In the nucleus, SMAD2/3:SMAD4 heterotrimer complex acts as a transcriptional regulator. The activity of SMAD2/3 complex is regulated both positively and negatively by association with other transcription factors (Chen et al. 2002, Varelas et al. 2008, Stroschein et al. 1999, Wotton et al. 1999). In addition, the activity of SMAD2/3:SMAD4 complex can be inhibited by nuclear protein phosphatases and ubiquitin ligases (Lin et al. 2006, Dupont et al. 2009).
View original pathway at Reactome.
Lin X, Duan X, Liang YY, Su Y, Wrighton KH, Long J, Hu M, Davis CM, Wang J, Brunicardi FC, Shi Y, Chen YG, Meng A, Feng XH.; ''PPM1A functions as a Smad phosphatase to terminate TGFbeta signaling.''; PubMedEurope PMCScholia
Sowa H, Kaji H, Hendy GN, Canaff L, Komori T, Sugimoto T, Chihara K.; ''Menin is required for bone morphogenetic protein 2- and transforming growth factor beta-regulated osteoblastic differentiation through interaction with Smads and Runx2.''; PubMedEurope PMCScholia
Lönn P, van der Heide LP, Dahl M, Hellman U, Heldin CH, Moustakas A.; ''PARP-1 attenuates Smad-mediated transcription.''; PubMedEurope PMCScholia
Sun Y, Liu X, Ng-Eaton E, Lodish HF, Weinberg RA.; ''SnoN and Ski protooncoproteins are rapidly degraded in response to transforming growth factor beta signaling.''; PubMedEurope PMCScholia
Dupont S, Zacchigna L, Cordenonsi M, Soligo S, Adorno M, Rugge M, Piccolo S.; ''Germ-layer specification and control of cell growth by Ectodermin, a Smad4 ubiquitin ligase.''; PubMedEurope PMCScholia
Feng XH, Lin X, Derynck R.; ''Smad2, Smad3 and Smad4 cooperate with Sp1 to induce p15(Ink4B) transcription in response to TGF-beta.''; PubMedEurope PMCScholia
Luo K, Stroschein SL, Wang W, Chen D, Martens E, Zhou S, Zhou Q.; ''The Ski oncoprotein interacts with the Smad proteins to repress TGFbeta signaling.''; PubMedEurope PMCScholia
Levy L, Howell M, Das D, Harkin S, Episkopou V, Hill CS.; ''Arkadia activates Smad3/Smad4-dependent transcription by triggering signal-induced SnoN degradation.''; PubMedEurope PMCScholia
Wong C, Rougier-Chapman EM, Frederick JP, Datto MB, Liberati NT, Li JM, Wang XF.; ''Smad3-Smad4 and AP-1 complexes synergize in transcriptional activation of the c-Jun promoter by transforming growth factor beta.''; PubMedEurope PMCScholia
Kang JS, Liu C, Derynck R.; ''New regulatory mechanisms of TGF-beta receptor function.''; PubMedEurope PMCScholia
Kawabata M, Inoue H, Hanyu A, Imamura T, Miyazono K.; ''Smad proteins exist as monomers in vivo and undergo homo- and hetero-oligomerization upon activation by serine/threonine kinase receptors.''; PubMedEurope PMCScholia
Chacko BM, Qin BY, Tiwari A, Shi G, Lam S, Hayward LJ, De Caestecker M, Lin K.; ''Structural basis of heteromeric smad protein assembly in TGF-beta signaling.''; PubMedEurope PMCScholia
Kaji H, Canaff L, Lebrun JJ, Goltzman D, Hendy GN.; ''Inactivation of menin, a Smad3-interacting protein, blocks transforming growth factor type beta signaling.''; PubMedEurope PMCScholia
Dupont S, Mamidi A, Cordenonsi M, Montagner M, Zacchigna L, Adorno M, Martello G, Stinchfield MJ, Soligo S, Morsut L, Inui M, Moro S, Modena N, Argenton F, Newfeld SJ, Piccolo S.; ''FAM/USP9x, a deubiquitinating enzyme essential for TGFbeta signaling, controls Smad4 monoubiquitination.''; PubMedEurope PMCScholia
Heldin CH, ten Dijke P.; ''SMAD destruction turns off signalling.''; PubMedEurope PMCScholia
Nagano Y, Mavrakis KJ, Lee KL, Fujii T, Koinuma D, Sase H, Yuki K, Isogaya K, Saitoh M, Imamura T, Episkopou V, Miyazono K, Miyazawa K.; ''Arkadia induces degradation of SnoN and c-Ski to enhance transforming growth factor-beta signaling.''; PubMedEurope PMCScholia
Xiao Z, Latek R, Lodish HF.; ''An extended bipartite nuclear localization signal in Smad4 is required for its nuclear import and transcriptional activity.''; PubMedEurope PMCScholia
Chen YG, Wang Z, Ma J, Zhang L, Lu Z.; ''Endofin, a FYVE domain protein, interacts with Smad4 and facilitates transforming growth factor-beta signaling.''; PubMedEurope PMCScholia
Qin BY, Chacko BM, Lam SS, de Caestecker MP, Correia JJ, Lin K.; ''Structural basis of Smad1 activation by receptor kinase phosphorylation.''; PubMedEurope PMCScholia
Schmierer B, Hill CS.; ''Kinetic analysis of Smad nucleocytoplasmic shuttling reveals a mechanism for transforming growth factor beta-dependent nuclear accumulation of Smads.''; PubMedEurope PMCScholia
Nakao A, Imamura T, Souchelnytskyi S, Kawabata M, Ishisaki A, Oeda E, Tamaki K, Hanai J, Heldin CH, Miyazono K, ten Dijke P.; ''TGF-beta receptor-mediated signalling through Smad2, Smad3 and Smad4.''; PubMedEurope PMCScholia
Kurisaki A, Kose S, Yoneda Y, Heldin CH, Moustakas A.; ''Transforming growth factor-beta induces nuclear import of Smad3 in an importin-beta1 and Ran-dependent manner.''; PubMedEurope PMCScholia
Bonni S, Wang HR, Causing CG, Kavsak P, Stroschein SL, Luo K, Wrana JL.; ''TGF-beta induces assembly of a Smad2-Smurf2 ubiquitin ligase complex that targets SnoN for degradation.''; PubMedEurope PMCScholia
Tang LY, Yamashita M, Coussens NP, Tang Y, Wang X, Li C, Deng CX, Cheng SY, Zhang YE.; ''Ablation of Smurf2 reveals an inhibition in TGF-β signalling through multiple mono-ubiquitination of Smad3.''; PubMedEurope PMCScholia
Canaff L, Vanbellinghen JF, Kaji H, Goltzman D, Hendy GN.; ''Impaired transforming growth factor-β (TGF-β) transcriptional activity and cell proliferation control of a menin in-frame deletion mutant associated with multiple endocrine neoplasia type 1 (MEN1).''; PubMedEurope PMCScholia
Sun Y, Liu X, Eaton EN, Lane WS, Lodish HF, Weinberg RA.; ''Interaction of the Ski oncoprotein with Smad3 regulates TGF-beta signaling.''; PubMedEurope PMCScholia
Koinuma D, Shinozaki M, Komuro A, Goto K, Saitoh M, Hanyu A, Ebina M, Nukiwa T, Miyazawa K, Imamura T, Miyazono K.; ''Arkadia amplifies TGF-beta superfamily signalling through degradation of Smad7.''; PubMedEurope PMCScholia
Melhuish TA, Gallo CM, Wotton D.; ''TGIF2 interacts with histone deacetylase 1 and represses transcription.''; PubMedEurope PMCScholia
Zhang S, Fei T, Zhang L, Zhang R, Chen F, Ning Y, Han Y, Feng XH, Meng A, Chen YG.; ''Smad7 antagonizes transforming growth factor beta signaling in the nucleus by interfering with functional Smad-DNA complex formation.''; PubMedEurope PMCScholia
Stroschein SL, Wang W, Zhou S, Zhou Q, Luo K.; ''Negative feedback regulation of TGF-beta signaling by the SnoN oncoprotein.''; PubMedEurope PMCScholia
Dai F, Duan X, Liang YY, Lin X, Feng XH.; ''Coupling of dephosphorylation and nuclear export of Smads in TGF-beta signaling.''; PubMedEurope PMCScholia
Wang W, Yang L, Hu L, Li F, Ren L, Yu H, Liu Y, Xia L, Lei H, Liao Z, Zhou F, Xie C, Zhou Y.; ''Inhibition of UBE2D3 expression attenuates radiosensitivity of MCF-7 human breast cancer cells by increasing hTERT expression and activity.''; PubMedEurope PMCScholia
Pestov NB, Ahmad N, Korneenko TV, Zhao H, Radkov R, Schaer D, Roy S, Bibert S, Geering K, Modyanov NN.; ''Evolution of Na,K-ATPase beta m-subunit into a coregulator of transcription in placental mammals.''; PubMedEurope PMCScholia
The TGF-beta/BMP pathway incorporates several signaling pathways that share most, but not all, components of a central signal transduction engine. The general signaling scheme is rather simple: upon binding of a ligand, an activated plasma membrane receptor complex is formed, which passes on the signal towards the nucleus through a phosphorylated receptor SMAD (R-SMAD). In the nucleus, the activated R-SMAD promotes transcription in complex with a closely related helper molecule termed Co-SMAD (SMAD4). However, this simple linear pathway expands into a network when various regulatory components and mechanisms are taken into account. The signaling pathway includes a great variety of different TGF-beta/BMP superfamily ligands and receptors, several types of the R-SMADs, and functionally critical negative feedback loops. The R-SMAD:Co-SMAD complex can interact with a great number of transcriptional co-activators/co-repressors to regulate positively or negatively effector genes, so that the interpretation of a signal depends on the cell-type and cross talk with other signaling pathways such as Notch, MAPK and Wnt. The pathway plays a number of different biological roles in the control of embryonic and adult cell proliferation and differentiation, and it is implicated in a great number of human diseases. TGF beta (TGFB1) is secreted as a homodimer, and as such it binds to TGF beta receptor II (TGFBR2), inducing its dimerization. Binding of TGF beta enables TGFBR2 to form a stable hetero-tetrameric complex with TGF beta receptor I homodimer (TGFBR1). TGFBR2 acts as a serine/threonine kinase and phosphorylates serine and threonine residues within the short GS domain (glycine-serine rich domain) of TGFBR1. The phosphorylated heterotetrameric TGF beta receptor complex (TGFBR) internalizes into clathrin coated endocytic vesicles where it associates with the endosomal membrane protein SARA. SARA facilitates the recruitment of cytosolic SMAD2 and SMAD3, which act as R-SMADs for TGF beta receptor complex. TGFBR1 phosphorylates recruited SMAD2 and SMAD3, inducing a conformational change that promotes formation of R-SMAD trimers and dissociation of R-SMADs from the TGF beta receptor complex. In the cytosol, phosphorylated SMAD2 and SMAD3 associate with SMAD4 (known as Co-SMAD), forming a heterotrimer which is more stable than the R-SMAD homotrimers. R-SMAD:Co-SMAD heterotrimer translocates to the nucleus where it directly binds DNA and, in cooperation with other transcription factors, regulates expression of genes involved in cell differentiation, in a context-dependent manner. The intracellular level of SMAD2 and SMAD3 is regulated by SMURF ubiquitin ligases, which target R-SMADs for degradation. In addition, nuclear R-SMAD:Co-SMAD heterotrimer stimulates transcription of inhibitory SMADs (I-SMADs), forming a negative feedback loop. I-SMADs bind the phosphorylated TGF beta receptor complexes on caveolin coated vesicles, derived from the lipid rafts, and recruit SMURF ubiquitin ligases to TGF beta receptors, leading to ubiquitination and degradation of TGFBR1. Nuclear R-SMAD:Co-SMAD heterotrimers are targets of nuclear ubiquitin ligases which ubiquitinate SMAD2/3 and SMAD4, causing heterotrimer dissociation, translocation of ubiquitinated SMADs to the cytosol and their proteasome-mediated degradation. For a recent review of TGF-beta receptor signaling, please refer to Kang et al. 2009.
Complex formed by RBL1 (p107), E2F4/5, DP1/2 and a trimer of phosphorylated R-SMADs (SMAD2/3) and SMAD4 (Co-SMAD) cooperatively binds to TIE (TGF-beta inhibitory element) and E2F sites in the MYC promoter and promotes cell-cycle independent inhibition of MYC transcription in response to TGF-beta stimulation (Chen et al. 2002).
The phosphorylated C-terminal tail of R-SMAD induces a conformational change in the MH2 domain (Qin et al. 2001, Chacko et al. 2004), which now acquires high affinity towards Co-SMAD i.e. SMAD4 (common mediator of signal transduction in TGF-beta/BMP signaling). The R-SMAD:Co-SMAD complex (Nakao et al. 1997) most likely is a trimer of two R-SMADs with one Co-SMAD (Kawabata et al. 1998). It is important to note that the Co-SMAD itself cannot be phosphorylated as it lacks the C-terminal serine motif.
ZFYVE16 (endofin) promotes SMAD heterotrimer formation. ZFYVE16 can bind TGFBR1 and facilitate SMAD2 phosphorylation, and it can also bind SMAD4, but the exact mechanism of ZFYVE16 (endofin) action in the context of TGF-beta receptor signaling is not known (Chen et al. 2007).
SKI and SKIL (SNO) are able to recruit NCOR and possibly other transcriptional repressors to SMAD2/3:SMAD4 complex, inhibiting SMAD2/3:SMAD4-mediated transcription (Sun et al. 1999, Luo et al. 1999, Strochein et al. 1999). Experimental findings suggest that SMAD2 and SMAD3 may target SKI and SKIL for degradation (Strochein et al. 1999, Sun et al. 1999 PNAS, Bonni et al. 2001), and that the ratio of SMAD2/3 and SKI/SKIL determines the outcome (inhibition of SMAD2/3:SMAD4-mediated transcription or degradation of SKI/SKIL). SKI and SKIL are overexpressed in various cancer types and their oncogenic effect is connected with their ability to inhibit signaling by TGF-beta receptor complex.
The phosphorylated R-SMAD:CO-SMAD complex rapidly translocates to the nucleus (Xu et al. 2000, Kurisaki et al. 2001, Xiao et al. 2003) where it binds directly to DNA and interacts with a plethora of transcription co-factors. Regulation of target gene expression can be either positive or negative. A classic example of a target gene of the pathway are the genes encoding for I-SMADs. Thus, TGF-beta/SMAD signaling induces the expression of the negative regulators of the pathway (negative feedback loop).
The nuclear R-SMAD:Co-SMAD complex recruits ubiquitin conjugating enzymes, such as UBE2D1 and UBE2D3, that ubiquitinate the complex and eventually lead to its proteasomal degradation. This provides an end point to the signaling pathway.
In the cytosol of human embryonic stem cells, WWTR1 (TAZ) binds heterotrimer composed of two R-SMADs (SMAD2 and/or SMAD3) and SMAD4. This interaction involves the C-terminus of WWTR1 (TAZ) and the MH1 domain of SMAD proteins.
In the nucleus, protein type 2C phosphatase, PPM1A, dephosphorylates SMAD2 and SMAD3, resulting in dissociation of SMAD2/3:SMAD4 heterotrimeric complexes.
TGF-beta-dependent nuclear accumulation of SMAD2/3 and SMAD4 is mediated by WWTR1 (TAZ). WWTR1 does not affect phosphorylation of SMAD2/3 or the formation of SMAD2/3:SMAD4 trimers.
Knocking down WWTR1 (TAZ) expression by siRNA treatment inhibits TGF-beta-dependent transcription of SERPINE1 (PAI-1 i.e. plasminogen activator inhibitor 1) in hepatocellular carcinoma cell line Hep G2. Chromatin immunoprecipitation (ChIP) confirmed binding of both WWTR1 and SMAD2/3 to the promoter of SERPINE1 gene in response to TGF-beta stimulation (Varelas et al. 2008). Binding of TGIF1 or TGIF2 to SMAD2/3:SMAD4 heterotrimer negatively regulates transcription of SERPINE1 (Wotton et al. 1999, Melhuish et al. 2001). SMAD7 can also bind PAI-1 promoter (and probably other TGF-beta-responsive promoters) and inhibit PAI-1 expression, probably by competing with SMAD2/3:SMAD4 binding (Zhang et al. 2007).
Knocking down WWTR1 (TAZ) expression by siRNA treatment inhibits TGF-beta-dependent transcription of SMAD7 in human hepatocellular carcinoma cell line Hep G2. Chromatin immunoprecipitation (ChIP) confirmed binding of both WWTR1 and SMAD2/3 to the promoter of SMAD7 gene in response to TGF-beta stimulation (Varelas et al. 2008). In fish, amphibian and avian species, ATP1B4 (aka X,K ATPase subunit beta m) functions as a subunit of Na/K ATPase, located in the plasma membrane. In mammals, this functionality is lost and ATP1B4 accumulates on the nuclear envelope where it can interact with SNW domain containing protein 1 (SNW1 aka Ski interacting protein, SKIP), a transcriptional regulator. This ATP1B4:SNW1 complex is able to modulate TGF beta mediated transcription by increasing mRNA levels of SMAD7, an inhibitor of TGF beta signalling (Pestov et al. 2007).
In response to TGF-beta stimulation, a complex composed of SMAD2/3:SMAD4 heterotrimer and RBL1 (p107), E2F4/5 and DP1/2 can be detected in the nucleus. Formation of this complex was confirmed by both co-immunoprecipitation of the endogenous complex from the human keratinocyte cell line HaCat and by protein interaction studies using tagged recombinant proteins. It is possible that cells contain pre-assembled cytosolic complexes of SMAD2/3, RBL1 (p107) and E2F4/5, that translocate to the nucleus after TGF-beta stimulation, when phosphorylated SMAD2/3 recruit SMAD4. The MH2 domain of SMAD3 establishes independent contacts with the N-termini of E2F4 (or E2F5) and unphosphorylated RBL1 (p107). RBL2 (p130), RB1 and E2F1 do not interact with SMAD2/3 (Chen et al. 2002).
CDK8 in complex with cyclin C (CDK8:CCNC) and CDK9 in complex with cyclin T (CDK9:CCNT) are able to phosphorylate the linker region of SMAD2 and SMAD3. In SMAD3, CDK8/CDK9 preferentially targets threonine residue T179, although serine residues S208 and S213 can also be phosphorylated. In SMAD2, CDK8/9 preferentially targets threonine residue T220 (corresponds to T190 in the short isoform of SMAD2, SMAD2-2). Phosphorylation of serine residues that correspond to serines S208 and S213 of SMAD3 has not been examined. Phosphorylation of the linker region of SMAD2 and SMAD3 by CDK8/CDK9 enhances transcriptional activity of SMAD2/3:SMAD4 complex, but also primes SMAD2 and SMAD3 for ubiquitination and subsequent degradation (Alarcon et al. 2009).
SMURF2 binds SMAD2/3:SMAD4 heterotrimer through ineraction with SMAD3. Phosphorylation of threonine T179 in the linker region of SMAD3 is critical for SMURF2 binding. SMURF2 also interacts with SMAD2 phosphorylated in the linker region.
SMURF2 monoubiquitinates SMAD3 on lysine residues in the MH2 domain. Lysines K333 and K378 are likely the major sites for monoubiquitination. Lysine K409 is also monoubiquitinated, and possibly lysine K341. Since lysines K333 and K378 are predicted to stabilize the interaction of SMAD3 with SMAD4, monoubiquitination of these lysine residues is expected to disrupt SMAD2/3:SMAD4 heterotrimer. SMURF2-mediated disruption of endogenous Smad2/3:Smad4 heterotrimers was demonstrated in mouse embryonic fibroblasts. SMURF2 also ubiquitinates SMAD2 phosphorylated in the linker region, but loss of Smurf2 has less impact on Smad2 ubiquitination than on Smad3 in vivo.
Transcriptional repressors TGIF1 and TGIF2 bind SMAD2/3:SMAD4 heterotrimer through interaction with SMAD2 and/or SMAD3. TGIF1 and TGIF2 recruit hystone deacetylase HDAC1 to SMAD2/3:SMAD4 heterotrimer.
MEN1 (menin), a transcription factor tumor suppressor mutated in a familial cancer syndrome multiple endocrine neoplasia type 1, binds SMAD2/3:SMAD4 heterotrimer through interaction with SMAD3. MEN1 likely acts as a trancriptional cofactor for SMAD2/3:SMAD4 and may be involved in transcriptional regulation of some SMAD2/3:SMAD4 target genes (Kaji et al. 2001, Sowa et al. 2004, Canaff et al. 2012).
After phosphorylated SMAD2/3 accumulate in the nucleus in response to TGF-beta stimulation, E3 ubiqutin ligases RNF111 (Arkadia) (Levy et al. 2007) and SMURF2 (Bonni et al. 2001) bind SKI/SKIL in complex with SMAD2/3:SMAD4 heterotrimer. The role of RNF111 was inferred from experiments that used recombinant mouse RNF111 and endogenous human SMADs and SKI/SKIL (Levy et al. 2007). The role of SMURF2 was inferred from experiments involving human proteins only (Bonni et al. 2001).
E3 ubiqutin ligases RNF111 (Arkadia) (Levy et al. 2007, Nagano et al. 2007) and SMURF2 (Bonni et al. 2001) ubiquitinate SKI/SKIL transcriptional repressors bound to activated SMAD2/3. The role of RNF111 was inferred from experiments that used recombinant mouse RNF111 and endogenous human SMADs and SKI/SKIL. The role of SMURF2 was inferred from experiments involving human proteins only.
SKI/SKIL ubiquitinated by RNF111 (Levy et al. 2007, Nagano et al. 2007) or SMURF2 (Bonni et al. 2001) are degraded in a proteasome dependent way, enabling transcription of SMAD2/3:SMAD4 target genes.
After ubiquitination by RNF111 (Arkadia), ubiquitinated SMAD7 is degraded in a proteasome dependent way. Therefore, RNF111 has a positive effect on trancription initiated by TGF-beta signaling. However, TGF-beta signaling ultimately results in a decrease of RNF111 mRNA level, enabling negative-feedback regulation of TGF-beta signal by SMAD7. RNF111 may fine tune the duration of cellular response to TGF-beta signal. Initially, RNF111 may enable signal propagation by inhibiting negative regulators of TGF-beta signaling, SKI/SKIL and SMAD7. Subsequent negative regulation of RNF111 expression by TGF-beta may allow the signaling cascade to be turned off (Koinuma et al. 2003).
RNF111 (Arkadia) polyubiquitinates SMAD7 (Koinuma et al. 2003). This was inferred from studies using recombinant mouse Smad7 and recombinant mouse Rnf111 expressed in human embryonic kidney cell line HEK293.
SMAD2/3:SMAD4 heterotrimer binds SMAD binding site in the promoter of JUNB transcription factor and in cooperation with AP-1 transcription factors, which bind to an adjacent promoter element, stimulates transcription of JUNB gene (Wong et al. 1999).
SMAD2/3:SMAD4:SP1 complex binds SP1 and SMAD promoter elements of CDKN2B (p15-INK4B) gene and stimulates transcription of CDKN2B. CDKN2B inhibits the action of cyclin-dependent kinases CDK4 and CDK6 and may be an effector of TGF-beta induced cell cycle arrest (Feng et al. 2000).
TGF-beta (TGFB1) stimulates formation of a complex of SP1 transcription factor and SMAD2/3:SMAD4 heterotrimer. SMAD2 and SMAD4 bind to SP1 directly, through their C- and N-terminus, respectively (Feng et al. 2000).
PARP1 ADP-ribosylates SMAD3 and SMAD4 in SMAD2/3:SMAD4 heterotrimer. ADP-ribosyl group is attached to glutamic acid residues E50 and E52 of SMAD3 and unknown amino acid residues of SMAD4. ADP-ribose monomer attached to SMAD3 and SMAD4 is subsequently extended to poly (ADP-ribosyl) chains (i.e. PAR chains) by PARP1, which is not shown here. ADP-ribosylation (PARylation) of SMAD3 and SMAD4 by PARP1 inhibits binding of SMAD2/3:SMAD4 heterotrimers to SMAD binding elements (SBEs) in promoters of SMAD-target genes.
In fish, amphibian and avian species, ATP1B4 (aka X,K-ATPase subunit beta-m) functions as a subunit of Na/K-ATPase, located in the plasma membrane. In mammals, this functionality is lost and ATP1B4 accumulates on the nuclear envelope where it can interact with SNW domain-containing protein 1 (SNW1 aka Ski-interacting protein, SKIP), a transcriptional regulator. This ATP1B4:SNW1 complex is able to modulate TGF-beta-mediated transcription by increasing mRNA levels of SMAD7, an inhibitor of TGF-beta signalling (Pestov et al. 2007).
TRIM33 (also known as Ecto, Ectodermin or Tif1-gamma) monoubiquitinates nuclear SMAD4 on lysine residue K519. This leads to disruption of heterotrimeric complexes composed of SMAD4 and two phosphorylated R-SMADs (SMAD2 and/or SMAD3). TRIM33 inhibits SMAD activity without affecting steady state levels of SMAD4 (Dupont et al. 2009, Dupont et al. 2005).
E3 ubiquitin protein ligase TRIM33 (also known as Ecto, Ectodermin or Tif1-gamma) binds to the SMAD heterotrimer, composed of SMAD4 and two phosphorylated R-SMADs (SMAD2 and/or SMAD3), in the nucleus (Dupont et al. 2009, Dupont et al. 2005, He et al. 2006).
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TGF-beta Receptor
ComplexTGF beta (TGFB1) is secreted as a homodimer, and as such it binds to TGF beta receptor II (TGFBR2), inducing its dimerization. Binding of TGF beta enables TGFBR2 to form a stable hetero-tetrameric complex with TGF beta receptor I homodimer (TGFBR1). TGFBR2 acts as a serine/threonine kinase and phosphorylates serine and threonine residues within the short GS domain (glycine-serine rich domain) of TGFBR1.
The phosphorylated heterotetrameric TGF beta receptor complex (TGFBR) internalizes into clathrin coated endocytic vesicles where it associates with the endosomal membrane protein SARA. SARA facilitates the recruitment of cytosolic SMAD2 and SMAD3, which act as R-SMADs for TGF beta receptor complex. TGFBR1 phosphorylates recruited SMAD2 and SMAD3, inducing a conformational change that promotes formation of R-SMAD trimers and dissociation of R-SMADs from the TGF beta receptor complex.
In the cytosol, phosphorylated SMAD2 and SMAD3 associate with SMAD4 (known as Co-SMAD), forming a heterotrimer which is more stable than the R-SMAD homotrimers. R-SMAD:Co-SMAD heterotrimer translocates to the nucleus where it directly binds DNA and, in cooperation with other transcription factors, regulates expression of genes involved in cell differentiation, in a context-dependent manner.
The intracellular level of SMAD2 and SMAD3 is regulated by SMURF ubiquitin ligases, which target R-SMADs for degradation. In addition, nuclear R-SMAD:Co-SMAD heterotrimer stimulates transcription of inhibitory SMADs (I-SMADs), forming a negative feedback loop. I-SMADs bind the phosphorylated TGF beta receptor complexes on caveolin coated vesicles, derived from the lipid rafts, and recruit SMURF ubiquitin ligases to TGF beta receptors, leading to ubiquitination and degradation of TGFBR1. Nuclear R-SMAD:Co-SMAD heterotrimers are targets of nuclear ubiquitin ligases which ubiquitinate SMAD2/3 and SMAD4, causing heterotrimer dissociation, translocation of ubiquitinated SMADs to the cytosol and their proteasome-mediated degradation. For a recent review of TGF-beta receptor signaling, please refer to Kang et al. 2009.
Annotated Interactions
ZFYVE16 (endofin) promotes SMAD heterotrimer formation. ZFYVE16 can bind TGFBR1 and facilitate SMAD2 phosphorylation, and it can also bind SMAD4, but the exact mechanism of ZFYVE16 (endofin) action in the context of TGF-beta receptor signaling is not known (Chen et al. 2007).
In fish, amphibian and avian species, ATP1B4 (aka X,K ATPase subunit beta m) functions as a subunit of Na/K ATPase, located in the plasma membrane. In mammals, this functionality is lost and ATP1B4 accumulates on the nuclear envelope where it can interact with SNW domain containing protein 1 (SNW1 aka Ski interacting protein, SKIP), a transcriptional regulator. This ATP1B4:SNW1 complex is able to modulate TGF beta mediated transcription by increasing mRNA levels of SMAD7, an inhibitor of TGF beta signalling (Pestov et al. 2007).
RNF111 may fine tune the duration of cellular response to TGF-beta signal. Initially, RNF111 may enable signal propagation by inhibiting negative regulators of TGF-beta signaling, SKI/SKIL and SMAD7. Subsequent negative regulation of RNF111 expression by TGF-beta may allow the signaling cascade to be turned off (Koinuma et al. 2003).