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Mol Cell. Author manuscript; available in PMC 2010 May 13.
Published in final edited form as:
Mol Cell. 2009 Nov 13; 36(3): 457–468.
doi: 10.1016/j.molcel.2009.09.043
PMCID: PMC2796330
NIHMSID: NIHMS159570
PMID: 19917253

Ubiquitin ligase Nedd4L targets activated Smad2/3 to limit TGF βsignaling

Sheng Gao,1 Claudio Alarcón,1 Gopal Sapkota,1,5 Sadia Rahman,1,2 Pan-Yu Chen,1 Nina Goerner,3 Maria J. Macias,3 Hediye Erdjument-Bromage,2 Paul Tempst,2 and Joan Massagué1,4
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Associated Data

Supplementary Materials

Summary

TGFβ induces phosphorylation of the transcription factors Smad2 and Smad3 at the C-terminus as well as at an interdomain linker region. TGFβ-induced linker phosphorylation marks the activated Smad proteins for proteasome-mediated destruction. Here we identify Nedd4L as the ubiquitin ligase responsible for this step. Through its WW domain Nedd4L specifically recognizes a TGFβ-induced phosphoThr-ProTyr motif in the linker region, resulting in Smad2/3 poly-ubiquitination and degradation. Nedd4L is not interchangeable with Smurf1, a ubiquitin ligase that targets BMP-activated, linker-phosphorylated Smad1. Nedd4L limits the half-life of TGFβ activated Smads, restricts the amplitude and duration of TGFβ gene responses, and in mouse embryonic stem cells limits the induction of mesoendodermal fates by Smad2/3-activating factors. Hierarchical regulation is provided by SGK1, which phosphorylates Nedd4L to prevent binding of Smad2/3. Previously identified as a regulator of renal sodium channels, Nedd4L is shown here to play a broader role as a general modulator of Smad turnover during TGFβ signal transduction.

Introduction

The cytokines of the transforming growth factor β (TGFβ) family signal through receptor serine/threonine kinases to control cell behavior and fate. These signals are propagated through the transcription factors Smad2 and Smad3 downstream of TGFβ, activin and nodal receptors, and Smads 1, 5 and 8 downstream of bone morphogenetic protein (BMP) receptors. The activated receptors directly phosphorylate Smad proteins at the C-terminal tail sequence SXS, creating a docking site for the shared co-factor Smad4 (Shi and Massagué, 2003). The resulting complex recruits DNA-binding proteins to target responsive gene enhancers, establishing the canonical TGFβ pathway (Feng and Derynck, 2005; Massagué, 2008).

We recently identified a second agonist-induced phosphorylation event that affects key residues in the interdomain linker region of receptor-activated Smad proteins (Sapkota et al., 2007; Sapkota et al., 2006). This event is a general and integral part of the TGFβ and BMP signaling pathways, and it promotes Smad transcriptional action followed by destruction of activated Smad proteins (Alarcón et al., 2009). Agonist-induced Smad linker phosphorylation differs in many respects from phosphorylation of this region in response to pathway antagonists. Acting through mitogen-activated protein kinases (MAPKs), mitogens such as EGF and FGF, and stress signals such as UV irradiation and osmotic stress cause linker phosphorylation of Smad proteins in the cytoplasm, triggering their cytoplasmic retention and proteasome-mediated degradation (Fuentealba et al., 2007; Grimm and Gurdon, 2002; Kretzschmar et al., 1997; Kretzschmar et al., 1999; Pera et al., 2003; Sapkota et al., 2007). As a result, antagonist-induced linker phosphorylation diminishes the capacity of Smad proteins to respond to TGFβ and BMP. In contrast, agonist-induced Smad linker phosphorylation takes place in the nucleus, is mediated by transcriptional cyclin-dependent kinases CDK8 and CDK9, and enhances Smad transcriptional action before triggering Smad turnover (Alarcón et al., 2009).

In the BMP/Smad1 pathway, the phosphorylated Smad1 linker sites recruit Smurf1 to trigger turnover of the activated Smad1 (Sapkota et al., 2007). Smurf1 contains WW protein interaction domains that interact with PPXY (PY) motifs (Ingham et al., 2004). Smad1, 2, 3, and 5 have a PY motif in the linker region. However, binding of Smurf1 to Smad1 additionally requires the phosphorylation of neighboring residues (Sapkota et al., 2007). Surprisingly, as we show here, Smurf1 and the closely related ubiquitin ligase Smurf2 are only minor participants in the recognition of TGFβ-induced linker phosphorylated Smad2/3.

Given the importance of the TGFβ signal transduction pathway, we sought to identify ubiquitin ligases that would specifically recognize the agonist-activated, linker-phosphorylated Smad proteins in this pathway. Our search led to the identification of Nedd4L as the principal ubiquitin ligase that selectively targets activated Smad2/3 for destruction. Nedd4L was previously identified as a regulator of kidney epithelial sodium channels in the distal nephron, by triggering ubiquitin-mediated endocytosis of channel subunits to the lysosome (Russo et al., 2005). Hypomorphic alleles of Nedd4L in human are associated with a reduced ability to down-regulate these sodium channels, which contributes to hypertension (Dahlberg et al., 2007; Russo et al., 2005). The present findings suggest a broader role for this ubiquitin ligase and provide a basis for differential regulation of signal termination in the TGFβ and BMP pathways.

Results

Nedd4L binds to TGFβ-activated Smad2/3

Addition of BMP or TGFβ to HaCaT human keratinocyte cells induced the phosphorylation of Smad1 or Smads2/3 not only at the C-tail but also at the linker region (Figure 1A). In comparison, EGF, UV irradiation (Figure 1A), and osmotic stress (Figure S4) induced only linker phosphorylation. In Smad1, linker phosphorylation creates a binding site for Smurf1, which triggers Smad1 degradation (Fuentealba et al., 2007; Sapkota et al., 2007). Accordingly, RNAi-mediated knockdown of Smurf1 extended the accumulation of tail-phosphorylated (activated) Smad1 following BMP stimulation (Figure 1B). Notably, the knockdown of Smurf1 or of the closely related Smurf2 (Figure S1A) did not prolong the accumulation of activated Smad2 following TGFβ stimulation (Figure 1B), though addition of proteasome inhibitor MG132 did (refer to Figure 5A below). The combined knockdown of Smurf1 and Smurf2 slightly dampened the decay of activated Smad2, suggesting that Smurfs play no major role in this process.

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Identification of Nedd4L as a TGFβ-dependent E3 ubiquitin ligase

(A) HaCaT cells were treated with different stimuli. Whole cell extracts were immunoblotted with the indicated antibodies. (B) HaCaT cells were transfected with small interfering RNA (siRNA) targeting indicated genes and treated with BMP or TGFβ for one hour. The agonists were then removed from the culture medium (zero time point). Whole cell extracts were harvested at indicated time points and immunoblotted with the indicated antibodies. (C) Alignment of the linker regions of the receptor activated Smads (R-Smad) in human (h) and Drosophila (d). The conserved PX(S/T)P MAPK kinase sites, proline-directed kinase sites (S/T)-P, and PPXY (PY) motifs are indicated with boxes of different colors. (D) HeLa-S3 cell extracts were subjected to affinity purification with the indicated bait. Some identified proteins were indicated. Contaminant heat-shock and cytoskeletal proteins (not marked) were also detected. (E) Schematic representation of four most closely related HECT domain E3 ubiquitin ligases. Three functional domains are shown: the N-terminal C2 domain, the protein-binding WW domains that vary in number and position among the proteins, and the C-terminal catalytic HECT domain. The numbers on the right indicate the sequence similarity to the Nedd4L protein when queried with full-length (FL) or WW domains of Nedd4L (NP_001138439) against the UniProtKB/Swiss-Prot database using FASTA program on the EMBL-EBI website. (F) HaCaT cells were treated with TGFβ for 1 h and lysed. The Smad2 immunoprecipitates or whole cell lysates were subjected to western immunoblotting with the indicated antibodies.

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Nedd4L mediates phospho-linker dependent Smad2/3 turnover in the cytoplasm

(A) HaCaT cells stably expressing shRNA targeting Nedd4L or pretreated with proteasome inhibitor MG132 were stimulated with TGFβ for 1 h. TGFβ was then removed from the culture medium and cells were lysed at the indicated time points after ligand removal. Cytosolic and nuclear fractions were immunoblotted with the indicated antibodies. (B) Control HaCaT cells and cells stably expressing an shRNA targeting Nedd4L were immunostained with anti-Nedd4L antibody. DAPI staining was used to visualize the nuclei. (C) Stable HaCaT cells described in (B) were harvested and subjected to cytosolic and nuclear fractionation. Cytosolic (C) and nuclear (N) fractions were immunoblotted for endogenous Nedd4L. Histone 1B and α-Tubulin were used as controls for nuclear and cytosolic fractions, respectively. (D) HaCaT cells were treated with TGFβ. Cytosolic and nuclear fractions of the cells were immunoblotted for endogenous Nedd4L. (E) HaCaT cells stably expressing HA-tagged Nedd4L were treated with TGFβ. Whole cell extract (WCE) or the cytosolic (C) and nuclear (N) fractions were subjected to anti-HA immunoprecipitation. The immunoprecipitates or lysates were immunoblotted with the indicated antibodies. (F) Activated, linker-phosphorylated Smad2/3 is mostly nuclear, but Nedd4L and the Nedd4L-Smad2/3 complex are mostly cytoplasmic.

Smurf1 binding to Smad1 requires the PY motif and the phosphorylation of one or more SerPro sites in the linker region (Figure 1C) (Sapkota et al., 2007). Compared to the linker region of Smad1 and its close homologue Smad5, the linker regions of Smads 2 and 3 have a different arrangement of SerPro and PY motifs (Figure 1C). These differences suggested that the TGFβ-activated, linker-phosphorylated Smads might be recognized by a distinct E3 ubiquitin ligase. To search for this component, a recombinant protein containing the linker region and the C-terminal MH2 domain of Smad3 was expressed in HEK293T cells, purified and coupled to agarose beads. Note that Smad3 protein overexpressed in these cells is heavily phosphorylated at all linker Ser/Thr-Pro sites (Figure S1B). HeLa-S3 cell extracts were incubated with these Smad3-coated beads and the bound proteins were eluted and visualized (Figure 1D). Mass spectrometry analysis of the excised bands identified various known Smad3 binding proteins including the transcription factors Smad4 (Lagna et al., 1996; Wu et al., 2000) and TIF1γ/TRIM33 (He et al., 2006), the DExD/H helicase family member DDX5 (Davis et al., 2008), and the nuclear membrane protein LEMD3/MAN1 (Pan et al., 2005) (Figure 1D). Other proteins identified included translation initiation factor eIF4B, nuclear export factor exportin 6 (XPO6) and, notably, the E3 ubiquitin ligase Nedd4L (Figure 1D).

Nedd4L belongs to the same E3 ubiquitin ligase family as the Smurf proteins. It consists of an N-terminal C2 calcium-binding domain, several WW domains for binding to target proteins, and a C-terminal HECT ubiquitin ligase domain (Figure 1E). Nedd4 is the family member with the highest level of sequence similarity to Nedd4L, followed by Smurf2 and Smurf1. Nedd4L was identified in multiple eluates from Smad3 beads but was not found in similar experiments using Smad1 as bait (data not shown). Co-immunoprecipitation of an endogenous Smad2-Nedd4L complex from TGFβ treated cells showed that this is a TGFβ-dependent interaction (Figure 1F).

Selective binding of Nedd4L and Smurf to linker-phosphorylated Smads

To directly compare the affinity and specificity of these interactions, we performed co-immunoprecipitation experiments using HEK293T cells overexpressing HA-tagged E3 ubiquitin ligases and Flag-tagged Smad proteins. As non-phosphorylated controls we used Smad1 and Smad3 constructs in which the conserved linker phosphorylation sites were eliminated by mutation (Thr to Val, and Ser to Ala; Figure 1C) (Kretzschmar et al., 1999; Sapkota et al., 2007). Protein binding to the resulting Smad1(mut) and Smad3(mut) constructs was considered phosphorylation-independent background binding. Nedd4L bound to Smad3 but not to Smad1, whereas Smurf1 bound to Smad1 and only weakly to Smad3 (Figure 2A; Figure S1C). Binding of Smurf2 to Smad1 or Smad3 was barely above background. Nedd4 did not bind (Figure 2A).

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Specific interaction of Nedd4L with linker-phosphorylated Smad2/3

(A) HEK293T cells were cotransfected with vectors encoding HA-tagged HECT ubiquitin ligases and either wild-type (WT) or linker phosphorylation site mutants (mut) of Flag-Smad3 or Smad1. Whole cell lysates were immunoprecipitated (IP) and subsequently immunoblotted with antibodies as shown. (B) Immobilized GST-Smad3 or GST-Smad1 phosphorylated by CyclinC-CDK8 or CyclinT-CDK9 was incubated with HEK293T cell lysates expressing HA-tagged ubiquitin ligases. Bound proteins were eluted from the beads and immunoblotted with anti-HA antibody. The amounts of GST fusion proteins were shown by Coomassie blue staining. The protein levels of ubiquitin ligases in HEK293T lysates used in (A) and (B) were determined by anti-HA immunoblot in the bottom panel. (C) As in (B), except that immobilized GST-Smad2 protein was phosphorylated in vitro by CyclinT-CDK9 and used as bait. (D) As in (B), immobilized GST-Smad3 protein was phosphorylated in vitro in the presence or absence of ATP and with addition of flavopiridol. (E) Scheme of Smad-E3 ubiquitin ligase interaction induced by TGFβ and BMP signaling. The linker and C-tail phosphorylation are depicted in orange and green, respectively.

To ascertain whether CDK8/9-mediated linker phosphorylation enables the binding of Nedd4L to Smad2/3 (Alarcón et al., 2009), we incubated purified GST-Smad fusion proteins with cyclinC-CDK8 or cyclinT-CDK9 and ATP, and then used the phosphorylated preparation in binding assays. CDK8/9 phosphorylated Smad3 displayed strong affinity for Nedd4L, weak affinity for Smurfs, and no affinity for Nedd4 (Figure 2B). CDK9 also conferred Nedd4L-binding affinity to Smad2 (Figure 2C), but not to Smad3(mut) (Figure S1D). In contrast, Smad1 phosphorylation by CDK8/9 conferred high affinity for Smurf1, low affinity for Smurf2, and no affinity for Nedd4L (Figure 2B). CDK9-induced Smad3-Nedd4L interaction is a phosphorylation dependent event that required ATP and was inhibited by flavopiridol, a CDK8/9 inhibitor (Figure 2D). Thus CDK8/9-mediated linker phosphorylation selectively targets Smad2/3 and Smad1/5 for interaction with Nedd4L and Smurf1, respectively (Figure 2E).

TGFβ induces phosphorylation of Smad2/3 T-PY motif and leads to Nedd4L interaction

We mapped the interaction domains of Nedd4L and Smad3 using a series of expression vectors encoding different fragments of Nedd4L and Smad3. When expressed in HEK293T cells, the second WW domain (WW2) of Nedd4L bound to Smad3 linker region, whereas the other three WW domains, the C2 domain, or the HECT domain did not (Figures 3A; Figure S2A–D).

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TGFβ induces Smad2/3 linker phosphorylation and leads to Nedd4L interaction

(A) Scheme of the Nedd4L-Smad3 interaction. Brackets indicate the critical interacting domains of Nedd4L and Smad3. (B) The dissociation constants of recombinant WW domains and synthetic Smad linker peptides were measured by isothermal titration calorimetry. Data were derived from triplicate experiments. Errors correspond to deviations from theoretical binding curves. (C) HaCaT cells were treated with TGFβ or EGF for the indicated length of time and lysed. Whole cell extracts were analyzed by western immunoblotting with the indicated antibodies. Anti-Smad3 pT179 cross-reacts with Smad2 pT220, allowing analysis of both phosphorylated species. (D) HaCaT cells stably expressing wild-type Flag-Smad3 were treated with TGFβ in the presence of SB431542, flavopiridol, or U0126. Whole cell extracts and Flag immunoprecipitates were immunoblotted with the indicated antibodies. (E) HaCaT cells stably expressing Flag-tagged Smad3, WT or mutant form, were treated with TGFβ or EGF. Whole cell extracts and Flag immunoprecipitates were immunoblotted with the indicated antibodies. (F) Summary of the phosphorylation events on Smad3 upon TGFβ and EGF treatments. The thickness of the arrows and the darkness of the colors indicate the strength of the phosphorylation of the specific sites. T1/2 for each phosphorylation is indicated in minutes.

Mutation of the PY motif (PPGY to AAGY mutation; Smad3(AY) construct) abolished this interaction, as did mutation of the four linker phosphorylation sites in the Smad3(mut) construct (Figure S2D). Using Smad3 constructs with individual mutations in these phosphorylation sites, or with mutation of all these sites but one, we determined that T179 is the only phosphorylation site required for the Smad3-Nedd4L interaction (Figure S3A). This is confirmed by interaction assays using Smad3 truncation mutants and GST fusion proteins (Figure S3B,C).

T179 (T220 in Smad2) lies directly upstream of the PY motif, suggesting that the WW2 domain of Nedd4L specifically recognizes a phosphothreonine-PY (pT-PY) motif in Smad2/3. We measured the affinity of the four individual Nedd4L WW domains for 13-amino acid short synthetic peptides, containing the T-PY motif of Smad2 or Smad3 with either a threonine or a phosphothreonine residue (Figure 3B). Isothermal titration calorimetry analysis revealed a high affinity of the WW2 domain for the pT-PY motif peptides (Kd=7.8 μM and 4.1 μM, respectively). This affinity is among the highest reported to date for a WW-PY domain interaction (Kanelis et al., 2006; Verdecia et al., 2000). The affinity of WW2 for the unphosphorylated T-PY motif was 7- to 15-fold lower (Figure 3B). The Nedd4L WW3 domain also preferentially bound to the phosphorylated T-PY motifs, but with lower affinity than the WW2 domain. The WW1 and WW4 domains bound even more weakly and with no preference for the phosphorylated T-PY motifs (Figure 3B). Interestingly, Smad1 (and Smad5) also contain a conserved T-PY motif (refer to Figure 1C). However, this threonine residue was not phosphorylated in vivo under any of the agonist or antagonist stimuli tested, and it was poorly phosphorylated by CDK8/9 in vitro (data not shown). The Smurf1 WW2 domain binds a synthetic peptide of 13 residues including the T-PY motif of Smad1 with a Kd of 32 μM and the phosphorylated Smad3 pT-PY motif with a Kd of 36 μM. These values agree with the observation that Smurf1 plays a minor role in Smad3 turnover and it requires contacts with the phosphorylated SerPro cluster for targeting Smad1.

Using Smad3 anti-phosphopeptide antibodies that specifically recognize four individual linker phosphorylation sites (Matsuura et al., 2004), we observed that TGFβ addition induced a rapid (t1/2=20 min) and pronounced phosphorylation of T179 shortly after C-tail phosphorylation (t1/2~10 min) (Figure 3C). This was followed by phosphorylation of the SerPro cluster residues S204, S208 and S213 (t1/2=40 min). In contrast, EGF addition induced rapid phosphorylation (t1/2=10 min) of T208 and T213, followed by phosphorylation of T204 and less prominently T179 (t1/2=30 min) (Figure 3C). A similar preference for phosphorylation of S204 and S208 was observed after UV irradiation or NaCl osmotic stress (Figure S4). The anti-Smad3 pT179 antibody cross-reacts with the corresponding residue in Smad2, pT220, and this cross-reaction revealed a rapid TGFβ-induced phosphorylation of this residue as well (Figure 3C).

To further analyze the contribution of these linker sites to the Smad3-Nedd4L interaction, we transduced vectors encoding Flag-tagged Smad3 into HaCaT cells that were stably depleted of endogenous Smad3 by RNAi-mediated knockdown. The addition of SB431542 (SB, an inhibitor of TGFβ type-I receptor kinases) (Inman et al., 2002a) or flavopiridol (Fla, a CDK inhibitor) (Sedlacek, 2001) prevented TGFβ induced linker phosphorylation, whereas only SB431542 blocked C-tail phosphorylation (Figure 3D). U0126 (U01, MEK inhibitor) (Favata et al., 1998) did not inhibit these TGFβ induced Smad3 phosphorylation events. Flavopiridol and SB431542 (Figure 3D) as well as the linker site mutation (Figure 3E) abolished the Smad3-Nedd4L interaction, as determined by co-immuoprecipitation of Smad3 proteins. EGF, which causes a strong MAPK-mediated phosphorylation of the SerPro cluster but a weak phosphorylation of T179, induced a weak Smad3-Nedd4L interaction (Figure 3E). These results indicate that TGFβ-dependent linker phosphorylation preferentially occurs at the threonine residue in the T-PY motif, enabling the recognition of activated Smad2/3 proteins by Nedd4L (Figure 3F).

Nedd4L-dependent poly-ubiquitination of linker-phosphorylated Smad3

To determine whether linker phosphorylation is required for Nedd4L-dependent poly-ubiquitination and turnover of Smad3, we took advantage of the spontaneous phosphorylation and Nedd4L interaction that occur when Smad3 is overexpressed in HEK293T cells. Coexpression with HA-Nedd4L resulted in poly-ubiquitination of Flag-Smad3 but not of a phosphorylation-defective Flag-Smad3 mutant, as determined by detection of epitope-tagged ubiquitin (Figure 4A). Nedd4L(DD), which has a C962A mutation in the HECT domain that renders it catalytically inactive (Debonneville et al., 2001), bound but did not ubiquitinylate Smad3 (Figure 4B; Figure S5). Nedd4L-dependent Smad3 poly-ubiquitination occurred in both the MH1 and MH2 domains (Figure 4C).

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Nedd4L mediates phospho-linker dependent Smad2/3 poly-ubiquitination

(A) HEK293T cells were cotransfected with vectors encoding Myc-tagged ubiquitin, HA-tagged Nedd4L, and Flag-tagged Smad3 (WT or mutant). Whole cell extracts were subjected to anti-Flag immunoprecipitation, followed by immunoblotting with anti-HA and anti-Myc antibodies. (B) As in (A), except that wild-type (WT) or catalytically inactive mutant (DD) form of HA-Nedd4L was cotransfected with WT Flag-Smad3 and Myc-ubiquitin. (C) As in (A), except that full-length (FL), MH1+linker (NL), or linker+MH2 (LC) fragments of Smad3 was cotransfected with WT HA-Nedd4L and Myc-ubiquitin. (D) Wild-type (WT) or catalytically inactive (mut) HA-SCP2 was cotransfected with HA-Nedd4L, Flag-Smad3, and Myc-ubiquitin in HEK293T cells. Whole cell extract and Flag immunoprecipitates were immunoblotted with indicated antibodies. (E) Control HaCaT cells or cells expressing shRNA targeting Nedd4L were transfected with Myc-tagged ubiquitin. 24 h post-transfection cells were stimulated with TGFβ for 1 h. The Smad2/3 immunoprecipitates or whole cell lysates were subjected to western immunoblotting with the indicated antibodies. (F) As in (E), except that HaCaT cells stably expressing Flag-tagged Smad3 in wild-type, AY, or mut forms were used.

Previous work showed that small C-terminal domain phosphatase-2 (SCP2) dephosphorylates the Smad3 linker region (Sapkota et al., 2006). When overexpressed, the wild-type SCP2 but not a catalytically inactive mutant caused a complete loss of Smad3 linker phosphorylation, and abolished the Nedd4L-mediated poly-ubiquitination of Smad3 (Figure 4D). In HaCaT cells, TGFβ stimulated the poly-ubiquitination of endogenous Smad2/3, which was inhibited by RNAi-mediated knockdown of Nedd4L (Figure 4E). Smad3 mutant lacking the PY motif (AY) or the linker phosphorylation sites (mut), did not exhibit the TGFβ dependent poly-ubiquitination, and accumulated at higher levels as tail-phosphorylated forms (Figure 4F).

Nedd4L-dependent turnover of activated Smad3

RNAi-mediated knockdown of Nedd4L in HaCaT cells prolonged the TGFβ-dependent accumulation of linker-phosphorylated and tail-phosphorylated (activated) Smad2 (Figure 5A; refer to Figure 5C for knockdown control and Figure 7A for more time points). This effect was observed in every cell line that we tested (Figure S6A). However, Nedd4L knockdown did not affect the accumulation of tail-phosphorylated Smad1 in response to BMP (Figure S6B). In Nedd4L knockdown cells (Figure S6C), activated Flag-Smad3(mut) accumulated to higher levels in response TGFβ than did activated Flag-Smad3 (Figure S6D). Nedd4L knockdown was almost as effective at preserving activated Smad2 as was the addition of the proteasome inhibitor MG132 (Figure 5A and Figure 7A). Note that these effects were detected in the pool of activated, tail-phosphorylated Smad2/3, but not in the larger pool of total Smad2/3 protein. Collectively, these results suggest that Nedd4L is the major mediator of degradative turnover of TGFβ-activated Smads.

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Nedd4L limits TGFβ/activin signaling in human keratinocyte cells and mouse embryonic stem cells

(A) Control HaCaT cells, cells expressing shRNA targeting Nedd4L, pretreated with proteasome inhibitor MG132, or pretreated with EGF, were stimulated with TGFβ for 1 h. TGFβ was then removed from the culture medium and cells were lysed at the indicated time points. Whole cell extracts were immunoblotted with the indicated antibodies. (B) The Smad2 pTail immunoblots of (A) were quantified using ImageJ. The intensity of each band was normalized to that of the band at zero time point after TGFβ removal. (C) Quantitative real-time PCR (qRT-PCR) analysis of the indicated TGFβ target genes. HaCaT cells expressing control shRNA or shRNA targeting Nedd4L were treated with TGFβ for 3 h, and total RNA was isolated at indicated time points for qRT-PCR analysis. Data show the mean ± S.D of quadruplicates and are representative of three independent experiments. (D) Mouse embryonic stem cells (mESCs) were infected with lentiviruses expressing control shRNA or two independent shRNAs against mNedd4L. These mESCs were treated with activin A or SB431542 for 3 h. Whole cell extracts were immunoblotted with the indicated antibodies. (E) Wild-type mESCs and cells stably expressing an shRNA targeting mNedd4L were immunostained with Nedd4L antibody. DAPI staining was used to visualize nuclei. (F) Control or mNedd4L knockdown mESCs were allowed to differentiate for four days on collagen IV-coated plates in the absence or presence of activin A. Expression levels of activin direct target gene, mLefty1, were measured by qRT-PCR. The bar graphs showed the fold induction by activin in each cell line. Data show the mean ± S.D of quadruplicates and are representative of two independent experiments. (G) Schematic of mouse embryonic stem cell differentiation under serum-free conditions. Addition of activin A specifically induces differentiation towards the definitive endoderm, anterior mesoderm, and axial mesoderm lineages. (H) As in (F), except that the fold induction of specific lineage markers are depicted in the bar graphs.

Nedd4L knockdown or the addition of MG132 increased the level of tail-phosphorylated Smad2 not only in the nucleus of TGFβ-stimulated cells but also in the cytoplasm (Figure 5A). Detection of Nedd4L by immunofluorescence staining of fixed HaCaT cells (Figure 5B) or by western immunoblotting of fractionated cell extracts (Figure 5C; Figure S6C) revealed that most of the Nedd4L protein is located in the cytoplasm. Addition of TGFβ did not affect this localization (Figure 5D). Furthermore, most of the complex between Nedd4L and linker-phosphorylated Smad2/3 was cytosolic (Figure 5E). These results suggest that the Nedd4L interaction with linker-phosphorylated Smads occurs as the pool of activated Smad cycles through the cytoplasm, although rapid cycling of Nedd4L through the nucleus is not ruled out (Figure 5F).

SGK1 phosphorylates Nedd4L and reduces interaction with phospho-Smad3

Flanking the Nedd4L WW2 domain there are two sites (Ser342 and Ser448) for phosphorylation by serum/glucocorticoid regulated kinase 1 (SGK1) (Figure 6A) (Debonneville et al., 2001; Snyder, 2005). SGK1 is a member of the PKB/Akt subfamily of protein kinases, and is regulated transcriptionally and post-translationally by various stimuli (Lang and Cohen, 2001). GST-Nedd4L fusion protein containing four WW domains was directly phosphorylated by SGK1 in vitro (Figure 6B). Ser to Ala mutation of either SGK1 site in Nedd4L reduced the phosphorylation, and mutation of both sites almost completely eliminated the phosphorylation (Figure 6B). SGK1-mediated Nedd4L phosphorylation significantly inhibited Smad3 binding, while Nedd4L double mutant was resistant to such inhibition with the Ser448 site accounting for most of this effect (Figure 6C). SGK1 also inhibited Nedd4L-Smad3 interaction when overexpressed in HEK293T cells, and again the Ser448 site accounted for this (Figure 6D). Knockdown of endogenous SGK1 in HaCaT cells (Figure S7) enhanced TGFβ-dependent Smad3-Nedd4L interaction (Figure 6E). Collectively, these results suggest that Nedd4L-Smad3 interaction is subjected to another regulation mediated by SGK1.

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SGK1 phosphorylates Nedd4L and inhibits Smad3 interaction

(A) Scheme of Nedd4L domains and locations of two potential SGK1 phosphorylation sites. (B) GST-Nedd4L WW proteins, WT or mutant at the indicated residues, were phosphorylated by activated SGK1 in vitro in the presence of 32P labeled ATP, and subjected to electrophoresis, autoradiography, and Coomassie blue staining. (C) Immobilized GST-Nedd4L WW proteins, WT or mutant, were phosphorylated in vitro by activated SGK1 and incubated with HEK293T lysates expressing Flag-Smad3. Bound proteins were eluted from the beads and immunoblotted with anti-Flag antibody. Protein was monitored by Coomassie blue staining. (D) HEK293T cells were cotransfected with vectors encoding Flag-tagged Smad3, HA-tagged Nedd4L with indicated mutations, and HA-tagged SGK1. Whole cell lysates were immunoprecipitated and immunoblotted with antibodies as shown. (E) HaCaT cells stably expressing Flag-tagged Smad3 were transfected with siRNA targeting endogenous SGK1. 24 h post-transfection cells were stimulated with TGFβ. The Flag immunoprecipitates or whole cell lysates were immunoblotted with the indicated antibodies. (F) Model depicting the molecular interactions and possible fates of linker-phosphorylated Smad2/3. Linker phosphorylation sites are depicted with small circles; red, major phosphorylation sites. The PPXY motifs are indicated with green bars.

In summary, TGFβ-activated Smad2/3 undergoes CDK8/9-mediated linker phosphorylation at a highly conserved threonine residue (T220 in Smad2, T179 in Smad3) before the PY motif, creating a docking site for Nedd4L binding, which leads to turnover of the pool of activated Smad2/3 (Figure 6F). SGK1 inhibits Nedd4L from binding to CDK8/9-phosphorylated Smad2/3. Smad2/3 inhibition by mitogen and stress-activated MAPK mainly involves other linker sites and yet unidentified ubiquitin ligases (Figure 6F).

Nedd4L limits TGFβ and activin/nodal signaling in differentiated cells and stem cells

To determine functional impact of Nedd4L on TGFβ signaling we examined the accumulation of tail-phosphorylated Smad2 and various Smad-dependent gene responses in HaCaT cells as a function of time. A 1-h pulse of TGFβ stimulation followed by removal of the ligand led to Smad2 C-tail phosphorylation with a decay t1/2~3h (Figure 7A,B). RNAi-mediated knockdown of Nedd4L or addition of MG132 to the cells markedly delayed this decay (t1/2>6h), whereas addition of EGF slightly accelerated it (t1/2=2h) (Figure 7A,B). The eventual decay in tail-phosphorylated Smad2 in MG132-treated cells (and in Nedd4L knockdown cells) may result from the action of C-tail phosphatases (Lin et al., 2006; Schmierer et al., 2008). In this context, the knockdown of Nedd4L expanded both the amplitude and the duration of three typical TGFβ gene responses, namely, the induction of connective tissue growth factor (CTGF), plasminogen-activator inhibitor 1 (PAI1), and SMAD7 (Figure 7C).

To investigate the influence of Nedd4L on the regulation of cell plasticity by the Smad2/3 pathway we focused on mouse embryonic stem cells (mESCs). mESCs express Nedd4L (Figure 7D,E) and their differentiation potential is highly sensitive to factors of the TGFβ family (Watabe and Miyazono, 2009). Activin addition to mESCs induced Smad2 C-tail phosphorylation, and this effect was magnified in mESCs with stable knockdown of Nedd4L obtained with independent shRNAs (Figure 7D; Figure S8A). Addition of the receptor kinase inhibitor SB431542 inhibited a trace basal level of C-tail phosphorylation (Figure 7D). Notably, Nedd4L knockdown led to elevated induction of the Smad2/3 target genes, Lefty1 and Lefty2 (Figure 7F, and data not shown).

When grown on collagen-IV coated plates in mitogen-poor medium, mESCs differentiate into posterior mesoderm and extraembryonic mesoderm fates. Activin A induces mESCs to differentiate into anterior mesoderm, axial mesoderm and definitive endoderm fates (Figure 7G) (Gadue et al., 2006; Willems and Leyns, 2008). During embryo development these processes are regulated by nodal factors that signal through activin/nodal receptors and Smad2/3 (Watabe and Miyazono, 2009). The increased sensitivity of Nedd4L-depleted mESCs to activation of this pathway was further manifested in a near doubling in the expression of marker genes for definitive endoderm (Sox17, Cxcr4), anterior mesoderm (Gsc, Lhx1) and axial mesoderm (Foxa2, Chrd), but not posterior mesoderm and extraembryonic mesoderm markers (Flk1, Evx1) (Figure 7H; Figure S8B). Collectively, these results suggest that Nedd4L acts to limit Smad signaling in the TGFβ and activin/nodal pathways.

Discussion

Smad2/3 linker phosphorylation events play different roles in different contexts: turnover of activated Smad proteins in the context of TGFβ action, and decrease of Smad signaling capacity in response to antagonists. The present identification of Nedd4L as the ubiquitin ligase that selectively targets activated, linker-phosphorylated Smad2/3 provides insights into the molecular basis for activation-coupled turnover of the central signal transduction component in the canonical TGFβ pathway (Figure 6F).

Three levels of selectivity in the Nedd4L-Smad2/3 interaction

The Nedd4L-Smad2/3 interaction is highly selective and distinct from related processes that target Smad1 in the BMP pathway or target Smad proteins by antagonist-activated pathways. The Nedd4L-Smad2/3 interaction incorporates three levels of selectivity. By depending on an activation-coupled phosphorylation event, Nedd4L distinguishes activated Smad2/3 from ground-state Smad2/3. Secondly, by specifically recognizing a phosphoT-PY motif, Nedd4L discriminates between Smad2/3 phosphorylated by CDK8/9 at this motif in response to TGFβ, and Smad2/3 phosphorylated by MAPKs elsewhere in the linker region in response to antagonistic signals. MAPK-phosphorylated Smad2/3 may be recognized by different ubiquitin ligases. Thirdly, Nedd4L discriminates between agonist-activated Smad2/3 and agonist-activated Smad1 (and presumably Smad5). BMP-induced linker phosphorylation of Smad1 also marks Smad1 for ubiquitin ligase binding and degradation. However, Smad1 is phosphorylated at a cluster of SerPro sites that are recognized by Smurf1 (Sapkota et al., 2007). The different configuration of phosphorylation sites in the linker regions of the TGFβ and BMP activated Smad proteins, and the differential binding properties of Smurf1 and Nedd4L, make these ubiquitin ligases non-interchangeable regulators of TGFβ and BMP signaling.

The basis for this selectivity lies with the second WW domain of Nedd4L and its specific affinity for the pT-PY motif of TGFβ-activated Smad2/3. Our evidence argues that the pT-PY motif is necessary and sufficient for Nedd4L binding to Smad2/3. The Nedd4L WW2 domain is highly conserved among vertebrates, and the Nedd4L-interacting motif of Smad2/3 is conserved through Drosophila. Interestingly, Nedd4L-Smad2/3 interaction is also regulated by phosphorylation of Nedd4L. We show that SGK1 specifically phosphorylates two Ser residues flanking the Nedd4L WW2 domain to decrease the interaction with Smad. SGK1 is regulated transcriptionally and post-translationally by many stimuli, such as glucocorticoid, serum factors, inflammatory cytokines, and TGFβ signaling itself (Lang and Cohen, 2001), providing protential entry points for the integration of these signals. The Nedd4L-Smad2/3 interaction is functionally distinct from a previously reported interaction with the inhibitory Smad family member Smad7, which in complex with Nedd4L targets TGFβ receptors and Smad4 for degradation (Kuratomi et al., 2005; Moren et al., 2005).

The present findings suggest a broader role for Nedd4L than previously thought. Nedd4L controls cell surface expression of kidney epithelial Na+ channels (ENaC) by inducing ubiquitin-mediated endocytosis and lysosome targeting of ENaC subunits (Russo et al., 2005). This interaction involves PY motifs in the cytoplasmic domain of ENaC. Interestingly, this interaction requires the WW3 and WW4 domains of Nedd4L, whereas the WW1 and WW2 domains show no appreciable affinity for ENaC subunits (Fotia et al., 2003; Snyder, 2005). Therefore, Nedd4L interacts with different targets through different WW domains and with different requirements on target phosphorylation. In sum, the Nedd4L-Smad2/3 interaction is a tightly regulated process, with a remarkable requirement for TGFβ-dependent phosphorylation of the linker region, and intriguing structural and biological implications.

Activation-coupled targeting of Smad2/3 by Nedd4L in the TGFβ pathway

TGFβ receptor-mediated phosphorylation of Smad proteins at the C-terminal tail allows their accumulation in the nucleus and their interaction with Smad4. However, full activation of Smad requires linker phosphorylation, which occurs in the context of the Smad transcriptional complex and is mediated by the flavopiridol-sensitive transcriptional kinases CDK8 and CDK9 (Alarcón et al., 2009). Thus linker phosphorylation simultaneously sets Smad2/3 for transcriptional action and for Nedd4L-mediated turnover, directly coupling these two functions.

The mechanisms that reset Smad proteins to the ground state at the end of the TGFβ-activated signaling cycle have been a matter of debate. Previous work provided evidence for ubiquitination/proteasome-mediated degradation of activated Smad2/3 (Lo and Massagué, 1999) as well as for phosphatase-mediated C-tail dephosphorylation of Smad proteins (Inman et al., 2002b; Lin et al., 2006). By identifying Nedd4L as the principal ubiquitin ligase targeting activated Smad2/3, the present work sheds light into this debate. RNAi-mediated depletion of Nedd4L in many human cell lines that we tested augments the accumulation of activated Smad following TGFβ stimulation as much as proteasome inhibition with MG132 does. This occurs without a detectable increase in the level of total Smad2/3, which is consistent with cell fractionation studies showing that tail-phosphorylated, nuclear Smad2/3 represents a small fraction of the total Smad2/3 pool in TGFβ-treated cells (Schmierer and Hill, 2005; Xu et al., 2002). Our evidence indicates that Nedd4L is predominantly localized in the cytoplasm. Activated, linker-phosphorylated Smad2/3 may encounter Nedd4L after dissociating from transcriptional complexes and shuttling back to the cytoplasm. We also note that even with Nedd4L knockdown or MG132 treatment, the level of tail-phosphorylated Smad2/3 eventually drops, likely reflecting the action of Smad phosphatases (Inman et al., 2002b; Lin et al., 2006).

Evidence that Nedd4L acts to constrain the signaling capacity of the TGFβ pathway is provided by the exaggerated TGFβ responsiveness of Nedd4L-depleted cells. RNAi-mediated depletion of Nedd4L in a human keratinocyte cell line augments not only the accumulation of tail-phosphorylated Smad2/3 in response to TGFβ, but also the amplitude and duration of typical TGFβ gene responses including the induction of CTGF, PAI1 and SMAD7. In mouse ES cells, Nedd4L depletion similarly increases the accumulation of tail-phosphorylated Smad2/3 and the induction of Lefty1 and Lefty2 in response to activin. In mouse ES cells these effects translate into a more robust induction of endodermal and anterior and axial mesodermal fates by activin.

Based on the present and previous evidence, it appears that the return of the TGFβ signal transduction machinery back to the ground state is mediated by a combination of Smad dephosphorylation and degradation processes. What determines the use of one process over the other in a particular cell context, and what specific roles each mechanism plays in physiological settings remain as important questions for future elucidation.

Experimental Procedures

Plasmids and antibodies

Bacterial and mammalian expression constructs encoding Smad1, Smad2, Smad3, Smurf1, and SCP2 were described previously (Kretzschmar et al., 1997; Kretzschmar et al., 1999; Sapkota et al., 2007; Sapkota et al., 2006). Note that the linker phosphorylation site mutant forms of Smad proteins, Smad1(mut) and Smad3(mut), were previously described as Smad1(EPSM) (Sapkota et al., 2007) and Smad3(EPSM) (Kretzschmar et al., 1999), respectively. Smad1, Smad1 pLinker (pS206), and Smad4 antibodies were previously described (Sapkota et al., 2007). Antibodies recognizing Smad3 pT179 (also recognizes Smad2 pT220 due to sequence similarity), pS204, pS208, pS213 were generous gifts from F. Liu (Rutgers).

Affinity purification of Hela-S3 extracts and protein identification

HEK293T cells were cultured in 150-mm dishes and transfected with pCI-Flag-Smad3(L+MH2) using Lipofectamine (Invitrogen). 48 h post-transfection cells were lysed and sonicated in lysis buffer (Sapkota et al., 2007). The cell lysates were cleared and Flag-tagged bait proteins were recovered on Flag-agarose beads by incubating at 4°C for 2 h. One liter of HeLa-S3 cells (National Cell Culture Center) was lysed by sonication in 3mL of lysis buffer and precleared with Flag-agarose beads. The supernatant was incubated with the Flag-agarose beads prebound with bait proteins at 4°C for 4 h. The bound proteins were eluted with Flag peptide (Sigma) following the supplier’s instructions, and were subjected to SDS-PAGE and Coomassie blue staining. Visible bands were excised from the gel and proteins identified by peptide mass fingerprinting coupled with mass spectrometric sequencing of selected peptides using matrix-assisted laser-desorption/ionization reflectron time of flight mass spectrometry (MALDI-reTOF MS) and MALDI-TOF/TOF (MS/MS) analysis as previously described (Winkler et al., 2002).

Mouse embryonic stem cell culture and differentiation

Mouse E14Tg2a feeder-free ES cells (mESCs) were cultured on gelatin-coated dishes in MEM medium (Invitrogen) supplemented with 10% FBS (Hyclone), 1mM sodium pyruvate (Invitrogen), 1% non-essential amino acids (Invitrogen), 2mM L-Glutamine (Invitrogen), 100unit/ml Penicillin/Streptomycin (Invitrogen), 1μg/ml Fungizone (Invitrogen), 0.1mM β-mercaptoethanol (Sigma), and 1000unit/mL LIF (Millipore).

To induce differentiation, mESCs were seeded on collagen IV-coated 6-well plates (BD) at 20,000/well in serum-free medium (SFM) containing N2 and B27 supplements (Gadue et al., 2006). mESCs were allowed to differentiate in the presence or absence of activin A (20ng/mL, R&D) for 4 days. Cells were then harvested for qRT-PCR analysis.

Other assays

Cell transfection, immunoprecipitation and western immunoblotting were performed as previously described (Sapkota et al., 2007). For immunostaining, HaCaT cells and mESCs were fixed in 4% PFA and immunostained with the indicated antibodies as previously described (Xu et al., 2002). Quantitative real-time PCR was carried out as previously described (Xi et al., 2008). The primer sequences for human genes were previously described (Gomis et al., 2006). The primer sequences for mouse genes are included in Supplementary Table 1. Other experimental procedures, including RNAi and isothermal titration calorimetry, are included in the Supplemental Experimental Procedures section.

Supplementary Material

01

Acknowledgments

We thank F. Liu and O. Staub for generous gifts of antibodies, Q. Xi for advice on the mESC experiments, A.I. Zaromytidou for critical reading of the manuscript, X. Jiang, W. He and D. Marenstein for reagents, E. Aragón for protein preparation, R. Prohens for help in setting up the ITC experiments, L. Fabrizio for help with mass spectrometry, and members of the Massagué laboratory for valuable discussions. We are grateful to Molecular Cytology Core Facility of MSKCC for technical help. This research was supported by NIH grant CA34610 (JM), CA08748 (PT), BFU2005-06276 and BFU2008-02795 (MJM). JM is an investigator of the Howard Hughes Medical institute.

Footnotes

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