Signaling by NODAL is essential for patterning of the axes of the embryo and formation of mesoderm and endoderm (reviewed in Schier 2009, Shen 2007). The NODAL proprotein is secreted and cleaved extracellularly to yield mature NODAL. Mature NODAL homodimerizes and can also form heterodimers with LEFTY1, LEFTY2, or CERBERUS, which negatively regulate NODAL signaling. NODAL also forms heterodimers with GDF1, which increases NODAL activity. NODAL dimers bind the NODAL receptor comprising a type I Activin receptor (ACVR1B or ACVR1C), a type II Activin receptor (ACVR2A or ACVR2B), and an EGF-CFC coreceptor (CRIPTO or CRYPTIC). After binding NODAL, the type II activin receptor phosphorylates the type I activin receptor which then phosphorylates SMAD2 and SMAD3 (R-SMADs). Phosphorylated SMAD2 and SMAD3 form hetero-oligomeric complexes with SMAD4 (CO-SMAD) and transit from the cytosol to the nucleus. Within the nucleus the SMAD complexes interact with transcription factors such as FOXH1 to activate transcription of target genes.
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Qin BY, Chacko BM, Lam SS, de Caestecker MP, Correia JJ, Lin K.; ''Structural basis of Smad1 activation by receptor kinase phosphorylation.''; PubMedEurope PMCScholia
Constam DB.; ''Regulation of TGFβ and related signals by precursor processing.''; 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
Xu L, Chen YG, Massagué J.; ''The nuclear import function of Smad2 is masked by SARA and unmasked by TGFbeta-dependent phosphorylation.''; 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
Fu G, Peng C.; ''Nodal enhances the activity of FoxO3a and its synergistic interaction with Smads to regulate cyclin G2 transcription in ovarian cancer cells.''; PubMedEurope PMCScholia
Nadeem L, Munir S, Fu G, Dunk C, Baczyk D, Caniggia I, Lye S, Peng C.; ''Nodal signals through activin receptor-like kinase 7 to inhibit trophoblast migration and invasion: implication in the pathogenesis of preeclampsia.''; PubMedEurope PMCScholia
Yeo C, Whitman M.; ''Nodal signals to Smads through Cripto-dependent and Cripto-independent mechanisms.''; PubMedEurope PMCScholia
Yanagisawa K, Uchida K, Nagatake M, Masuda A, Sugiyama M, Saito T, Yamaki K, Takahashi T, Osada H.; ''Heterogeneities in the biological and biochemical functions of Smad2 and Smad4 mutants naturally occurring in human lung cancers.''; 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
Reissmann E, Jörnvall H, Blokzijl A, Andersson O, Chang C, Minchiotti G, Persico MG, Ibáñez CF, Brivanlou AH.; ''The orphan receptor ALK7 and the Activin receptor ALK4 mediate signaling by Nodal proteins during vertebrate development.''; PubMedEurope PMCScholia
Zhong Y, Xu G, Ye G, Lee D, Modica-Amore J, Peng C.; ''Nodal and activin receptor-like kinase 7 induce apoptosis in human breast cancer cell lines: Role of caspase 3.''; 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
Dai F, Duan X, Liang YY, Lin X, Feng XH.; ''Coupling of dephosphorylation and nuclear export of Smads in TGF-beta signaling.''; PubMedEurope PMCScholia
Kumar A, Novoselov V, Celeste AJ, Wolfman NM, ten Dijke P, Kuehn MR.; ''Nodal signaling uses activin and transforming growth factor-beta receptor-regulated Smads.''; PubMedEurope PMCScholia
Chen X, Weisberg E, Fridmacher V, Watanabe M, Naco G, Whitman M.; ''Smad4 and FAST-1 in the assembly of activin-responsive factor.''; PubMedEurope PMCScholia
Xu G, Zhong Y, Munir S, Yang BB, Tsang BK, Peng C.; ''Nodal induces apoptosis and inhibits proliferation in human epithelial ovarian cancer cells via activin receptor-like kinase 7.''; PubMedEurope PMCScholia
Wu JW, Hu M, Chai J, Seoane J, Huse M, Li C, Rigotti DJ, Kyin S, Muir TW, Fairman R, Massagué J, Shi Y.; ''Crystal structure of a phosphorylated Smad2. Recognition of phosphoserine by the MH2 domain and insights on Smad function in TGF-beta signaling.''; PubMedEurope PMCScholia
Chen X, Rubock MJ, Whitman M.; ''A transcriptional partner for MAD proteins in TGF-beta signalling.''; PubMedEurope PMCScholia
Zhou S, Zawel L, Lengauer C, Kinzler KW, Vogelstein B.; ''Characterization of human FAST-1, a TGF beta and activin signal transducer.''; PubMedEurope PMCScholia
Chen C, Shen MM.; ''Two modes by which Lefty proteins inhibit nodal signaling.''; PubMedEurope PMCScholia
Bondestam J, Huotari MA, Morén A, Ustinov J, Kaivo-Oja N, Kallio J, Horelli-Kuitunen N, Aaltonen J, Fujii M, Moustakas A, Ten Dijke P, Otonkoski T, Ritvos O.; ''cDNA cloning, expression studies and chromosome mapping of human type I serine/threonine kinase receptor ALK7 (ACVR1C).''; PubMedEurope PMCScholia
Jörnvall H, Reissmann E, Andersson O, Mehrkash M, Ibáñez CF.; ''ALK7, a receptor for nodal, is dispensable for embryogenesis and left-right patterning in the mouse.''; PubMedEurope PMCScholia
Bianco C, Adkins HB, Wechselberger C, Seno M, Normanno N, De Luca A, Sun Y, Khan N, Kenney N, Ebert A, Williams KP, Sanicola M, Salomon DS.; ''Cripto-1 activates nodal- and ALK4-dependent and -independent signaling pathways in mammary epithelial Cells.''; PubMedEurope PMCScholia
DaCosta Byfield S, Major C, Laping NJ, Roberts AB.; ''SB-505124 is a selective inhibitor of transforming growth factor-beta type I receptors ALK4, ALK5, and ALK7.''; PubMedEurope PMCScholia
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Yeo CY, Chen X, Whitman M.; ''The role of FAST-1 and Smads in transcriptional regulation by activin during early Xenopus embryogenesis.''; 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
Piccolo S, Agius E, Leyns L, Bhattacharyya S, Grunz H, Bouwmeester T, De Robertis EM.; ''The head inducer Cerberus is a multifunctional antagonist of Nodal, BMP and Wnt signals.''; 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
Munir S, Xu G, Wu Y, Yang B, Lala PK, Peng C.; ''Nodal and ALK7 inhibit proliferation and induce apoptosis in human trophoblast cells.''; PubMedEurope PMCScholia
Either FURIN or PACE4 endoproteases cleave the 321 amino acid NODAL proprotein to yield the 110 amino acid NODAL mature protein. In cultured mouse cells the CRIPTO coreceptor at the plasma membrane recruits both NODAL proprotein and FURIN or PACE4 endoprotease.
NODAL binds a receptor comprising a type I activin receptor (ACVR1B or ACVR1C), a type II activin receptor (ACVR2 or ACVR2B), and a EGF-CFC coreceptor (CRIPTO or CRYPTIC). Though NODAL is able to signal via the ACVR1C (ALK7) receptor (Reissman et al. 2001), experiments in mouse indicate NODAL signaling via ALK7 is dispensable during embryogenesis (Jornvall et al. 2004).
As inferred from the response of the activin receptor to activin, the type II component of the NODAL receptor phosphorylates the type I component in response to NODAL binding. Experiments with human proteins in frog oocytes show NODAL can signal via the CRIPTO:ACVR1B(ALK4):ACVR2 complex (Yeo and Whitman 2001).
LEFTY1 and LEFTY2 are able to inhibit NODAL signaling by binding the EGF-CFC coreceptor (CRIPTO or CRYPTIC) and thereby preventing the coreceptor from interacting with other components of the NODAL receptor.
As inferred from mouse (Chen and Shen 2004) both LEFTY1 and LEFTY2 can bind NODAL and inhibit NODAL signaling. The stoichiometry of the resulting LEFTY:NODAL complex is unknown.
As inferred from Xenopus (Piccolo 1999) and as observed in human cells (Aykul et al. 2015) CERBERUS binds NODAL and inhibits NODAL signaling. CERBERUS and NODAL are believed to bind as dimer to form a tetrameric complex (Aykul et al. 2015)
NODAL receptors signal by phosphorylating SMAD2 and SMAD3 (Bondestam et al. 2001, Kumar et al. 2001, DaCosta Byfield et al. 2004). As in TGF-beta signaling, Smad anchor for receptor activation (SARA) may bind and present SMAD2 and SMAD3 for phosphorylation but this has not yet been demonstrated in NODAL signaling.
As inferred from the response of the activin receptor to activin, the type II component of the NODAL receptor phosphorylates the type I component in response to NODAL binding. As inferred from mouse and frog (Xenopus) NODAL can signal via the ACVR1C (ALK7) type I activin receptor (Reissman et al. 2001) though this may be dispensable for development in mouse (Jornvall et al. 2004).
SMAD2 and SMAD3 do not bind DNA efficiently. They must interact with DNA-binding proteins to activate transcription. FOXH1 interacts with phospho-SMAD2 and phospho-SMAD3 complexed with CO-SMAD (SMAD4) at promoters containing the Activin Response Element (Zhou et al. 1998, Yanagisawa et al. 2000, inferred from Xenopus in Chen et al. 1996, Chen et al. 1997, Yeo et al. 1999). Follicle-stimulating hormone beta subunit (FSHB) and the Lim1 homeobox gene (LXH1) are examples of genes regulated by Activin.
FOXO3 (FOXO3A) interacts with phospho-SMAD2 and phospho-SMAD3 complexed with CO-SMAD (SMAD4) at a promoter containing the FoxO3a-binding Element (Fu and Peng 20110).
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).
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).
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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).