Posttranslational Regulation of Smads

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Pinglong Xu,1 Xia Lin,2 and Xin-Hua Feng1,2,3

1Life Sciences Institute and Innovation Center for Cell Signaling Network, Zhejiang University, Hangzhou, Zhejiang 310058, China 2Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, Texas 77030 3Department of Molecular & Cellular Biology, Baylor College of Medicine, Houston, Texas 77030 Correspondence: [email protected]

Transforming growth factor b (TGF-b) family signaling dictates highly complex programs of gene expression responses, which are extensively regulated at multiple levels and vary depending on the physiological context. The formation, activation, and destruction of two major functional complexes in the TGF-b signaling pathway (i.e., the TGF-b receptor complexes and the Smad complexes that act as central mediators of TGF-b signaling) are direct targets for posttranslational regulation. Dysfunction of these complexes often leads or contributes to pathogenesis in cancer and fibrosis and in cardiovascular, and autoimmune diseases. Here we discuss recent insights into the roles of posttranslational modifications in the functions of the receptor-activated Smads in the common Smad4 and inhibitory Smads, and in the control of the physiological responses to TGF-b. It is now evident that these modifications act as decisive factors in defining the intensity and versatility of TGF-b responsiveness. Thus, the characterization of posttranslational modifications of Smads not only sheds light on how TGF-b controls physiological and pathological processes but may also guide us to manipulate the TGF-b responses for therapeutic benefits.

he transforming growth factor b (TGF-b) main of the receptor upon TGF-b1 binding Tfamily consists of structurally related poly- provides the basis for signal transduction. Li- peptide dimers that are involved in a diversity of gand binding stabilizes the interaction of TGF- physiological processes in all metazoans. They b type II receptor (TbRII) dimers with dimers regulate cell functions, cell and tissue differen- of the TGF-b type I receptor, TbRI, also called tiation, and morphogenesis. As a result, dereg- activin receptor-like kinase 5 (ALK-5), enabling ulation of TGF-b family activities leads to or TbRII to phosphorylate serines and threonines contributes to the pathogenesis of cancer and in the short juxtamembrane Gly-Ser (GS) mo- ﬁbrosis, cardiovascular and autoimmune dis- tifs of TbRI receptors (Souchelnytskyi et al. eases, and developmental defects. 1996). This phosphorylation event triggers con- The signal transduction pathway activated formational changes in TbRI that allow a sur- by TGF-b family proteins appears quite face of TbRI to interact with a basic patch of the straightforward. Viewing TGF-b1 as the proto- Mad homology 2 (MH2) domain of the recep- type, phosphorylation in the cytoplasmic do- tor-regulated Smads (R-Smads) and phosphor-

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P. Xu et al.

ylation of R-Smads by TbRI on distal two ser- activated in response to stimulation of TGF-b, ines of the conserved carboxy-terminal SSXS nodal, and activin, whereas Smad1, Smad5, motif (Huse et al. 2001; Shi and Massague´ and Smad8 respond to bone-morphogenetic 2003). Subsequently, two phosphorylated R- proteins (BMPs) and growth and differentia- Smads form a heterotrimeric complex with tion factors (GDFs) (Heldin and Moustakas the common Smad4 (co-Smad). The resulting 2012). Characterized by a highly conserved ami- trimeric complex is rapidly translocated into no-terminal MH1 and carboxy-terminal MH2 the nucleus where it elicits transcriptional re- domains, and separated by a variable serine- sponses (Feng and Derynck 2005; Massague´ and proline-rich linker region, these R-Smads et al. 2005; Schmierer and Hill 2007). The act as main effectors for TGF-b family signaling Smad complex associates with a wide range of (Shi and Massague´ 2003). Like many key signal- DNA-binding transcription factors and cofac- ing mediators, the expression level and activity tors (e.g., coactivators and corepressors) to de- of R-Smads are under stringent control, partic- liver diverse transcriptional responses on the ularly through PTMs including (de)phosphor- DNA in a context-dependent manner. Besides ylation, (de)ubiquitylation, sumoylation, (de)- the canonical Smad-dependent pathway, ligand acetylation, methylation, and ADP-ribosylation binding to cognate receptor complexes also (Fig. 1). leads to activation of non-Smad signaling pathways, such as mitogen-activated protein kinase (MAPK) pathways and the phosphoinositide 3- kinase (PI3K)-Akt-mTOR pathway (Derynck Phosphorylation and Dephosphorylation and Zhang 2003). These non-Smad pathways of R-Smads play an important role in some TGF-b-regulat- Phosphorylation of the SSXS Motif by TGF-b ed processes, such as the generation of regula- Type I Receptor and Other Kinases tory Treg (T) and TH17 cells (Korn et al. 2009). All signaling mediators in the TGF-b path- R-Smads are activated through ligand-induced way are subject to extensive posttranslational phosphorylation by the type I receptors of modifications (PTMs). Taking TGF-b receptors two serines in their carboxy-terminal SSXS mo- as examples, a variety of modifications have tif. The TGF-b-specific type I receptor TbRI/ been identified in recent years. These include ALK-5 phosphorylates Smad2 and Smad3, phosphorylation (Lawler et al. 1997; Galliher whereas the BMP-specific type I receptors and Schiemann 2007; Lee et al. 2007b), ubiqui- ALK-3/BMPRIA (BMP receptor, type IA) and tylation, sumoylation (Kang et al. 2008), ned- ALK-6/BMPRIB phosphorylate Smad1, 5, and dylation (Zuo et al. 2013), and ectodomain 8. This phosphorylation leads to the dissocia- shedding (Atfi et al. 2007; Liu et al. 2009a; Xu tion of R-Smads from their type I receptors, and and Derynck 2010; Mu et al. 2011), which con- homo- or heterodimerization of phosphorylat- tribute to the spatial and temporal regulation in ed R-Smads. After association of two R-Smads TGF-b responsiveness and function in distinct with Smad4, the R-Smad/Smad4 complexes contexts. PTMs of TGF-b receptors have been translocate into the nucleus, in which they elicit reviewed elsewhere (Xu et al. 2012). We now transcriptional responses by interacting with a focus our discussion on the current knowledge variety of transcription factors and transcrip- of the dynamic regulation of distinct Smads by tional coregulators. PTMs, and its pivotal roles in controlling TGF- In addition to this canonical model of TGF- b family functions. b signaling through Smad2 and Smad3, some TGF-b family ligands were shown to initiate unexpected phosphorylation of a noncanonical POSTTRANSLATIONAL MODIFICATIONS R-Smad. Specifically, in addition to the ex- OF R-Smads pected Smad2 and Smad3 activation, TGF-b The eight Smads identified in vertebrates com- induces Smad1 and Smad5 phosphorylation. prise five R-Smads. Smad2 and Smad3 are In endothelial cells, the TGF-b-specific type I

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PP P Ac PPPP PAK2 TβR1 ERK p300 CaMK II GRK2 ERK CaMK II PAK2 P/CAF CDK8/9 Araf PAK4 ERK PPM1A MPK38 MTMR4 Figure 1. Defined modifications and modifying enzymes of Smad2 and Smad3. Domain structures are indicated. Blue triangles represent modifications that promote Smad signaling, whereas red triangles (upside down) represent modifications that attenuate Smad signaling. 3 Downloaded from http://cshperspectives.cshlp.org/ on September 26, 2021 - Published by Cold Spring Harbor Laboratory Press

P. Xu et al.

receptor TbRI/ALK-5 as well as the BMP family type I TGF-b family receptor kinases. For exam- type I receptor ALK-1 are essential for TGF- ple, Mps1, a kinase that regulates mitotic pro- b-induced Smad1 and Smad5 phosphoryla- gression, can phosphorylate the SSXS motif tion, together with the accessory receptor en- of Smad2 and Smad3, although it is unclear doglin (Goumans et al. 2002, 2003; Lebrin whether it activates the Smad pathway (Zhu et al. 2004). Although TbRI/ALK-5-mediated et al. 2007). The oncogenic kinase p21 pro- Smad2 and Smad3 activation leads to inhibition tein-activated kinase 4 (PAK4) can phosphory- of cell migration and proliferation, ALK-1-me- late Ser465 in the SSXS motif of Smad2, which diated Smad1 and Smad5 activation results in leads to Smad2 proteasomal degradation. Ac- opposite effects. Therefore, a fine balance be- cordingly, a marked increase in phospho-S465 tween TbRI/ALK-5 and ALK-1 signaling de- Smad2 level, concomitant with a lower total fines the roles of TGF-b in endothelium (Gou- Smad2 protein level, has been observed in gas- mans et al. 2002, 2003; Lebrin et al. 2004). A tric cancer tissues (Wang et al. 2014a). The similar scenario is also observed in dermal fi- physiological significance of R-Smad phos- broblasts, in which TGF-b stimulates Smad1 phorylation and activation by these kinases re- phosphorylation and Smad1-mediated tran- mains to be further explored. scription of collagen and other target genes, depending on both TbRI/ALK-5 and ALK-1, Dephosphorylation of the SSXS Motif and activation of extracellular signal regulated kinases 1 and 2 (Erk1/2) MAP kinases (Pannu With the carboxy-terminal SSXS phosphoryla- et al. 2007). Additionally, TbRI/ALK-5 and tion of R-Smads as the first essential step in canonical Smad1 activation by BMP family activating Smad-mediated transcription, some type I receptors are required for TGF-b-induced mechanisms inhibit the activation of R-Smads Smad1 and Smad5 phosphorylation in kerati- to terminate R-Smad functions. These include nocytes and fibroblasts. The phosphorylation phosphatase-mediated dephosphorylation, ubiq- of Smad1 and Smad5 endows carcinoma cells uitylation-dependent degradation, and nuclear with a motile and invasive phenotype, and an- export of R-Smads. A low level of continuous chorage-independent growth (Bharathy et al. dephosphorylation allows for disassemblyof the 2008; Daly et al. 2008). More strikingly, TGF- Smad complex and subsequent nuclear export b-induced Smad1 and Smad5 phosphorylation of R-Smads (Inman et al. 2002; Xu et al. 2002b). was shown to be independent of BMP receptors Several phosphatases mediate phospho-SSXS and essential for the TGF-b-induced migration dephosphorylation. PPM1A (protein phos- þ þ of mammary epithelial cells (Liu et al. 2009b; phatase, Mg2 /Mn2 -dependent, 1A)/PP2Ca Wrighton et al. 2009b). These studies there- (protein phosphatase-2Ca), the prototype of fore reveal the functional significance of the PPM family phosphatases, preferentially binds cross talk between TGF-b ligand and the nor- and dephosphorylates the phospho-SSXS mo- mally BMP-regulated R-Smads, and the roles of tif of not only Smad2 and Smad3, but also of TGF-b-induced Smad1 and Smad5 phosphor- the BMP-specific Smad1 and Smad5 (Duan ylation in a cell type-dependent manner, be- et al. 2006; Lin et al. 2006). PPM1A limits the cause TGF-b stimulates, but does not inhibit, duration of the R-Smad activity in the nucleus the proliferation of endothelial cells under cer- in both the TGF-b- and BMP-activated Smad tain conditions (Goumans et al. 2003), and pathways, and thus attenuates TGF-b- and TGF-b appears to be essential for anchorage- BMP-regulated transcriptional responses, and independent growth of epithelial cells (Daly TGF-b/nodal-stimulated effects on cell growth et al. 2008). arrest and early embryogenesis of zebrafish In addition to the canonical phosphoryla- (Duan et al. 2006; Lin et al. 2006). tion by type I receptor kinase, the carboxy-ter- Interestingly, phosphatase and tensin ho- minal SSXS motif of R-Smads can also be phos- molog (PTEN), a negative regulator of the phorylated by other kinases distinct from the PI3K/Akt pathway, serves as a cofactor of

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Posttranslational Regulation of Smads

PPM1A to enhance the abrogation of Smad2/3 forTGF-bsignalingandTGF-b-inducedgrowth phosphorylation by stabilizing PPM1A, sug- inhibition of epithelial cells (Shukla et al. 2009). gesting an intriguing cross talk between sphin- Dephosphorylation and subsequent disas- golipid and TGF-b signaling pathways (Bu et al. sembly of the Smad complexes often lead 2008). MAN1, also known as LEMD3, an inte- to nuclear export of R-Smads (Inman et al. gral protein of the inner nuclear membrane, 2002; Xu et al. 2002a; Shi and Massague´ 2003; associates with PPM1A in the dephosphory- Schmiererand Hill 2005). A mathematical model lation of Smad2 and Smad3 (Bourgeois et has been proposed for how Smad phosphoryla- al. 2013). Inactivation of the gene encoding tion and dephosphorylation, complex forma- MAN1/LEMD3 leads to developmental anom- tion, and nucleocytoplasmic shuttling work alies in a mouse model, whereas heterozygous together to transduce TGF-b signals (Schmierer loss-of-function mutations in humans cause et al. 2008). The nuclear transport cofactor Ran- sclerosing bone dysplasia (Bourgeois et al. binding protein 3 (RanBP3) directly recognizes 2013). In addition, the dual-specificity protein the dephosphorylated Smad2 and Smad3, and phosphatase MTMR4, identified as a phospha- mediates their nuclear export in a Ran-depen- tase that dephosphorylates PI3P, was also re- dent manner to inhibit TGF-b responses in ported to dephosphorylate the phospho-SSXS mammalian cells and Xenopus embryos (Dai motif of Smad2 and Smad3 in endosomes and et al. 2009). RanBP3L was found to be respon- to repress the TGF-b response (Yu et al. 2010). sible for recognition and nuclear export of Using RNAi-mediated gene silencing screening, Smad1, Smad5, and Smad8, which regulate the protein pyruvate phosphatase (PDP) (Chen osteogenesis of mesenchymal stem cells (Chen et al. 2006) and small carboxy-terminal domain et al. 2015). Remarkably, PPM1A can also de- phosphatases (SCPs) (Knockaert et al. 2006) phosphorylate RanBP3 to enable efficient nu- were found to increase the carboxy-terminal clear export of Smad2 and Smad3 (Dai et al. phosphorylation of Drosophila Mad, the homo- 2011). Besides nuclear export of dephosphory- log of Smad1 and Smad5, or Smad1. Therefore, lated R-Smads, activated R-Smads also undergo the expression of PDPenhances decapentaplegic faster degradation in the nucleus (Lo and Mas- (Dpp)- or BMP-stimulated target gene expres- sague´ 1999; Izzi and Attisano 2004; Mavrakis sion, whereas the expression of SCPs leads to et al. 2007). induction of a secondary dorsal axis in Xenopus The complexity of phosphorylation and de- (Knockaert et al. 2006). However, ectopically phosphorylation of the SSXS motif by multiple increased expression of mammalian PDPs or kinases and phosphatases highlights the critical SCPs does not affect phospho-Smad1 dephos- role of SSXS phosphorylation in TGF-b family phorylation (Duan et al. 2006). Another SCP signaling. Further studies are required to better family phosphatase, SCP4/CTDSPL2, physical- define how the Smad kinases and phosphatases ly interacts with and dephosphorylates Smad1, exert their activities and how these processes are Smad5, and Smad8, thus attenuating BMP-in- controlled by cellular contexts. duced transcriptional responses and osteogenic differentiation in C2C12 cells (Zhao et al. 2014). Linker Region Phosphorylation of R-Smads Phosphatase PP2A under hypoxic conditions can dephosphorylate the phospho-SSXS motif Besides rapidly inducing phosphorylation of R- of Smad3, but not Smad2, thus suggesting a Smads at their carboxy-terminal SSXS motif, mechanism by which a hypoxia condition regu- TGF-b also induces linker phosphorylation, at lates TGF-b responses (Heikkinen et al. 2010b). three other sites, in Smad3 but with a slower Remarkably, CLIC4, a multifunctional pro- kinetics (Millet et al. 2009). The linker region tein that shuttles between the cytoplasm and nu- of R-Smads, that is, the less conserved sequence cleus, can associate with activated Smad2 and between the amino-terminal MH1 domain and Smad3 to protect them from dephosphorylation the carboxy-terminal MH2 domain, can also be of their phospho-SSXS motif, which is required phosphorylated on Ser/Thr in response to oth-

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P. Xu et al.

er growth factors and by various kinases, which hormone (PTHLH), which promotes osteoclast consequently regulate R-Smad activities (Der- maturation for osteolytic colonization (Wang ynck and Zhang 2003; Feng and Derynck 2005). et al. 2014b). As a result, silencing the expres- Given the flexible nature of the R-Smad linker sion of DLC1 in cancer cells promotes bone region, different phosphorylation patterns may metastasis and leads to osteolysis and accelerat- impose conformational interfaces that may af- ed death in mice. Additionally, a with no lysine fect protein–protein interactions and functions (WNK) family kinase WNK1, originally identi- of both MH1 and MH2 domains (Sapkota et al. fied as a kinase upstream of Erk5 MAPK (Lee 2007). Mitogens and hyperactive Ras lead to et al. 2007a), and murine protein serine-threo- Erk1/2-mediated linker phosphorylation of nine kinase 38 (MPK38) (Seong et al. 2010), Smad3 at Ser204, Ser208, and Thr179. Muta- also directly phosphorylate Smad2. Thus, de- tion of these sites often promotes activation pletion of WNK1 increases the accumulation of target genes by Smad3, suggesting that the of activated Smad2 in the nucleus and enhances MAPK-mediated Smad3 linker phosphoryla- the transcription of TGF-b target genes, where- tion is inhibitory for Smad3 activity (Kretzsch- as knockdown of MPK38 inhibits TGF-b mar et al. 1999; Lehmann et al. 2000), although signaling, and TGF-b-induced apoptosis and oppositely stimulating effects have also been re- growth arrest in epithelial cells (Seong et al. ported (Lehmann et al. 2000). Studies in mice 2010). GRK2, a kinase that together with b-ar- carrying mutations in the potential MAPK restins desensitizes many G protein–coupled phosphorylation sites of Smad1 also support receptors by regulating their internalization an inhibitory role of linker phosphorylation and trafficking, also associates with Smad2 and on Smad1, because mice bearing these mu- Smad3 and phosphorylates their linker regions tants show defects in gastric epithelial homeo- (Ho et al. 2005). GRK2-mediated linker phos- stasis (Aubin et al. 2004). Additional kinases, phorylation inhibits TGF-b ligand-induced car- for example, casein kinase (CK) 1g2, CK11, cal- boxy-terminal SSXS phosphorylation through cium/calmodulin-dependent protein kinase II an as-yet-unknown mechanism, and prevents (CaMK II), protein kinase C (PKC), and MAP nuclear translocation and transcriptional activ- kinase kinase-1 (MEKK-1), also phosphory- ity of the Smad complex, as well as TGF-b-stim- late the R-Smad linker regions and down-regu- ulated apoptosis and growth inhibition in pri- late Smad-dependent transcriptional responses mary hepatocytes (Ho et al. 2005). Additionally, (Wrighton and Feng 2008). In contrast, phos- a Raf family member of kinase, Araf, was also phorylation of the linker region of Smad3 by found to phosphorylate Ser253 in the linker re- p38 MAPK and Rho-associated, coiled coil ki- gion of Smad2 and to attenuate Smad2 signaling nase (ROCK) (Ser204, Ser208, and Ser213), or by accelerating its degradation (Liu et al. 2013). by c-Jun amino-terminal kinase (JNK) (Ser208 Thus, silencing Araf expression in zebrafish re- and Ser213), enhances the transcriptional activ- sults in increased accumulation of mesendo- ity of Smad3 to generate a more robust TGF-b derm precursors and a dorsalized phenotype. response (Engel et al. 1999; Mori et al. 2004; In the nucleus, Smad3 is phosphorylated by Kamaraju and Roberts 2005). All of these data the cyclin-dependent kinases (CDK)2 and illustrate the direct cross talk between the TGF- CDK4 at Thr8, Thr179, and Ser213. This phos- b/Smad signaling pathway and other signaling phorylation inhibits the transcriptional acti- pathways. vity of Smad3 and the antiproliferative effect Furthermore, deleted in liver cancer 1 of TGF-b (Matsuura et al. 2004). It thus con- (DLC1), a StAR-related lipid transfer protein, fers cancer-cell resistance to TGF-b-induced whose gene is frequently inactivated in tumor growth-inhibitory effects. Remarkably, a sig- cells and is seen as a candidate tumor suppressor nificant increase in Ser208/Ser213 phosphory- gene, blocks Rho-ROCK-mediated Smad3 link- lation of Smad3 is associated with late stage er phosphorylation and inhibits the TGF-b-in- colorectal tumors, indicating that Smad3 linker duced expression of parathyroid hormone-like phosphorylation may contribute to the tumor-

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Posttranslational Regulation of Smads

promoting role of TGF-b (Yamagata et al. 2005). con et al. 2009; Aragon et al. 2011). The switch Taken together, the linker region of R-Smads is from initial activation to subsequent destruc- emerging as an important and critical regulatory tion appears to result from the recognition of platform for fine-tuning of TGF-b responses Smad phosphoserines by WW (Trp-Trp) do- and cross talk with other signaling pathways. mains of YAP or Pin1 and the E3 ubiquitylat- ing ligases (Aragon et al. 2011). For example, CDK8- and CDK9-mediated Smad3 linker Linker Region Phosphorylation and phosphorylation creates binding sites for WW Dephosphorylation and R-Smad Degradation domains of Pin1, whereas subsequent phos- Glycogen synthase kinase 3 (GSK3), a down- phorylation by GSK3 switches off Pin1 binding stream effector of Wnt signaling, and the CDK but generates binding sites for Smurf1 WW do- family kinases CDK8 and CDK9 phosphorylate mains (Aragon et al. 2011). and mark activated Smad2 or Smad3 for pro- Cancer cells often possess high levels of teasomal destruction (Alarcon et al. 2009; Mil- MAPK and CDK activities that lead to a higher let et al. 2009; Wang et al. 2009). Following phosphorylation level in the R-Smad linker phosphorylation, the PPXY motif in the linker region. A comprehensive study indicates that region is recognized by the E3 ubiquitin ligase mutation in the Smad3 linker phosphorylation neural precursor cell expressed, developmen- sites markedly inhibits primary tumor growth, tally down-regulated 4-like (Nedd4L), also but significantly increases lung metastasis of known as Nedd4-2, which then polyubiquity- breast cancer cells, which is in line with the in- lates Smad2 or Smad3 and targets them for hibitory role of TGF-b in tumor initiation and proteosome-mediated degradation (Gao et al. TGF-b’s promotion of tumor migration (Bae 2009). Thus, linker phosphorylation limits the et al. 2014). This is also in consistent with earlier half-life of activated Smads, and the amplitude findings that mutation of CDK2, CDK4, and and duration of TGF-b responses, as it is appar- Erk phosphorylation sites in the linker regions ent from the induction of mesendodermal fates of Smad2 and Smad3 enhances the TGF-b re- in mouse embryonic stem cells (mESCs). Sim- sponse and functions (Kretzschmar et al. 1999; ilarly, the duration of Smad1 activation is con- Matsuura et al. 2004, 2005). trolled by sequential linker phosphorylation The discovery of R-Smad linker phospha- at the conserved MAPK and GSK3 recogni- tases further illustrates the complexity of linker tion sites, and in turn controls the intensity of phosphorylation and supports the notion that BMP signals during embryonic pattern forma- linker phosphorylation and dephosphorylation tion (Fuentealba et al. 2007). Notably, CDK8 are dynamic processes that are subject to tight and CDK9 also phosphorylate the linker re- regulation. For example, the SCP family phos- gion of Smad1, enabling the recruitment of phatases SCP1, 2, and 3 can remove the linker the transcription factor Yes-associated protein and MH1 domain phosphorylations of Smad2 (YAP), an effector of the Hippo pathway. They and Smad3, with no effect on the carboxy-ter- also phosphorylate the linker region of Smad2 minal SSXS phosphorylation of Smad3, and and Smad3, allowing recruitment of the pepti- hence reverse the inhibitory effect of these phos- dylprolylisomerase Pin1 that isomerizes phosphorylations (Sapkota et al. 2006; Wrighton phoserine/threonine-proline motifs and thus et al. 2006). Likewise, SCP1, SCP2, and SCP3 enhances Smad-mediated transcription. YAP can also dephosphorylate the linker of Smad1, consequently promotes Smad1-dependent thereby, suggesting their critical roles in regulat- transcription and controls BMP-mediated sup- ing TGF-b family signaling and, consequently, pression of neural differentiation of mESCs their biological effects, such as TGF-b-stimulat- (Alarcon et al. 2009). This phosphorylation is ed cell growth inhibition, and TGF-b/BMP- ultimately recognized by the E3 ubiquitin ligase regulated transcriptional responses in mammal- Smad ubiquitin regulatory factor 1 (Smurf1) or ian cells and in Xenopus embryos (Sapkota et al. Nedd4L and leads to Smad degradation (Alar- 2006; Wrighton et al. 2006).

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Phosphorylation of the MH1 and MH2 degradation, including the homologous E6- Domains of R-Smads AP carboxy-terminal (HECT) family E3 ligases Smurf1, Smurf2, Nedd4-2/Nedd4L, and In association with the scaffolding protein Axin, WWP1 (WW domain-containing protein 1), GSK3b phosphorylates the MH1 domain of the the RING-finger family E3 ligase SCF complex nonactivated form of Smad3, which results in (Rbx1, Skp1, Cullins, F-box proteins) and Ar- Smad3 ubiquitylation and subsequent degrada- kadia (also known as RNF111), and the U-box tion, and thus defines the basal level and activ- family E3 ligase carboxyl terminus of Hsc70- ity of Smad3, and the cell sensitivity to TGF-b interacting protein (CHIP) (Soond and Chan- (Guo et al. 2008a). In contrast, Erk-dependent try 2011; Tang and Zhang 2011). phosphorylation of Smad2 at the amino-ter- The ubiquitin E3 ligases named “Smad minal Thr8 site enhances the transcriptional ubiquitin regulatory factor (Smurf )” were the activity of Smad2 by an unknown mechanism first identified ubiquitin E3 ligases for Smads, (Funaba et al. 2002). Additionally, PKG (cGMP- and have been well studied. They target R- dependent protein kinase) in endothelial cells Smads, inhibitory Smads (I-Smads), as well as (Saura et al. 2005) and CK1g2 in mouse embry- some Smad-associated proteins for proteaso- onic fibroblasts or epithelial cells (Guo et al. mal degradation (Feng and Lin 2006; Izzi and 2008b) induce the phosphorylation of Smad3 Attisano 2004). Specifically, Smurf1 ubiquity- MH2 domains at Ser388 or Ser418 to enhance lates the BMP-specific Smad1 and Smad5, and Smad ubiquitylation and degradation. Addi- marks them for degradation without affecting tionally, PAK2 induces Smad2 phosphoryla- TGF-b-specific Smad2 and Smad3 (Zhu et al. tion at Ser417 to block its association with 1999). The related Smurf2 shows broader sub- TbRI (Yan et al. 2012), all leading to inhibi- strate specificity and physically associates with tion of TGF-b-stimulated gene expression. Smad1 and Smad2, resulting in their degrada- Whether additional phosphorylation sites in tion (Lin et al. 2000; Zhang et al. 2001). Besides the R-Smads and other kinases are involved in Smurf1 and Smurf2, distantly related members these activities remains to be determined. of WW-containing HECT family E3 ligases such as WWP1/Tiul1 and Nedd4-2/Nedd4L Ubiquitylation and Deubiquitylation also control Smad stability (Komuro et al. of R-Smads 2004; Seo et al. 2004; Kuratomi et al. 2005). In contrast, Itch/AIP4, a Smurf2-like E3 ligase, Ubiquitin E3 Ligases for Polyubiquitylation enhances TGF-b signaling and TGF-b-induced and Degradation of R-Smads cell growth arrest through an as yet undefined Ubiquitylation, that is, the covalent attachment mechanism (Bai et al. 2004). of a chain of ubiquitin polypeptides onto the Surprisingly, Smad3 is not targeted for de- polypeptide substrate results from the sequen- struction by Smurf proteins although it has high tial actions of E1, E2, and E3 ubiquitin ligases, affinity for Smurf2, but instead is targeted by a and defines the turnover and functions of di- different family of ubiquitin E3 ligases (i.e., verse proteins in a wide range of biological pro- Skp1-cullin-F-box [SCF] proteins) (Fukuchi cesses (Kerscher et al. 2006). Like many other et al. 2001; Ray et al. 2005). For example, the transcription regulators, R-Smads are regulated interaction of Smad3 with ROC1/Rbx1, the by ubiquitylation and ubiquitin-like modi- Ring subunit of SCFbTrCP complex, is partly re- fications that serve to control the subcellular sponsible for Smad3 degradation (Fukuchi et al. localization, stability, and activity of Smads. 2001). Smad3 is also targeted by CHIP,a U-box TGF-b-induced Smad2 activation is followed family E3 ligase, which ubiquitylates and de- by polyubiquitylation, which targets nuclear grades Smad3 independently of TGF-b stimu- Smad2 for degradation and terminates its sig- lation and reduces the TGF-b response, includ- naling (Lo and Massague´ 1999). Several ubiq- ing the control of cell proliferation by TGF-b uitin ligases have been implicated in R-Smad (Xin et al. 2005). Additionally, during mouse

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Posttranslational Regulation of Smads

embryogenesis, Arkadia/RNF111, a nuclear expression of Smurf2 parallels the ubiquityla- RING-domain E3 ubiquitin ligase, which was tion and degradation of Smad2 in rat glomeruli, initially shown to ubiquitylate Smad7 and the leading to glomerulonephritis (Togawa et al. Smad corepressor SnoN (Levy et al. 2007), also 2003). However, increased expression of Smurf1 directly interacts with phospho-Smad2 and and Smurf2 may also lead to Smad7 degrada- -Smad3, and enhances their levels in mouse tion, which, in turn, increases the TGF-b re- embryonic cells. This association is followed sponse and plays a pathogenic role in the pro- by phospho-Smad2 and Smad3 ubiquitylation gression of renal fibrosis (Fukasawa et al. 2004). that leads to their proteasomal degradation, thus resulting in down-regulation of nodal sig- Role and Regulation of R-Smad naling (Mavrakis et al. 2007). Ubiquitylation by Phosphorylation As the best studied Smad E3 ligases, the functions of Smurfs in Smad signaling are also As mentioned, TGF-b-induced linker phos- supported by observations in pathophysiologi- phorylation marks activated Smads for pro- cal contexts. Transgenic analysis supports the teasomal destruction, mediated by the activity role of Smurf1 in the destruction of BMP-spe- of the E3 ubiquitin ligase Nedd4L (Gao et al. cific Smad1 and Smad5. Osteoblast-specific ex- 2009). Nedd4L thus limits the half-life of TGF- pression of a Smurf1 transgene in mice indeed b-activated Smads and restricts the intensity leads to a significant reduction in bone mass and duration of TGF-b signaling, and the in- during postnatal life (Zhao et al. 2004). How- duction of mesendodermal fates in mESCs. ever, transgenic mice with increased expression Similarly, Smurf1 selectively binds to Smad1, of Smurf1 in chondrocytes fail to show clear following phosphorylation of its linker region, abnormalities (Horiki et al. 2004). These data further illustrating the interplay between phos- suggest that Smurf1 plays a specific role in bone phorylation and polyubiquitylation in mouse development. Unexpectedly, targeted disrup- osteoblast differentiation and Xenopus neural tion of the Smurf1 gene in mice does not affect development (Sapkota et al. 2007). Additionally Smad-mediated TGF-b or BMP signaling, and Smad3 phosphorylation at Ser418 in its MH2 Smurf1-deficient mice are born normally (Ya- domain by CK1g2 targets activated Smad3 mashita et al. 2005). However, these mice show for ubiquitylation-dependent degradation and an age-dependent increase of bone mass and decreases TGF-b responsiveness (Guo et al. enhanced activities of osteoblasts (Yamashita 2008b), although the responsible E3 ubiquitin et al. 2005). Additionally, mouse lung branch- ligase has not been identified. Ubiquitylation- ing morphogenesis can be inhibited by adeno- mediated degradation occurs preferentially viral expression of Smurf1 in airway epithelial with the nonactivated form of Smad3. For ex- cells (Shi et al. 2004a). Mice that lack a func- ample, GSK3b-dependent phosphorylation of tional Smurf2 gene display defects in planar cell nonactivated Smad3 at Thr66 in the MH1 do- polarity and extension movement, through main is essential for ubiquitylation and subse- changes in noncanonical Wnt signaling path- quent degradation of Smad3 (Guo et al. 2008a). way, but without obvious changes in TGF-b This ubiquitylation leads to reduction of total family signaling (Narimatsu et al. 2009). There- Smad3 level, thus decreasing the sensitivity of fore, the in vivo significance of Smurf proteins cells to TGF-b. The sequential roles and inter- in TGF-b family signaling is complex and re- play of phosphorylation and ubiquitylation quires further investigation. provide extensive control of TGF-b signaling. Increased expression of Smurf2 correlates R-Smads can also function as adaptors of with reduced levels of Smads under various dis- E3 ubiquitin ligase to facilitate the ubiquity- ease conditions. For example, increased Smurf2 lation and degradation of associated proteins expression with a concomitantly reduced level by the E3 ligase. For example, TGF-b induces of phospho-Smad2 is observed in esophageal the formation of a ternary complex consisting cancers (Fukuchi et al. 2002). Similarly, elevated of Smad2, SnoN, and Smurf2, in which Smad2

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P. Xu et al.

serves as an adaptor that enables the Smurf2 E3 or CDK9, in combination with an adjacent PY ligase to target SnoN for degradation (Bonni motif in the MH2 domain is a prerequisite for et al. 2001). This degradation relieves SnoN- this monoubiquitylation. mediated suppression of TGF-b targets in the absence of TGF-b ligand. The degradation of SnoN is also regulated by anaphase-promoting Deubiquitylation of R-Smads complex (APC) (Stroschein et al. 2001; Wan Ubiquitylation is a reversible process. Deubiq- et al. 2001). In this case, Smad3 binds to both uitylating enzymes (DUBs) oppose the effects SnoN and APC, and cooperates with the APC of ubiquitylation on Smads. Ubiquitin specific activator Cdc20 homologue-1 (CDH1) to pro- peptidase (USP15) is a DUB that removes mote SnoN ubiquitylation and degradation monoubiquitylation of Smad3 and thus en- by APC. In another example, Smad3 physically hances the occupancy of target promoters by associates with the cell-cycle regulator Cdc25A Smad complexes (Inui et al. 2011). Silencing bTrCP and the SCF E3 ligase in response to TGF- the expression of USP15 consequently impairs bTrCP b, thus facilitating SCF -mediated Cdc25A the recruitment of TGF-b-activated Smad com- ubiquitylation and degradation (Ray et al. plexes to regulatory DNA sequences of tar- 2005). Thus, the stabilization of Cdc25A ob- get genes and inhibits TGF-b response in cell served in various cancers might be related to growth arrest, cell migration, and Xenopus em- dysregulation of the TGF-b/Smad3 pathway. bryonic germ layers induction and patterning (Inui et al. 2011). Cylindromatosis (CYLD), Monoubiquitylation of R-Smads turban tumor syndrome, a DUB in the NF-kB pathway, also inhibits TGF-b signaling and lung Monoubiquitylation, whereby a protein is mod- fibrosis by decreasing the stability of Smad3 as a ified by covalent attachment of a single ubiqutin result of the CYLD-mediated Akt deubiquity- at a single or multiple lysines, regulates the sig- lation and the release of Akt-mediated inhibi- naling function of the substrates. Smad2 and tion of GSK3b-CHIP-induced Smad3 degra- Smad3 are monoubiquitylated in response to dation (Lim et al. 2012). Interestingly, OTU TGF-b; however, the effect of this monoubiqui- domain-containing ubiquitin aldehyde-bind- tylation differs depending on context. The ing protein 1 (OTUB1) a member of ovarian HECT family E3 ligase Itch/AIP4 forms a com- tumor (OTU) family of DUBs, inhibits TGF- plex with Smad2 and the activated form of b-induced Smad2 and Smad3 ubiquitylation TbRI, and monoubiquitylates Smad2, which by blocking the E2 ubiquitin-conjugating en- in turn enhances Smad2 interaction with TbRI zyme, independently of its catalytic activity and TGF-b-induced cell growth arrest (Bai et al. (Herhaus et al. 2013). These observations sug- 2004). In contrast, Smad3 was shown to be gest a dynamic balance of monoubiquitylation monoubiquitylated in its MH1 domain at mul- and deubiquitylation in the control of R-Smad tiple lysine residues, possibly by Smurf2 or activities. Nedd4, and these modifications interfere with Smad3 binding to the regulatory promoter sequences of target genes and block Smad3-me- Other Posttranslational Modifications diated cell growth arrest, cell migration, and of R-Smads embryonic germ layers induction and pattern- Sumoylation of R-Smads ing in Xenopus (Inui et al. 2011). Different from controlling Smad1 and Smad2 stability through Sumoylation covalently attaches a small ubi- polyubiquitylation, Smurf2-mediated Smad3 quitin-like modifier (SUMO) polypeptide that monoubiquitylation of the MH2 domain inter- structurally resembles ubiquitin to a lysine res- feres with the formation of functional Smad3 idue of protein substrate, and occurs primarily complexes (Tang et al. 2011). Interestingly, on perinuclear and nuclear proteins including Thr179 phosphorylation of Smad3 by CDK8 transcription factors (Kerscher et al. 2006). Su-

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Posttranslational Regulation of Smads

moylation usually does not lead to degradation (PARP-1), which results in dissociation of of targeted proteins, but often regulates their Smad3/4 complexes from DNA. As a result, subcellular localization, protein associations, PARP-1 attenuates Smad-mediated TGF-b and functions. Smad3 was found to associate responses, and cells with PARP-1 overexpres- with a protein inhibitor of activated STATy sion show impaired Smad3-mediated gene re- (PIASy, also known as PIAS4), a nuclear ma- sponses and TGF-b-induced epithelial–mesen- trix-associated SUMO E3 ligase, and to be su- chymal transition (EMT) (Lo¨nn et al. 2010). moylated leading to a suppressed activity of Thus, ADP-ribosylation of Smad proteins may Smad3 (Imoto et al. 2003). Intriguingly, knock- serve as another mechanism to control the inten- down of Ubc9, the only SUMO E2 ligase, sity and duration of Smad-mediated transcrip- reduces the accumulation of phospho-Smad3 tion. Whether ADP-ribosylation also occurs on during the pathogenesis of fibrosis and has other R-Smad molecules is to be determined. an antifibrosis effect in either TbRI or bleomycin-induced dermal fibrosis model, suggesting a POSTTRANSLATIONAL REGULATION functional significance of sumoylation in ca- OF THE Co-Smad, Smad4 nonical TGF-b signaling (Khodzhigorova et al. 2012). Smad4, the common Smad for all R-Smads that consequently controls Smad signaling of Acetylation of R-Smads the entire TGF-b family signaling, functions as a transcription co-activator that associates In addition to phosphorylation and ubiquityla- with R-Smads and enhances the effectiveness tion, Smad2 and Smad3 are also acetylated of R-Smad-mediated transcription (Feng et al. at lysine residues in response to TGF-b. The 1998; Heldin and Moustakas 2012). The gene acetylation results from TGF-b-induced associ- encoding Smad4 is frequently altered in cancers ation of the transcription coactivators cAMP- and other diseases, which can lead to impaired response element-binding (CREB)-protein responsiveness to TGF-b-imposed growth inhi- (CBP) and p300, which possess intrinsic acetyl- bition. Like R-Smads, the activities of Smad4 transferase activity, with Smad2 and Smad3. are stringently modulated by PTMs, including Smad2 is primarily acetylated in its MH1 phosphorylation, ubiquitylation, and sumoly- domain, whereas Smad3 is acetylated in its ation, all of which define the Smad4 interac- MH2 domain, although both acetylations en- tome, its subcellular localization, and its stabil- hance Smad-mediated transcription (Simons- ity (Fig. 2). son et al. 2006; Inoue et al. 2007; Tu and Luo 2007). Thus, coactivator-mediated acetylation of R-Smads in the nucleus represents an addi- Phosphorylation of Smad4 tional avenue to modulate TGF-b signaling. The Constitutive phosphorylation of Smad4 has detailed characterization and underlying mech- been observed in cells, although not all the anisms of how acetylation alters the activity of phosphorylation sites have been identified. the R-Smad complex and changes the selectivity Erk kinases phosphorylate Smad4 specifically of Smad partners/coregulators are still not clear. at Thr276 in the linker region, and this phosphorylation stabilizes the binding of the coac- ADP-Ribosylation of R-Smads tivators p300 and CBP to the heteromeric Smad ADP-ribosylation is another type of protein complex (Roelen et al. 2003). Therefore, the modification that adds one or more ADP-ribose substitution of Thr276 to alanine leads to moieties to arginine residue by the ADP-ri- decreased nuclear localization of Smad4 and bosyltransferase, and regulates various cell sig- a reduced Smad-mediated transcriptional re- naling and cellular processes. Smad3 can be sponse. In addition to constitutive phosphory- ADP-ribosylated in its MH1 domain through lation, Smad4 phosphorylation is also regulated the action of poly (ADP-ribose) polymerase-1 by signal stimulation (Wrighton et al. 2009a).

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P Phosphorylation

U Monoubiquitylation

S Sumoylation PIASy PIAS1 TIF1γ LKB1 PIASxβ ERK MPK38 Unknown USP9x odSrn abPrpc Biol Perspect Harb Spring Cold P S P P U U T77 K113 K159 T277 S343 K507 K519

Smad4 MH1 Linker SAD MH2

Figure 2. Deﬁned modiﬁcations and modifying enzymes of Smad4. Triangle symbols are as in Figure 1. o:10.1101 doi: / cshperspect.a022087 Downloaded from http://cshperspectives.cshlp.org/ on September 26, 2021 - Published by Cold Spring Harbor Laboratory Press

Posttranslational Regulation of Smads

For example, the liver kinase B1 (LKB1), also activity of SCFbTrCP1 is facilitated by the direct known as STK11, which regulates cell polarity association of Smad4 with Jun activating bind- and is required in the control of cell metabo- ing protein 1 (Jab1), a component of the COP9 lism, phosphorylates Thr77 in the MH1 do- signalosome (Wan et al. 2004). Another mem- main of Smad4. This phosphorylation inter- ber of the SCF family E3 ligases, SCFSkp2, has no feres with Smad4 binding to either TGF-b or effect on the stability of wild-type Smad4 but BMP-specific promoter sequences, and inhibits only targets cancer-derived Smad4 mutants for Smad-mediated gene expression and TGF-b- increased ubiquitylation and accelerated degra- induced EMT (Moreń et al. 2011). In contrast, dation (Liang et al. 2004a). The HECT family phosphorylation of Smad4 by the serine/thre- E3 ligases, the Smurfs, polyubiquitylate Smad4 onine kinase MPK38, known to activate p53 and promote the proteasomal degradation of function, is thought to enhance TGF-b signal- Smad4 (Moreń et al. 2005). Because Smad4 ing and TGF-b-induced apoptosis and cell lacks a PY motif and cannot directly associate growth arrest (Seong et al. 2010). with HECT domain E3 ligases, I-Smads serve Phosphorylation of several cancer-associat- as adaptors for the recruitment of Smurfs to ed Smad4 mutants appears to be a prerequisite Smad4. Furthermore, a single-subunit RING for ubiquitylation of Smad4 by the SCF com- E3 ligase TRIM33/TIF1g, also named ectoder- plex, and leads to their accelerated proteolysis min, has similarly been shown to specifically (Liang et al. 2004a). For example, the R100T, bind to Smad4, and reduce the level of nuclear G65V, and L43S mutation in the MH1 domain Smad4 in the ectoderm by targeting Smad4 for of Smad4 leads to enhanced phosphorylation ubiquitylation and proteosomal degradation. of Smad4 by JNK/p38 MAPK, and consequent- Thus, overexpression of TRIM33/ectodermin ly increased affinity of mutant Smad4 for results in a low level of TGF-b signaling and SCFbTrCP and SCFSkp2 (Liang et al. 2004a). Sim- indirectly promotes the mesodermal fate (Du- ilar mutations of Smad4 in acute myelogenous pont et al. 2005). However, TRIM33/TIF1g was leukemia or pancreatic cancer cause a rapid pro- also reported to compete with Smad4 in bind- teasomal degradation of Smad4, mediated by ing TGF-b-activated Smad2 and Smad3, and to SCFbTrCP (Yang et al. 2006). Additionally, hy- direct Smad4-independent transcription that peractive Ras has also been shown to drive the regulates erythroid differentiation (He et al. degradation of Smad4 in intestinal epithelial 2006). The PHD-bromodomain of TRIM33/ cells through Erk MAPK activity and an uniden- TIF1g also facilitates its binding to H3K9me3 tified ubiquitin E3 ligase, which represses TGF- and H3K18ac at the promoters of mesendo- b-activated transcriptional responses (Saha derm regulators, which subsequently displaces et al. 2001). the chromatin-compacting factor HP1g (het- erochromatin protein 1g), and makes nodal-responsive DNA elements accessible to Smad Ubiquitylation of Smad4 complexes. At the same time, recruitment of RNA polymerase II to this DNA-Smad complex Polyubiquitylation and Degradation site enhances the expression of mesendoderm of Smad4 regulators goosecoid (Gsc) and Mix-like homeo- Like R-Smads, Smad4 is also targeted for poly- domain protein 1 (Mixl1), and thus controls ubiquitylation that often leads to its degra- the mesendodermal differentiation of embry- dation. Smad4 stability is controlled by three onic stem cells (ESCs) (Agricola et al. 2011; families of E3 ligases: RING-finger SCF com- Xi et al. 2011). Like SCFbTrCP and SCFSkp2, plex, HECT-domain, and PHD-finger E3 li- TRIM33/TIF1g also shows increased expres- gases (Liang et al. 2004a; Wan et al. 2004; sion in tumors, suggesting that destruction Dupont et al. 2005). SCFbTrCP1 promotes ubiq- of Smad4 may play a key role in tumorigenesis. uitylation and degradation of Smad4, and In addition, the presence of more than one antagonizes TGF-b function. The E3 ligase E3 ligase targeting Smad4 for degradation

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P. Xu et al.

highlights the critical role of Smad4 in me- Other Posttranslational Modifications diating TGF-b signaling in normal or diseased of Smad4 cells. Sumoylation of Smad4 SUMO-1 and Ubc9, the only E2 sumoylation Monoubiquitylation of Smad4 ligase, were identified as Smad4-interacting Monoubiquitylation also regulates the signal- proteins in yeast two-hybrid screens (Lee et al. ing and transcription functions of Smad4. 2003; Lin et al. 2003b). Furthermore, Smad4 TRIM33/TIF1g was shown to monoubiquity- is sumoylated on Lys113 and Lys159, and se- late Smad4 at Lys519 in the MH2 domain (Mo- quence comparison suggests that sumoylation reń et al. 2003), thus preventing Smad4 associ- of Smad4 is highly conserved throughout evo- ation with activated Smad2/3 (Dupont et al. lution (Lee et al. 2003; Lin et al. 2003b). 2009). Smad4 Lys519 monoubiquitylation im- The effects of sumoylation on Smad4 are posed by TRIM33/TIF1g can be removed by multifaceted. Smad4 sumoylation may prevent the deubiquitylase FAM/USP9x, which conse- Smad4 from ubiquitylation, thus protecting quently restores Smad4 function in the contexts Smad4 from degradation to enhance TGF-b- of TGF-b-induced growth inhibition and cell induced Smad signaling. Accordingly, the su- migration (Dupont et al. 2009). Interestingly, moylated form of Smad4 confers enhanced three naturally occurring amino acid mutations growth inhibition and transcriptional responses in Smad4 that were detected in cancer cells are in mammalian cells and Xenopus embryos (Lee located near this monoubiquitylation site in et al. 2003; Lin et al. 2003a,b). However, Smad4 Smad4, and result in defective monoubiquity- sumoylation was also reported to repress Smad- lation of Smad4, which may lead to Myhre syn- mediated transcription by recruiting Daxx (Fas drome, a developmental retardation disorder death domain–associated protein), initially (Le Goff et al. 2012). In contrast, FOXM1, a identified as Fas-interacting protein to regulate forkhead box family transcription factor that cell apoptosis, to Smad4 at regulatory promoter possesses strong oncogenic activity, interacts sequences, thus inhibiting the intrinsic transac- with Smad3 and interferes with TRIM33/ tivation capability of Smad4 (Long et al. 2004; TIF1g binding to the Smad3/4 complex. The Chang et al. 2005). This apparent contradiction reduced binding of TRIM33/TIF1g to the might be because of the use of different promot- Smad complex attenuates Smad4 monoubiqui- er sequences as models, and needs further study tylation by TRIM33/TIF1g, and thus stabilizes in physiological contexts. Remarkably, the ac- Smad3/4 complex formation and facilitates tivity of TRIM33/TIF1g as transcriptional co- TGF-b-dependent promotion of cancer metas- repressor and as a Smad4 substitute in TGF-b tasis (Xue et al. 2014). On the other hand, signaling under some circumstances (Dupont et Smad4 monoubiquitylation at Lys507 may en- al. 2009) also requires sumoylation of its Smad- hance the complex formation of Smad4 with interacting domain to regulate TGF-b signaling activated R-Smads (Moreń et al. 2003). This and inhibit TGF-b-regulated EMT (Fattet et Lys507, conserved among all members of the al. 2013). Smad4 sumoylation requires a PIAS Smad family, resides in the L3 loop region of protein as the E3 ligase, and PIAS1, PIASxb, and Smad4’s MH2 domain and is responsible for PIASy were shown to bind to Smad4 and pro- Smad4 recognition of the phospho-SSXS motif mote Smad4 sumoylation (Ohshima and Shi- of R-Smads during Smad heterotrimer forma- motohno 2003; Liang et al. 2004b; Long et al. tion. The substitution of Lys507 with Arg blocks 2004). Consistent with these observations, the nuclear accumulation and transcriptional PIAS1 mutants that lack a functional RING do- responses of Smad4 (Moreń et al. 2003). How- main but retain the Smad4-binding domain, ever, the E3 ligase(s) that catalyze(s) Smad4 can block Smad4 sumoylation and TGF-b tran- monoubiquitylation at Lys507 have not been scriptional responses in a dominant-negative identified. manner (Liang et al. 2004b).

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Posttranslational Regulation of Smads

Intriguingly, Smad4 sumoylation is lost in and SCFbTrCP E3 ligases, which are also onco- several cancer-derived Smad4 mutants, espe- genic and often are up-regulated in cancer cially those with missense mutations in MH1 cells (Liang et al. 2004a). As mentioned, these domain (Lin et al. 2003a). As mentioned, these Smad4 mutants show increased phosphoryla- cancer mutants show enhanced phosphorylation by JNK and p38 MAPKs, leading to en- tion, preferentially by JNK or p38 MAPK, which hanced ubiquitylation and degradation. There- results in an increased affinity for SCFSkp2/ fore, pharmacological inhibition of JNK or p38 SCFbTrCP E3 ligases and subsequent ubiquityla- MAP kinases can prevent the degradation of tion and degradation (Liang et al. 2004a; Wan Smad4 mutants to the same extent as proteaso- et al. 2004; Wan et al. 2005). These observations mal inhibitors (Liang et al. 2004a). The acceler- support the notion that the cancer-associated ated degradation of these cancer-derived mu- mutations in the MH1 domain can switch tants may be a mechanism to ensure complete Smad4 from being sumoylated to being ubiqui- removal of residual tumor suppressor activity tylated, and lead to loss of Smad4 activity. of Smad4. In conclusion, the function and stability of Smad4 are extensively regulated by PTM, which provides high versatility to Smad ADP-Ribosylation of Smad4 signaling. Similar to Smad2 and Smad3, Smad4 is also ADP-ribosylated by PARP-1 in its DNA-binding REGULATION OF I-Smads MH1 domain, which interferes with Smad4 DNA binding and attenuates Smad-mediated The expression and activities of the two I-Smads transcription and induction of EMT (Lo¨nn in vertebrates, Smad6 and Smad7, represent a et al. 2010). Stimulated dissociation of Smad critical level of negative control of TGF-b sig- complexes from DNA by PTMs represents an naling. I-Smads shuttle continuously between intriguing mode for terminating Smad sig- nucleus and cytoplasm allowing for an inhibi- naling. It is worthwhile to further investigate tory feedback loop in multiple compartments whether other modifications lead to similar sce- (Heldin and Moustakas 2012). Several mecha- narios in regulating TGF-b family signaling. nisms enable I-Smad-mediated inhibition of TGF-b family signaling, including competitive interference with activation of R-Smads by ac- Naturally Occurring Mutations in Smad4 tivated type I receptors (Hayashi et al. 1997; Consistent with its critical role in mediating Imamura et al. 1997; Nakao et al. 1997), inhi- TGF-b function, alterations of the SMAD4 bition of formation of Smad complexes (Hata gene frequently occur in human cancers and et al. 1998), and direct repression of target gene other diseases. Although many SMAD4 muta- promoters in the nucleus (Bai et al. 2000; Lin tions or deletions affect the sequence encoding et al. 2003c; Zhang et al. 2007). Additionally, the MH2 domain and render Smad4 inactive Smad7 binding to TbRI allows for recruitment in driving Smad signaling, a number of can- of the E3 ubiquitin ligases Smurf1, Smurf2, or cer-associated SMAD4 missense mutations, WWP1 to the receptor complex, resulting in primarily in the sequence encoding the MH1 ubiquitylation and degradation of TbRI or domain, cause an accelerated degradation of BMP type I receptors (Kavsak et al. 2000; Ko- Smad4 (Moreń et al. 2000; Xu and Attisano muro et al. 2004). Binding of Smad7 to the type 2000). For example, the R100T missense mu- I receptor can also recruit the GADD34-PP1c tation of Smad4 in pancreatic cancers results in phosphatase complex to reverse activation of a faster degradation, whereas, likewise, Smad4 TbRI, thereby terminating TGF-b responses, with MH1 domain mutations, such as L43S, such as TGF-b-induced cell-cycle arrest (Shi G65V, and P130L, are also unstable (Xu and et al. 2004b). The phosphatase PP1 regulatory Attisano 2000). These cancer cell-derived subunit GADD34 has been reported to play an Smad4 mutants show higher affinity for SCFSkp2 important role in endoplasmic reticulum (ER)

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stress-induced cell death (Kojima et al. 2003). It ducible factor 1a), which contributes to malig- will be interesting to determine the connection nant cell invasiveness (Heikkinen et al. 2010a). between TGF-b signaling and ER stress-in- Smad7 expression is additionally controlled duced cellular responses (Tanjore et al. 2011). by miRNAs, which reflects another layer of In addition to repression of TGF-b signaling, complexity in the regulation of I-Smad expres- Smad7 can also act as a transcription factor in sion. miR-106b-25, which derives from a poly- the nucleus (Pulaski et al. 2001; Zhang et al. cistron that is frequently activated by genomic 2007), and an adaptor protein essential for amplification in multiple cancer types and TGF-b-induced activation of p38 MAPK sig- suppresses p21Cip1 expression, inhibits Smad7 naling. Consequently, Smad7 promotes TGF- expression and enhances TGF-b signaling in b-induced, Smad3-independent inactivation epithelial cells and human breast cancer cells. of GSK3b, nuclear accumulation of b-catenin, miR-106b-25 expression is up-regulated by and recruitment of the tumor suppressor ade- Six1, a metastatic regulator. The miR-106b-25 nomatous polyposis coli (APC) to the microtu- cluster targets Smad7 mRNA, resulting in in- bule plus end, all of which might control TGF- creased TGF-b type I receptor levels and TGF- b-stimulated migratory response (Ekman et al. b signaling, and induction of EMT (Smith et al. 2012). Reflecting the functional diversity of 2012). Similarly, miR-21, a miRNA that targets I-Smads, the level and activity of Smad6 and some tumor suppressor genes such as PTEN Smad7 are extensively regulated both at the and Bcl2, also decreases Smad7 expression and transcriptional level and through PTMs (Fig. 3). enhances TGF-b signaling (Liu et al. 2010; Smith et al. 2012). During induction of lung fibrosis, TGF-b directly induces the expression Complexity in the Regulation of I-Smad of miR-21, which in turn inhibits the expression Gene Expression of Smad7. This miR-21-mediated decline of Distinct from other Smads, the expression of I- Smad7 expression represses Smad7-mediated Smads is regulated by a number of hormones, negative feedback of TGF-b signaling, allowing growth factors and cytokines, which conse- for prolonged TGF-b effects in inducing fibro- quently also control TGF-b signaling. As a di- genic activation of pulmonary fibroblasts. rect negative feedback mechanism, TGF-b directly induces Smad7 expression resulting from binding of Smad3/4 complexes to Smad7 gene Ubiquitylation and Deubiquitylation regulatory sequences (Derynck and Zhang of I-Smads 2003). Activation of Erk, JNK or p38 MAPK Polyubiquitylation and Degradation signaling all result in transcriptional activation of I-Smads of the Smad7 gene, thus dampening TGF-b signaling (Guo and Wang 2009). Some cytokines, Besides regulation at the transcriptional level, such as interferon-g or interleukin-7, induce I-Smad levels are tightly regulated at the protein Smad7 expression through activation of Jak/ level, mainly through ubiquitylation and degra- STAT signaling (Ulloa et al. 1999; Huang et al. dation. Several E3 ligases, such as Smurfs and 2002), whereas tumor necrosis factor-a or in- WWP1/2, have been implicated in this regula- terleukin-1b induce Smad7 expression through tion (Soond and Chantry 2011). In the case of the NF-kB pathway (Bitzer et al. 2000), leading Smurf2, the Smad7-Smurf2 complex is formed to inhibition of TGF-b/Smad-mediated tran- in the nucleus through a paired interaction be- scription responses. As a result, recombinant tween a WW motif in Smurf2 and a PY motif interleukin-7 alleviates bleomycin-induced pul- in Smad7, allowing for direct association of monary fibrosis in vivo (Huang et al. 2002). the amino-terminal sequence of Smad7 and Additionally, hypoxia also induces Smad7 ex- the HECT domain of Smurf2 (Ogunjimi et al., pression, mediated by activation of expression 2005). This complex is subsequently exported of the transcription factor HIF1a (hypoxia in- from the nucleus and translocated to the plasma

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P Phosphorylation

U Monoubiquitylation

M Methylation SIRT1 onSeptember26,2021-PublishedbyColdSpringHarborLaboratoryPress HDAC1 CYLD Ac p300 MPK38 odSrn abPrpc Biol Perspect Harb Spring Cold Acetylation Unknown Unknown Ac P P U K64 K70 S249 K360 K374 T96

Smad7 N domain Linker MH2 o:10.1101 doi:

Smad6 N domain Linker MH2 otrnltoa euaino Smads of Regulation Posttranslational / cshperspect.a022087 R74 R81 S435 K174

M U P

PRMT1 UBE2O PrKX

Figure 3. Deﬁned modiﬁcations and modifying enzymes of Smad6 and Smad7. Triangle symbols are as in Figure 1. 17 Downloaded from http://cshperspectives.cshlp.org/ on September 26, 2021 - Published by Cold Spring Harbor Laboratory Press

P. Xu et al.

membrane (Suzuki et al. 2002; Tajima et al. TbRI to up-regulate TGF-b-dependent tran- 2003). Smad7 is then degraded by Smurf2 in scriptional responses (Wicks et al. 2005; conjunction with TGF-b receptor activation, Eichhorn et al. 2012). Notably, USP15 amplifi- most likely as Smurf2 targets the receptor- cation confers poor prognosis of glioblastoma Smad7 complex for degradation. Through asso- patients, whereas depletion or inhibition of ciation with Axin, Smad7 is also targeted by USP15 reduces the oncogenic capacity of pa- Arkadia/RNF111 E3 ligase, which results in tient-derived glioma-initiating cells caused Smad7 degradation and increased TGF-b sig- by repressed TGF-b signaling (Eichhorn et al. naling (Liu et al. 2006). In colorectal cancer, 2012). Intriguingly, Smad6 can also recruit A20, the effect of Arkadia on Smad7 degradation a DUB for TRAF6, allowing A20 to abolish and subsequently enhanced TGF-b signaling K63-linked polyubiquitylation of TRAF6 and may provide a basis for the tumor suppressor thus prevent TGF-b-induced activation of p38 functions of Arkadia (Sharma et al. 2011). In MAPK and JNK signaling and apoptosis in he- addition, Jab1/CSN5, a component of the patocytes (Jung et al. 2013). In conclusion, the COP9 signalosome complex, also interacts ubiquitylation and deubiquitylation of I-Smads with Smad7 and facilitates Smad7 nuclear ex- further show the complexity of the control of port for degradation, thereby relieving the in- TGF-b responses by I-Smads. hibitory role of Smad7 on TGF-b target gene responses in the nucleus (Kim et al. 2004). Other Posttranslational Modifications of I-Smads Monoubiquitylation of I-Smads Acetylation and Deacetylation of I-Smads Monoubiquitylation of I-Smads also regulates I-Smad activity. During adipogenesis, the puta- The activity of Smad7 is also modulated by acet- tive E2 ubiquitin-conjugating enzyme UBE2O ylation and deacetylation. Interaction of Smad7 is found to interact with Smad6, and func- with the transcriptional coactivator p300, a his- tions as an E2–E3 hybrid to monoubiquitylate tone acetyltransferase, results in direct Smad7 Smad6 at Lys174 (Zhang et al. 2013). This mod- acetylation on Lys64 and Lys70 through the in- ification impairs Smad6 binding to BMP type I trinsic acetyltransferase activity of p300 (Gro¨n- receptor, thus potentiating BMP-7 signaling roos et al. 2002). As these same lysine residues and BMP-7-induced adipocyte differentiation. are also targeted by ubiquitylation, their acetylation prevents Smurf-mediated ubiquitylation and subsequent degradation of Smad7 (Gro¨n- Deubiquitylation of I-Smads roos et al. 2002). These two lysines are not pres- Deubiquitylation of Smad7 occurs through ent in Smad6, indicating a mechanistic differ- various DUBs. In addition to indirectly regulat- ence in ubiquitylation-dependent regulation ing Smad3 stability (Lim et al. 2012), as already between Smad6 and Smad7 (Gro¨nroos et al. discussed, the K63-specific deubiquitylase 2002). Furthermore, the histone deacetylases CYLD forms a complex with Smad7 and de- HDAC1 and SIRT1 can interact with the MH1 ubiquitylates Smad7 at K360 and K374, which domain of Smad7 and reverse Smad7 acetyla- dampens Smad7-facilitated activation of TAK1 tion, thus enhancing Smurf1-mediated Smad7 and p38, and impairs Treg differentiation (Zhao ubiquitylation and degradation, and conse- et al. 2011). Two other DUBs, namely, ubiquitin quently attenuating TGF-b-induced mesangial carboxyl-terminal hydrolase 37 (UCH37), also cell apoptosis (Simonsson et al. 2005; Kume known as UCHL5, and USP15, can interact with et al. 2007). The competition between acetyla- Smad7 through the MH1 domain on Smad7, tion and ubiquitylation likely controls Smad7 and thus are recruited to TbRI, where UCH37 stability inside the nucleus. In addition, the and USP15 remove the polyubiquitin chains presence of Smad7 in the nucleus and its acet- attached to TbRI by Smurfs, and thus stabilize ylation by p300 suggest that Smad7 may func-

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Posttranslational Regulation of Smads

tion as a transcription factor (Itoh et al. 1998). induced biological responses is evolutionarily Smad7 shows TGF-b-independent transactiva- conserved as PRMT1 regulates wing develop- tion activity, which is regulated by Ser249 phos- ment in Drosophila (Xu et al. 2013). Therefore, phorylation by a yet unidentified kinase (Pulas- arginine methylation, which is mostly known to ki et al. 2001). Consistent with the possible role regulate gene expression through histone meth- of I-Smads in transcription, Smad6 can directly ylation, can also regulate the TGF-b and BMP recruit the corepressor CtBP to the Id1 promot- signaling pathways by direct modification of I- er, or perhaps other BMP-responsive promot- Smads. ers, to regulate BMP signaling (Lin et al. 2003c). CONCLUDING REMARKS Phosphorylation of I-Smads Although TGF-b family signaling through Only a few observations of I-Smad phosphory- Smads is a conceptually simple and linear path- lation have been reported. PrKX, a kinase that way, it allows for amazing versatility and context controls macrophage differentiation, can phos- dependence of the TGF-b family responses. phorylate Smad6 in its MH2 domain, and this Like many key regulatory proteins, the expres- phosphorylation is required for phorbolmyris- sion and activities of Smads are regulated at tate acetate (PMA)-induced differentiation of different levels including by PTMs (Fig. 4). In- HL-60 cell into macrophages (Glesne and Hu- creasing evidence emphasizes the fundamental berman 2006). Additionally, MPK38-mediated roles of phosphorylation, ubiquitylation, su- phosphorylation of Smad7 on Thr96 promotes moylation, and acetylation in Smad regulation. redistribution of Smad7 to the cytoplasm Through their effects on the activity, stability, (Seong et al. 2010), whereas Ser249 phosphor- and interactome of Smads, these PTMs finely ylation by an unknown kinase enhances the tune TGF-b family responses and define the transcriptional activity of Smad7 without af- intensity and versatility of TGF-b family re- fecting TGF-b signaling (Pulaski et al. 2001). sponsiveness in pathophysiological processes. Moving forward, systematic studies under physiological conditions are needed to explore Methylation of I-Smads additional modification sites of all Smads, the Monomethylation, asymmetric, and symmetric corresponding modifying enzymes, and their dimethylation of arginines in proteins often regulation and functional consequences. Such regulates protein function in a temporal–spa- studies also require sensitive modern method- tial fashion. Based on mass spectrometric anal- ologies, such as mass spectrometry, chromatin- ysis, Smad6 was shown to be methylated by the immunoprecipitation (ChIP)-seq, deep RNA arginine methyltransferase PRMT1 (Inamitsu sequencing, and bioinformatics, in combina- et al. 2006). Arg methylation of Smad6 by tion with detailed mechanistic studies. Finally, PRMT1 serves as a first critical step in initiating it is vitally important to identify additional and BMP signaling through Smads. Specifically, in novel modifications, such as lipidation, nitro- response to BMP, PRMT1-mediated methyla- sylation, neddylation, and carbon-chain modi- tion of Arg74 and Arg81 residues in Smad6 fications, and reveal the synergistic or opposing promotes the release of Smad6 from the BMP effects of different types of modifications at receptor complex, allowing for Smad1 activa- multiple sites. Because most modifications are tion through carboxy-terminal phosphoryla- reversible, identification and characterization of tion by BMP type I receptors. Thus, signaling respective enzymes, and the interacting proteins initiation through Arg methylation may ex- that control their catalytic activities, will pro- plain the slow kinetics of Smad activation, and vide further insights into the functions of these defines PRMT1 as a positive regulator of BMP- modifications in TGF-b pathophysiology. In induced Smad activation (Xu et al. 2013). conclusion, advances in characterizing of novel Notably, the involvement of PRMT1 in BMP- modifiers and modification modes will reveal a

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P. Xu et al.

(De)ubiquitylation: Smurf2, WWP1, 2, Arkadia, Jab1/CSN5 (De)ubiquitylation: CYLD, UCH37, USP15 I-Smad (De)acetylation: PrKX,PrPrKrKKXXX,, MPK38MPMPKK33838 p300 PP Methylation: PRMT1 RII RI

P (De)phosphorylation: P ERK, JNK, p38, MPK38 (De)ubiquitylation: Smad4 R-Smad SCFs, Jab1/CSN5, FAM/USP9x P P P Sumoylation: PIAS1, PIASxβ, PIASy P Degradation Degradation P P ADP-Ribosylation: PARP-1 P P (De)phosphorylation: p38, JNK, Rock, MPK38, (De)phosphorylation: WNK1, MPS1, SCP1, 2, 3 LKB1 (De)ubiquitylation: (De)ubiquitylation: Itch/AIP4, USP15, OTUB1 Ectodermin/TIFγ (De)acetylation: CBP/p300

(De)phosphorylation: PPM1A, PP2A, MTMR4 PDP, SCP4, CDKs, GSK3, Araf, PKG, CK1s, CaMKII, PAK2, 4, GRK2 P (De)ubiquitylation: P Smurf1, 2, Nedd4-2, WWP1, SCF, Coregulator ROC1, Arkadia ADP-Ribosylation: TFs or PARP-1

Figure 4. Roles of posttranslational modiﬁcations (PTMs) in transforming growth factor b (TGF-b) family signaling. Modiﬁcations and modifying enzymes that promote or inhibit Smad signaling are indicated.

clearer picture of the activities of TGF-b family review. The research described in the authors’ proteins. laboratories was partly supported by grants from the Ministry of Science and Technology (MOST) (2012CB966600, 2015CB553800), ACKNOWLEDGMENTS Natural Science Foundation of China (NSFC) This review of recent advances requires that we (91540205, 31571447, 31090360), Department limit ourselves to selected observations to illus- of Defense (DoD) (1W81XWH-15-1-0650), trate the roles of Smad posttranslational modi- National Institutes of Health (NIH) (R01G ﬁcations. Many exciting ﬁndings could not be M63773, R01AR053591, R01CA108454) to included. We therefore apologize to those re- X.-H.F.; MOST (2015CB553800) and NSFC searchers whose work was not included in this (81472665) to P.X.; NIH (R01DK073932) to

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Posttranslational Regulation of Smads

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Posttranslational Regulation of Smads

Pinglong Xu, Xia Lin and Xin-Hua Feng

Cold Spring Harb Perspect Biol published online October 6, 2016

Subject Collection The Biology of the TGF-β Family

TGF-β Family Signaling in Early Vertebrate TGF-β Family Signaling in Mesenchymal Development Differentiation Joseph Zinski, Benjamin Tajer and Mary C. Mullins Ingo Grafe, Stefanie Alexander, Jonathan R. Peterson, et al. Bone Morphogenetic Protein−Based Therapeutic TGF-β1 Signaling and Tissue Fibrosis Approaches Kevin K. Kim, Dean Sheppard and Harold A. Jonathan W. Lowery and Vicki Rosen Chapman TGF-β Family Signaling in Ductal Differentiation Bone Morphogenetic Proteins in Vascular and Branching Morphogenesis Homeostasis and Disease Kaoru Kahata, Varun Maturi and Aristidis Marie-José Goumans, An Zwijsen, Peter ten Dijke, Moustakas et al. TGF-β Signaling in Control of Cardiovascular TGF-β Family Signaling in Epithelial Function Differentiation and Epithelial−Mesenchymal Marie-José Goumans and Peter ten Dijke Transition Kaoru Kahata, Mahsa Shahidi Dadras and Aristidis Moustakas TGF-β Family Signaling in Tumor Suppression TGF-β Family Signaling in Connective Tissue and and Cancer Progression Skeletal Diseases Joan Seoane and Roger R. Gomis Elena Gallo MacFarlane, Julia Haupt, Harry C. Dietz, et al. Targeting TGF-β Signaling for Therapeutic Gain The TGF-β Family in the Reproductive Tract Rosemary J. Akhurst Diana Monsivais, Martin M. Matzuk and Stephanie A. Pangas Regulation of Hematopoiesis and Hematological TGF-β Family Signaling in Drosophila Disease by TGF- β Family Signaling Molecules Ambuj Upadhyay, Lindsay Moss-Taylor, Myung-Jun Kazuhito Naka and Atsushi Hirao Kim, et al. TGF-β Family Signaling in Neural and Neuronal Signaling Cross Talk between TGF-β/Smad and Differentiation, Development, and Function Other Signaling Pathways Emily A. Meyers and John A. Kessler Kunxin Luo For additional articles in this collection, see http://cshperspectives.cshlp.org/cgi/collection/

For additional articles in this collection, see http://cshperspectives.cshlp.org/cgi/collection/