Pin1 Down-regulates Transforming Growth Factor-β (TGF-β) Signaling by Inducing Degradation of Smad Proteins*

Transforming growth factor-β (TGF-β) is crucial in numerous cellular processes, such as proliferation, differentiation, migration, and apoptosis. TGF-β signaling is transduced by intracellular Smad proteins that are regulated by the ubiquitin-proteasome system. Smad ubiquitin regulatory factor 2 (Smurf2) prevents TGF-β and bone morphogenetic protein signaling by interacting with Smads and inducing their ubiquitin-mediated degradation. Here we identified Pin1, a peptidylprolyl cis-trans isomerase, as a novel protein binding Smads. Pin1 interacted with Smad2 and Smad3 but not Smad4; this interaction was enhanced by the phosphorylation of (S/T)P motifs in the Smad linker region. (S/T)P motif phosphorylation also enhanced the interaction of Smad2/3 with Smurf2. Pin1 reduced Smad2/3 protein levels in a manner dependent on its peptidyl-prolyl cis-trans isomerase activity. Knockdown of Pin1 increased the protein levels of endogenous Smad2/3. In addition, Pin1 both enhanced the interaction of Smurf2 with Smads and enhanced Smad ubiquitination. Pin1 inhibited TGF-β-induced transcription and gene expression, suggesting that Pin1 negatively regulates TGF-β signaling by down-regulating Smad2/3 protein levels via induction of Smurf2-mediated ubiquitin-proteasomal degradation.

(BMPs), are multifunctional proteins that regulate proliferation, differentiation, migration, and apoptosis (1). Ligation of TGF-␤ family members by type I and type II serine/threonine kinase receptors transduces the signals to intracellular Smad proteins (2)(3)(4). After TGF-␤ binding, the TGF-␤ type II receptor (T␤R-II) phosphorylates the TGF-␤ type I receptor (T␤R-I), which in turn phosphorylates the TGF-␤-specific receptor-regulated Smads Smad2 and Smad3 at C-terminal SXS motifs. Phosphorylated Smad2 and Smad3 form a complex with Smad4, a common partner Smad. This complex translocates into the nucleus, where it regulates the transcription of target genes in cooperation with transcriptional activators and/or repressors.
Smad proteins possess a conserved N-terminal Mad homology 1 (MH1) domain and a C-terminal MH2 domain connected by a linker region. The MH1 domain binds DNA, whereas the MH2 domain is important for receptor binding, nuclear import, and transcription (5). The linker region contains several serine and threonine residues that can be phosphorylated by kinases. Mitogen-activated protein kinases (MAPKs), which are activated by growth factors and cytokines, modulate Smad signaling (3,4,6,7). In the BMP signaling pathway, extracellular signal-regulated kinase (Erk) phosphorylates the linker region of Smad1 to inhibit Smad1 nuclear accumulation and transcriptional activity (8). Smad1 mutant mice lacking the MAPK phosphorylation sites revealed their importance in BMP signaling (9). In the TGF-␤ signaling pathway, activated MAPKs inhibit TGF-␤ signaling by inducing the cytoplasmic retention of Smad2 and/or Smad3 via phosphorylation of the linker region (8, 10 -12). Conversely, Kamaraju and Roberts (13) reported that phosphorylation of the Smad3 linker region by p38 MAPK was required for maximal transcriptional activation and growth inhibition by TGF-␤.
TGF-␤ family signaling is regulated by proteasomal proteolysis, which is mediated by HECT (homologous to the E6-accessory protein C terminus)-type E3 ubiquitin family ligases, Smad ubiquitin regulatory factor 1 (Smurf1), and Smurf2, a Smurf1related protein (14 -19). Smurf1, which was originally identified as an E3 ubiquitin ligase whose WW domain interacts with the PY motif in the linker region of Smad1 and Smad5, induces degradation of Smads (19). Sapkota et al. (20) reported that phosphorylation of the Smad1 linker region facilitates the interaction with Smurf1 to enhance Smurf1-dependent polyu-biquitination of Smad1. Therefore, phosphorylation of the linker region of receptor-regulated Smads is crucial in the regulation of TGF-␤ family signaling. The molecular mechanism by which phosphorylation of receptor-regulated Smad linker regions exerts these effects remains unclear.
Here we identified Pin1, a peptidyl-prolyl cis-trans isomerase (PPIase), as a novel binding partner for Smad2/3. Pin1 interacted with phosphorylated (S/T)P (p(S/T)P) motifs in the linker regions of Smad2 and Smad3. This interaction inhibited TGF-␤ signaling by down-regulating Smad2/3 protein levels via the induction of Smurf2-mediated ubiquitin-proteasomal degradation. Our findings revealed that Pin1 participates in the complex regulation of the Smad2/3 linker region; thus, altered expression of Pin1 in cancer cells may adversely affect TGF-␤ signaling.
Immunoprecipitation, Immunoblotting, and Ubiquitination Assay-We transfected cDNAs into cells using FuGENE 6 (Roche Applied Science) according to the manufacturer's protocols. After 24 h, cells were lysed in 1% IGEPAL CA630 in lysis buffer and incubated with antibodies for 1.5-16 h at 4°C. Immune complexes were precipitated with protein G-Sepharose beads for 30 min and analyzed by immunoblotting. Endogenous proteins were immunoprecipitated with anti-Smad2/3 antibody, which had been preincubated for 6 h with Dynabeads M-280 sheep anti-mouse IgG (Invitrogen). To assess ubiquitination, 293T cells were preincubated with 2.5 M lactacystin (Wako, Osaka, Japan) 24 h prior to cell harvest to inhibit proteasomal degradation.
Quantification of Immunoblotting Data-We used the Multi Gauge version 3.0 software (Fujifilm, Tokyo, Japan) for quantification of immunoblotting data. All quantification data were obtained from raw image data acquired by LAS-3000 mini luminoimage analyzer.
Luciferase Assay-Cells were transiently transfected with the indicated combinations of promoter-reporter constructs and expression plasmids. The total amount of transfected DNA was adjusted to a constant quantity using empty vector. After 24 -48 h, cells were lysed, and luciferase activity was measured with the Dual-Luciferase reporter system (Promega, Madison, WI). pGL4.75-SV40-hRluc (Promega) was used for normalization. (CAGA) 9 -MLP-Luc2 was generated from (CAGA) 9 -MLP-Luc (27) by replacing the plasmid backbone of pGL3 (Promega) with that of pGL4 (Promega).

RESULTS AND DISCUSSION
Pin1 Interacts with Smad2 and Smad3 but Not Smad4-We previously reported the identification of several Smad2-binding proteins, such as JunB and c-Ski, by yeast-two-hybrid screening of a human cDNA library with full-length Smad2 as bait (29). This screen also determined that Pin1, a PPIase, interacted with Smad2 (data not shown). To confirm the interaction between Pin1 and Smad proteins, we incubated GST-Pin1 with lysates from COS7 cells transfected with Myc 6 -epitope-tagged Smad2 (Myc 6 -Smad2), Myc 6 -Smad3, or Myc 6 -Smad4. GST pull-down served to isolate Pin1; we then assayed for associated Smads by immunoblotting with an anti-Myc antibody. GST-Pin1 bound Smad2 and Smad3 but not Smad4 (Fig. 1A); this interaction was potentiated by co-expression of constitutively active TGF-␤ type I receptor (c.a.T␤R-I).
To determine the site of Pin1 binding to Smad3, we generated various deletion mutants of FLAG-epitope-tagged Smad3 (FLAG-Smad3). In a GST pull-down assay, Pin1 interacted with full-length and the linker ϩ MH2 domain Smad3 but not with the MH2 or MH1 ϩ linker domains of Smad3 (supplemental Fig. S1), suggesting that both the linker and the MH2 domains of Smad3 are important for the Pin1-Smad3 interaction. Pin1 specifically binds the p(S/T)P motifs of substrates through its WW domain (30); there are four conserved (S/T)P motifs in the linker regions of Smad2 and Smad3. These four (S/T)P motifs are phosphorylated by MAPKs (10,12). TGF-␤ stimulation induces phosphorylation of both the C terminus and the linker region via MAPKs activation (7). We confirmed an increase in the phosphorylation of these (S/T)P motifs in the Smad2 and Smad3 linker regions following ectopic expression of constitutively active Ras (RasG12V) or c.a.T␤R-I (supplemental Fig. S2). Ectopic expression of RasG12V and c.a.T␤R-I also synergistically enhanced the interaction of Pin1 with Smad2/3 (supplemental Fig. S3). To determine the contribution of these p(S/T)P motifs to the Smad3-Pin1 interaction, we mutated the Smad3 (S/T)P motifs and examined the interaction of these mutants with Pin1. Single and double point mutants (Smad3T179A, S204A/S208A, S213A) both exhibited reduced binding activity (Fig. 1B). A mutant lacking all four (S/T)P motifs (Smad3-4A) failed to interact with GST-Pin1 (Fig. 1B), suggesting that the four p(S/T)P motifs in the Smad3 linker region are essential for the interaction of Pin1 with Smad3.
We next tested whether Pin1 binds Smad3 in vivo. Myc 6 -Smad3 and either FLAG-tagged wild-type Pin1 (FLAG-Pin1WT) or a FLAG-tagged PPIase mutant of Pin1 (FLAG-Pin1CA) were co-transfected into COS7 cells with RasG12V and c.a.T␤R-I. We detected the interaction of Smad3 and Pin1 by immunoprecipitation followed by immunoblotting. FLAG-Pin1CA and, to a lesser extent, FLAG-Pin1WT interacted with Smad3 in vivo (Fig. 1C). We confirmed the endogenous inter-action of Pin1 with Smad2/3 in the human breast cancer cell line MDA-MB-231 (Fig. 1D).
Pin1 Reduces Smad3 Protein Levels in a Manner Dependent on Its PPIase Activity-The phosphorylation-dependent interactions of Pin1 cause conformational changes in substrates that typically affect stability and function of substrate proteins (31,32). We thus examined the effect of Pin1 on Smad2/3 protein levels. We performed experiments in the presence of c.a.T␤R-I because it enhanced the effect of Pin1 on the Smad3 steadystate level (supplemental Fig. S4). Co-expression of Pin1WT decreased Smad3 protein levels in a dose-dependent manner ( Fig. 2A). Co-expression of Pin1 mutant lacking the WW domain (Pin1WA), which failed to bind to Smads (supplemen-
Pin1 Enhances the Interaction of Smad2/3 with Smurf2 and Induces Smurf2-mediated Ubiquitination-MAPKs inhibit BMP signaling by phosphorylating the linker region of Smad1 to induce Smurf1-mediated ubiquitin-dependent degradation of Smad1 (20). We examined the effect of Smad linker phosphorylation on the interaction of Smurf2 with Smad2 and Smad3. Because Smurf2WT, but not Smurf2CA, a catalytic inactive mutant of Smurf2, induced degradation of Smad2 and Smad3 (supplemental Fig. S8), we performed the experiment using Smurf2CA. Co-expression of either RasG12V or c.a.T␤R-I enhanced interaction of FLAG-Smurf2CA with Myc 6 -Smad2 and Myc 6 -Smad3 (Fig. 3A). Binding enhancement seemed to result from linker phosphorylation as Myc 6 -Smad2/3-4A failed to bind to FLAG-Smurf2CA even in the presence of both RasG12V and c.a.T␤R-I. We confirmed the endogenous interaction of Smad2/3 with Smurf2 in the presence of TGF-␤ and EGF (Fig. 3B). The Smurf2-mediated ubiquitina- tion of Myc 6 -Smad2/3-4A was profoundly reduced (data not shown). Thus, linker phosphorylation of Smad2/3 was required for Smurf2-mediated ubiquitination. We examined the effect of Pin1 on the interaction of Smurf2 with Smad2/3 in MEFs. MEFs deficient in Pin1 (Pin1 Ϫ/Ϫ MEFs) and Pin1 Ϫ/Ϫ MEFs infected with a Pin1-encoding adenovirus (21) were lysed and subjected to immunoprecipitation with an anti-Smad2/3 antibody. Restoration of Pin1 expression in Pin1 Ϫ/Ϫ MEFs enhanced the interaction of Smurf2 with Smad2/3 in the presence of exogenous TGF-␤ and EGF (Fig. 3C). These results suggest that phosphorylation of the Smad linker region enhances the interaction of Smurf2 with Smad2/3. We examined the effect of Pin1 on Smurf2mediated ubiquitination of Smad3 in 293T cells expressing abundant Pin1 (data not shown). We transfected Myc 6 -Smad3, FLAG-ubiquitin (Fig. 3D, FLAG-Ub), and HA-Smurf2 into 293T cells with or without an siRNA specific for Pin1. Smad3 ubiquitination was detected by immunoprecipitation with an anti-FLAG antibody followed by immunoblotting with an anti-Myc antibody. Pin1 knockdown reduced the ubiquitination of Smad3 induced by Smurf2 in vivo (Fig. 3D), suggesting that endogenous Pin1 enhances the ubiquitination of Smad3 by Smurf2.
Pin1 is a member of parvulin family of PPIases that specifically recognizes p(S/T)P motifs. The enzymes catalyze the cis-trans isomerization of bound peptide at a site preceding proline (30,33). Our observations thus suggest that Pin1 induces conformational change in the linker regions of Smad2/3 through binding to the phosphorylated (S/T)P motifs, which likely enables Smurf2 to interact with Smad2/3 via the PY motif and cause their degradation.
The phosphorylation of (S/T)P motifs by proline-directed kinases is a major regulatory mechanism for the control of various cellular processes. Pin1 serves as a post-phosphorylation regulatory factor in multiple signaling pathways. It has been reported that Smurf1 negatively regulates BMP signaling through ubiquitin-dependent degradation of Smad1/5. Smurf1 binds to Smad1 via PY motif, and the interaction is enhanced by phosphorylation of (S/T)P motifs (20). In the study, Sapkota et al. (20) suggested that phosphorylation of (S/T)P motifs enables Smurf1 binding by unmasking the PY motif or by providing additional Smurf1 docking sites that cooperate with the PY motif. In the present study, we suggest that phosphorylation of (S/T)P motifs facilitates binding of Smads to Pin1 and conformational change in Smads by Pin1, and the conformational change induces interaction of Smads with Smurfs. Although we cannot exclude the possibility of other mechanisms by which Pin1 regulates TGF-␤ signaling, down-regulation of Smad proteins would be one of the major mechanisms of inhibition of TGF-␤ signaling by Pin1.
Pin1 is overexpressed in many cancer tissues. Such overexpression often correlates with increases in cyclin D1 and ␤-catenin (34 -37). On the other hand, dysregulation of TGF-␤ signaling is often observed in cancers. Our study adds a new role to Pin1 and provides new insight into the mechanisms by which cancers impair TGF-␤ signaling.