad7GADD34 complex via Smad7
Because Smad7 acts as an antagonist in the TGFß signaling pathway by binding to the TßRI (Wrana et al., 1994a,b; Wieser et al., 1995; Feng and Derynck, 1997; Attisano and Wrana, 2002), and GADD34 is a targeting subunit of PP1 (He et al., 1996; Hollander et al., 1997; Novoa et al., 2001; Liebermann and Hoffman, 2002), the interaction of Smad7 with GADD34 implicates a negative regulatory mechanism via the dephosphorylation of TßRI. Recent studies have revealed that PP1 negatively regulates decapentaplegic signaling in Drosophila melanogaster by affecting the phosphorylation state of TßRI (Bennett and Alphey, 2002). We therefore examined whether TGFß mediates the binding of GADD34PP1c serine/threonine phosphatase to its substrate, TßRI, because GADD34 is a target regulatory subunit of the PP1 holoenzyme. First, we examined whether GADD34 forms complexes with TßRI and Smad7 in mammalian cells. Immunoprecipitation assays were performed in TGFß-responsive MvlLu cells treated with or without TGFß-1. TGFß-induced endogenous TßRISmad7GADD34 complexes were immunoprecipitated with either anti-TßRI or anti-GADD34 antibody (Fig. 2, a and b). This complex is further confirmed by a sequential immunoprecipitation. COS1 cells were first cotransfected with TßRIHA and PP1c with or without FlagSmad7 and GADD34. After 2 h of stimulation with TGFß-1, the cells were lysed, subjected to first immunoprecipitation with Flag antibody, and the resultant precipitates were eluted from the protein GSepharose bead by Flag peptide competition and then subjected to second immunoprecipitation with HA antibody. The final precipitates were immunoblotted with antibodies against all these components (Fig. 2 c). The results indicated that the triple components complex, Smad7TßRIGADD34, were formed along with PP1c. To identify the region of Smad7 that binds GADD34, a series of truncated Smad7 truncation constructs were generated for a yeast two-hybrid assay (Fig. 2 d). The results indicate that the COOH terminus is responsible for the binding of Smad7 to GADD34. Immunoprecipitation experiments further corroborate the mapping results from yeast two-hybrid assays (Fig. 2 e). Together, the results demonstrate that TßRI forms complexes with GADD34 and that TGFß enhances this interaction via Smad7, whose expression induced by TGFß enhances the complex formation (Fig. 2, a and b).
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Discussion |
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It seems that there are three mechanisms by which Smad7 negatively regulates TGFß signaling: (1) mechanical blockage of R-Smad's phosphorylation, (2) proteasomal degradation, and (3) dephosphorylation of TßRI. Smad7 was initially found involved in the regulation of a variety of physiological and pathological processes such as shear stress in the vascular epithelium (Topper et al., 1997; Ishisaki et al., 1998; Kleeff et al., 1999; Nakao et al., 1999). It functions as an intracellular receptor antagonists by binding stably to activated TßRI to prevent phosphorylation of R-Smads. The physical blocking of TßRI requires the interaction between Smad7 and TßRI, which is also the initial step of the PP1c complexmediated dephosphorylation. This mechanism may explain why Smad7 binds to phosphorylated TßRI with a much higher affinity than to dephosphorylated TßRI. TGFß ignites the phosphorylation TßRI with subsequent signaling and induction of Smad7, which further initiates the formation of GADD34PP1 complex for TßRI dephosphorylation. Therefore, it is likely that the phosphorylation state of TßRI regulates the interaction between Smad7 and TßRI. So, dominant-negative GADD34 inhibits the dephosphorylation of TßRI, which leads to a longer stable association of Smad7 with phosphorylated TßRI, inhibiting TGFß signaling. This could be the reason that dominant-negative GADD34 with an absent PP1c binding site appears not efficiently rescue the inhibition of TGFß signaling in transcriptional response and cell cycle assays. This observation is further confirmed by the fact that the blocking of GADD34 expression by RNAi eliminates the effect of dnGADD34 in the UV light irradiation experiment. Physical blockage is only one step of Smad7 inhibition, and each Smad7 molecule can only inhibit one TßRI receptor in the physical blockage model. Whereas, the involvement of PP1c dephosphorylation, Smad7 could inhibit TßRI much more efficiently through enzymatic activity. In the third proteasome degradation mechanism, Smad7 was found to act as an adaptor protein to bind to Smurf2 to form an E3 ubiquitin ligase that targets TßRI for its degradation (Kavsak et al., 2000). This is an irreversible and terminal destruction of TßRI, a different level of regulation.
Because cytokines such as interferon and TNF
also induce Smad7 expression, and TßRI is occasionally phosphorylated by constitutively active type II receptor (TßRII; Ventura et al., 1994; Chen et al., 1995) or other kinases in the absence of ligands (Topper et al., 1997; Ulloa et al., 1999; Zhang and Derynck, 1999; Bitzer et al., 2000), there is a basal level of induced Smad7 expression. Importantly, the interaction between ubiquitin ligase Smurf2 and Smad7 is induced by IFN
. Therefore, the mechanism by which Smad7 targets TßRI for degradation is important for the turnover of TßRI and IFN
-dependent inhibition of TGFß signaling (Kavsak et al., 2000). Our data show this basal level Smad7 still mediates the recruitment of PP1 holoenzyme to minimize the background signaling initiated by random, promiscuous phosphorylation of TßRI, which may in turn be important for maintenance of cell function. Taken together, these data imply that Smad7, in different cellular contexts, differentially regulates cellular activity by a preferential mechanism, although all three mechanisms may act simultaneously to contribute to the final response of the cell.
GADD34 was initially reported to be induced by various types of cellular stress and DNA damage, such as UV light irradiation and unfolded proteins, and its function in overcoming a protein synthesis checkpoint is supported by the fact that the (1)34.5 domain necessary for averting the total shutoff of protein synthesis in herpes simplex virusinfected cells maps to the COOH-terminal domain of the
(1)34.5 protein. This region is highly homologous to the corresponding domains in MyD116 and GADD34 (He et al., 1998). Moreover, GADD34 has been implicated in the dephosphorylation of eIF2
in a negative feedback loop that inhibits stress-induced gene expression and that might promote recovery from translational inhibition in the unfolded protein response (Novoa et al., 2001). The involvement of GADD34 in our proposed complex implies that the regulation of TGFß signaling by this PP1 complex may play an important role in stress-induced cell response. Interestingly, GADD34 and Smad7 expression can simultaneously be induced by UV light irradiation (Hollander et al., 1997; Quan et al., 2001). Furthermore, cellular stress caused by UV light irradiation has been known to confer TGFß resistance in Mv1Lu cells (Quan et al., 2001). UV lightinduced TGFß resistance in Mv1Lu cells is likely attributable to up-regulated expressions of Smad7 and GADD34 and that disruption of this up-regulation will resensitize the cell to TGFß signaling. Blocking expression of GADD34 and Smad7 with RNAi not only restored TGFß signaling in UV lightirradiated cells, but rescued the suppressed expression of downstream gene, PAI-1. Dephosphorylation of TßRI by Smad7-mediated PP1 complex is a quick reversible mechanism and it plays a very important role in regulating TGFß signaling in certain cellular context, such as cellular stress, DNA damage, and induced growth arrest, which further indicates the diversity of cell growth regulation under different cellular context. It will be of substantial interest to investigate the role of our proposed complex in the tumorigenesis of some TGFß-resistant tumors, developmental events, and other TGFß-mediated disorders.
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Materials and methods |
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Immunoprecipitation and immunoblotting
Cells transfected by LipofectAMINE (GIBCO BRL) were lysed with radioimmune precipitation buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl, 1% Nonidet-P-40, 0.5% sodium deoxycholate, 0.1% SDS) containing protease inhibitors (as described above for cell homogenization) and phosphatase inhibitors (10 mM sodium orthovanadate, 1 µM OA, and 50 mM sodium ß-glycerophosphate). Lysates were immunoprecipiated by incubation with the appropriate antibodies, followed by adsorption to protein G Sepharose. Immunoprecipitates were separated by SDS-PAGE, blotted onto a PVDF (Bio-Rad Laboratories) membrane, and visualized by enhanced chemiluminescence (ECL Kit; Amersham Biosciences). For mapping the interaction domains between Smad7 and GADD34 in mammalian cells, a series of different deletion constructs were epitope tagged with Flag or HA and subcloned into pcDNA3. All immunoprecipitation and blotting antibodies were obtained from commercial sources: monoclonal antiFlag M2 and antiß-actin (Sigma-Aldrich), anti-HA (Babco), polyclonal anti-TßRI (Genex Bioscience, Inc.), polyclonal goat anti-Smad7, polyclonal anti-GADD34, and monoclonal anti-PP1 (Santa Cruz Biotechnology, Inc.).
RNAi
To silence endogenous Smad7 and GADD34 expression, single-stranded 21-nt RNAs directed against Smad7 and GADD34 were chemically synthesized and purified (Ambion). The target sequences were 5'-AGGUCACCACCAUCCCCACUU-3' and 5'-GUCAAUUUGCAGAUGGCCAUU-3', respectively. siRNA duplexes were generated and transfected into cells using the SilencerTM siRNA transfection kit (Ambion) according to the manufacturer's instructions. The amount of transfected siRNA was kept constant by addition of scrambled dsRNA provided by the manufacturer.
In vitro phosphorylation and dephosphorylation
GST-TßRI was purified from bacterial lysates by absorption to glutathioneagarose beads as described elsewhere (He et al., 1996). GSTTßRI beads were washed with phosphorylation buffer (25 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 2 mM MnCl2, 0.4 mM EDTA, 1 mM dithiothreitol, 2 mM orthovanadate, 10 mM NaF, 5 mM ß-glycerophosphate, and 10 µM ATP) containing a protease inhibitor mixture (1 mM phenylmethylsulfonyl fluoride and 10 µg/ml antipain, chymostatin, leupeptin, and pepstatin A). 50 µCi of [32P]ATP was then added to the mixture and incubated for 20 min at 30°C with anti-HA precipitates of TßRIIHA-transfected cells and protein GSepharose beads. The protein GSepharose beads and particulate material were pelleted at 14,000 g for 20 min, washed again with dephosphorylation buffer (20 mM Tris-HCl, pH 7.4, 50 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 0.8 mM ATP), and incubated in the same buffer with different precipitates from cells transfected with the indicated genes in the presence or absence of 0.05 µM recombinant PP1 catalytic subunit (
isoform from rabbit; Sigma-Aldrich) or its inhibitors (1.0 µM OA and 50 nM I-1; Sigma-Aldrich). Phosphorylation status was analyzed on an 8.5% SDS-PAGE gel and autoradiography. Phosphatase activity of the precipitates was determined by phosphorylation status of the supposed substrate, TßRIHA.
Metabolic 32P labeling and in vivo phosphorylation state detection
36 h after transfection with different combination of genes, cells were washed twice with phosphate-free DME containing 2% dialyzed fetal calf serum, incubated in the same medium for 4 h, and then labeled with 1 mCi/ml [32P]orthophosphate (PerkinElmer) for an additional 2 h at 37°C in the absence or presence of TGFß-1. The cells were washed again with the same medium and incubated with regular DME/ 2% FBS for another 2 h with or without PP1 inhibitor treatment. The 32P-labeled cells were then washed with ice-cold PBS and lysed with radioimmunoprecipitation assay buffer. TßRIHA was immunoprecipitated with anti-HA as described above. The resultant precipitates were separated by 8.5% SDS-PAGE. Gels were dried and exposed to Biomax Mr or MS film (Eastman Kodak Co.). After autoradiographic analysis, dried gels were rehydrated with transfer buffer, and transferred onto PVDF membranes. For equal loading confirmation, the transfected TßRIHA was visualized by the ECLPlus Western blotting detection system (Amersham Biosciences).
Transcriptional response assay
Mv1Lu cells were transiently transfected either with 3TP-Lux alone or together with indicated constructs using LipofectAMINE (GIBCO BRL) transfection. Total DNA was kept constant by the addition of pcDNA3 plasmid. 24 h after transfection, cells were incubated overnight with or without 4 ng/ml TGFß-1. Luciferase activity was measured using the Dual Luciferase assay kit (Promega) according to the manufacturer's instructions.
Cell cycle analysis
Transfected cells were harvested in PBS containing 0.1% BSA and then washed once in PBS containing 1% FBS, centrifuged, resuspended in 0.5 ml of PBS, and fixed by adding 5 ml of cold absolute ethanol. Fixed cells were stored at 4°C until the time of analysis. Immediately before analysis on the flow cytometer, the fixed cells were centrifuged at 1,600 rpm for 5 min, washed once with PBS/1% FBS, and then incubated at 37°C for 2 h in propidium iodide/RNase A solution (10 µg/ml propidium iodide in 0.76 mM sodium citrate at pH 7.0; 100 ng/ml RNase A in 10 mM Tris-HCl, 15 mM NaCl at pH 7.5) diluted into PBS/1% FBS. Cells were first sorted for cotransfected GFP and then DNA contents were quantified. FACS® sorting was performed on a FACStar® machine and analyzed with CellQuest program.
UV light irradiation
Subconfluent cells were incubated in serum-free medium overnight. The next morning, the media were removed and cells were covered with a thin layer of PBS and irradiated with UV light (20 mJ/cm2) using four FS24T12UVB-HO bulbs. A Kodacel filter was used to eliminate wavelengths below 290 nm (UVC). The irradiation intensity was monitored with an IL400A radiometer and a SED240/UVB/W photodetector (International Light). After irradiation, the PBS was replaced with the original media. Cellular viability 24 h after UV light irradiation was near 100% based on cell morphology and number. For luciferase assay, the day before UV light irradiation, cells were transfected with 3-TP luciferase reporter construct and TGFß-1 (4 ng/ml) was added after UV light irradiation overnight before luciferase activity assay. siRNA was transfected the day before UV light irradiation.
Immunolocalization
After UV light irradiation and ligand stimulation, cells were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 on ice in blocking buffer (10%BSA in PBS), and labeled with antibodies in PBS with 2% BSA. GADD34 was visualized by immunostaining with rabbit antibody against GADD34 (Santa Cruz) and goat antirabbit Texas redconjugated IgG (Amersham Biosciences). For Smad2 translocation observation, TGFß-1 (4 ng/ml) was added for 2 h after UV light irradiation, and phosphorylated Smad2 was visualized by immunostaining with rabbit antibody against phosphorylated Smad2 at 465 and 467 residues (Biosource International) and goat antirabbit fluorescein-conjugated IgG. For PAI-1 induction observation, immunostaining performed with antibodies (rabbit antiPAI-1 from Santa Cruz; goat antirabbit fluorescein-conjugated IgG from Amersham Biosciences) 24 h after UV light irradiation. Digital pictures were taken with an Olympus, IX TRINOC camera under Olympus, IX70 Inverted Research Microscope (Olympus) with objective lenses of Hoffman Modulation Contrast ®, HMC 10 LWD PL FL, 0.3NA /1, OPTICS INC at room temperature, and proceeded with MagnaFire® SP imaging software (Optronics).
Online supplemental material
Cultured cells were homogenized and fractionated as described previously (Chan and Leder, 1996). Fractions were collected and analyzed by Western blotting. Online supplemental material available at http://www.jcb.org/cgi/content/full/jcb.200307151/DC1.
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Acknowledgments |
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This work was supported by grants DK52434, DK57501, and DK60913 from the National Institutes of Health; DAMD17-02-1-0265 from the Department of Defense and sub-2000-408 from NASA (to X. Cao); and P30AR46301 from the Center for Metabolic Disease (to J. McDonald).
Submitted: 23 July 2003
Accepted: 17 November 2003
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