Phosphorylation status at Smad3 linker region modulates transforming growth factor-β-induced epithelial-mesenchymal transition and cancer progression

Abstract

Smad3, a major transcription factor in transforming growth factor-β (TGF-β) signaling, plays critical roles in both tumor-suppressive and pro-oncogenic functions. Upon TGF-β stimulation, the C-terminal tail of Smad3 undergoes phosphorylation that is essential for canonical TGF-β signaling. The Smad3 linker region contains serine/threonine phosphorylation sites and can be phosphorylated by intracellular kinases, such as the MAPK family, cyclin-dependent kinase (CDK) family and glycogen synthase kinase-3β (GSK-3β). Previous reports based on cell culture studies by us and others showed that mutation of Smad3 linker phosphorylation sites dramatically intensifies TGF-β responses as well as growth-inhibitory function and epithelial-mesenchymal transition (EMT), suggesting that Smad3 linker phosphorylation suppresses TGF-β transcriptional activities. However, recent discoveries of Smad3-interacting molecules that preferentially bind phosphorylated Smad3 linker serine/threonine residues have shown a multitude of signal transductions that either enhance or suppress TGF-β responses associated with Smad3 turnover or cancer progression. This review aims at providing new insight into the perplexing mechanisms of TGF-β signaling affected by Smad3 linker phosphorylation and further attempts to gain insight into elimination and protection of TGF-β-mediated oncogenic and growth-suppressive signals, respectively.

Keywords: EMT; Smad3; TGF-β; breast cancer; metastasis.

Figure 1

Smad3 linker phosphorylation by intracellular kinases attenuates transforming growth factor‐β (TGF‐β)‐induced Smad3 transcriptional activity. A, Serine/threonine residues at Smad3 C‐terminal and linker region are depicted together with responsible kinases, including CDK family, ERK, JNK, p38 MAPK, and glycogen synthase kinase‐3β (GSK‐3β). TGF‐β induces Smad3 phosphorylation at three sites in the linker region, threonine 179 (T179), serine 204 (S204), and serine 208 (S208), along with the two C‐terminal residues, serine 423 (S423) and serine 425 (S425). Even in the absence of TGF‐β, the linker region in cancer cells often becomes phosphorylated at a certain level (pink) by intracellular kinases that are constitutively activated, but less weakly as compared to that in the presence of TGF‐β (red). B, In the breast cancer cell line (MCF‐10CA1a.cl1, hereafter CA1a), treatment with a pan‐cyclin‐dependent kinase (CDK) inhibitor flavopiridol lowered levels of TGF‐β‐induced Smad3 linker phosphorylation at T179, S204, and S208. Although CDK family does not directly phosphorylate S204, it can provide a priming site (pS208) for phosphorylation by GSK‐3. GSK‐3 is responsible for phosphorylation at S204. Flavopiridol, therefore, can reduce the level of GSK‐3β‐catalyzed phosphorylation at S204 together with that of CDK‐catalyzed phosphorylation at T179, S208, and S213 (not shown). Level of Smad3 linker phosphorylation is inversely correlated with TGF‐β‐induced Smad3 transcriptional activity. EGF, epidermal growth factor; FGF, fibroblast growth factor; HGF, hepatocyte growth factor; IGF, insulin‐like growth factor; PDGF, platelet‐derived growth factor

Figure 2

Mutation of Smad3 linker phosphorylation sites markedly enhances transforming growth factor‐β (TGF‐β)‐induced epithelial‐mesenchymal transition (EMT) and Smad3 transcriptional activity, whereas mutation of C‐terminal phosphorylation sites blocks all TGF‐β responses. A, Schematic depiction of Smad3 wild‐type (Smad3), Smad3 mutants at the linker phosphorylation sites (EPSM), C‐terminal phosphorylation sites (3SA), and both C‐terminal and linker phosphorylation sites (EPSM/3SA). For the construction of Smad3 mutants, serine and threonine residues are replaced with non‐phosphorylatable amino acids, valine (V) and alanine (A), respectively. B, Adenoviruses constitutively expressing Smad3 (Ad‐Smad3), EPSM (Ad‐EPSM), 3SA (Ad‐3SA), or EPSM/3SA (Ad‐EPSM/3SA) are infected into the breast cancer cell line (CA1a). TGF‐β‐induced Smad3 transcriptional activity is upregulated by infection of Ad‐EPSM as compared to that of Ad‐Smad3, suggesting that Smad3 linker phosphorylation attenuates TGF‐β responses. C, Phase‐contrast pictures of the breast cancer cell line (CA1a) infected with Ad‐Smad3, Ad‐EPSM, Ad‐3SA, and Ad‐EPSM/3SA in the presence (10 ng/mL) or absence of TGF‐β1. Although Ad‐Smad3‐ and Ad‐EPSM‐infected breast cancer cells undergo TGF‐β‐induced EMT, Ad‐EPSM‐infected cells show a far more prominent TGF‐β‐induced EMT as compared to Ad‐Smad3‐infected counterparts. Even in the absence of TGF‐β1, Ad‐EPSM‐infected cells show mild features of EMT. Both Ad‐3SA and Ad‐EPSM/3SA infection completely abrogated TGF‐β‐induced EMT. Scale bar, 50 μm

Figure 3

Smad3 linker phosphorylation negatively regulates transforming growth factor‐β (TGF‐β)/Smad3 signaling. Blockade of Smad3 linker phosphorylation (shown as ××××) by Smad3 mutant at linker phosphorylation sites (EPSM) markedly intensifies TGF‐β‐induced epithelial‐mesenchymal transition (EMT) as well as Smad3 transcriptional activation, indicating that Smad3 linker phosphorylation suppresses TGF‐β responses (shown by a hammer‐headed line under the word Linker). Although the Smad3 pathway plays a principal role in TGF‐β‐induced EMT, cooperation of the non‐Smad3 signaling pathways is indispensable for target gene expression. Transcriptional cofactors that form complexes with Smad3 are activated through Wnt/glycogen synthase kinase‐3β (GSK‐3β), Ras/ERK, p38, JNK, and PI3K/Akt pathways. EMT‐promoting transcriptional cofactors include Snail, Zeb, Twist, β‐catenin, AP‐1, Foxc2, TCF, and SP1. They are required for suppression of epithelial markers, such as E‐cadherin and upregulation of mesenchymal markers, such as vimentin (not shown). Arrow‐ and hammer‐headed lines represent activation and inhibition, respectively, in signaling pathways. Upward arrow‐headed line (red) indicates augmentation of TGF‐induced EMT, metastasis, and invasion by mutation of Smad3 linker phosphorylation sites; CDK, cyclin‐dependent kinase; EGF, epidermal growth factor; FGF, fibroblast growth factor; HGF, hepatocyte growth factor; IGF, insulin‐like growth factor; PDGF, platelet‐derived growth factor

Figure 4

Regulation of transforming growth factor‐β (TGF‐β) signaling by molecules associated with phosphorylated serine/threonine residues at the linker region of Smad3. A, Phosphorylation of Smad3 linker serine/threonine residues by cyclin‐dependent kinase (CDK)8/9 first provides binding sites for peptidyl‐prolyl cis‐trans isomerase NIMA‐interacting 1 (Pin1). Subsequent phosphorylation by glycogen synthase kinase‐3β adds binding sites for neural precursor cell expressed developmentally downregulated gene 4‐like (Nedd4L). Pin1 induces peak TGF‐β responses in a cell context‐dependent way. Nedd4L enhances proteasomal degradation of Smad3 by poly‐ubiquitination. TGF‐β responses, therefore, are first activated and later suppressed by a sequential on‐off switch. B, Pin1 recognizes phosphorylated serine/threonine‐proline motifs, catalyzes peptidyl‐prolyl cis‐trans isomerization of Smad3, and helps promote cell motility and migration concomitantly with N‐cadherin expression in a TGF‐β‐dependent way. C, Pin1 facilitates Smad3 interaction with Smad ubiquitin regulatory factor 2 (Smurf2). Smurf2 induces poly‐ubiquitination and leads to proteasomal degradation of Smad3. Smurf2 has also been shown to induce mono‐ubiquitination at the MH2 domain of Smad3. The mono‐ubiquitination blocks access of Smad3 to the TGF‐β type I receptor and inhibits the formation of active Smad3‐cofactor complexes required for TGF‐β/Smad3 transcriptional activation. D, Poly(rC)‐binding protein 1 (PCBP1) activated by epidermal growth factor (EGF) or its family member TGF‐α binds to the phosphorylated Smad3 linker region. The Smad3‐PCBP1 complex induces alternate exon exclusion on CD44 pre‐mRNA in response to TGF‐β, generating cancer‐promoting CD44s isoforms. Alternative splicing by Smad3 is now known to occur in a wide variety of gene products other than CD44 protein. Arrow‐ and hammer‐headed lines indicate acceleration and suppression, respectively. Colored upward and downward arrow‐headed lines represent augmentation and suppression, respectively

Figure 5

Smad3 linker phosphorylation modulates transforming growth factor‐β (TGF‐β)‐induced epithelial‐mesenchymal transition (EMT), metastasis, and cancer progression mediated through Smad3‐ and non‐Smad3 signaling pathways. Phosphorylated Smad3 linker serine/threonine residues (shown by ○○○○) provide binding sites for peptidyl‐prolyl cis‐trans isomerase NIMA‐interacting 1 (Pin1), neural precursor cell expressed developmentally downregulated gene 4‐like (Nedd4L), Smad ubiquitin regulatory factor 2 (Smurf2), and poly(rC)‐binding protein 1 (PCBP1). Active complexes thus formed regulate transforming growth factor‐β (TGF‐β) signaling and cancer progression. Pin1 enhances TGF‐β responses transiently or promotes cell migration. Nedd4L induces Smad3 proteasomal degradation by poly‐ubiquitination. Pin1 further facilitates the binding of Smurf2 and Smad3, leading to poly‐ubiquitination‐mediated degradation. Smurf2 also suppresses formation of the Smad3 complex with Smad4 by mono‐ubiquitination of Smad3, leading to suppression of TGF‐β signaling. The PCBP1‐Smad3 complex promotes alternative splicing and produces varied gene products, including CD44s, which favor cancer progression and metastasis. Arrow‐ and hammer‐headed lines indicate activation and inhibition, respectively, in signaling networks. CDK, cyclin‐dependent kinase; EGF, epidermal growth factor; FGF, fibroblast growth factor; GSK‐3β, glycogen synthase kinase‐3β; HGF, hepatocyte growth factor; IGF, insulin‐like growth factor; PDGF, platelet‐derived growth factor