SMAD4, also calledSMAD family member 4,Mothers against decapentaplegic homolog 4, orDPC4 (Deleted in Pancreatic Cancer-4) is a highly conserved protein present in allmetazoans. It belongs to theSMAD family oftranscription factor proteins, which act as mediators of TGF-β signal transduction. TheTGFβ family of cytokines regulates critical processes during the lifecycle of metazoans, with important roles during embryo development, tissue homeostasis, regeneration, and immune regulation.[5]
SMAD 4 belongs to theco-SMAD group (common mediator SMAD), the second class of the SMAD family. SMAD4 is the only known co-SMAD in most metazoans. It also belongs to theDwarfin family of proteins that modulate members of theTGFβ protein superfamily, a family of proteins that all play a role in the regulation of cellular responses. Mammalian SMAD4 is ahomolog of theDrosophila protein "Mothers against decapentaplegic" named Medea.[6]
SMAD4 interacts with R-Smads, such asSMAD2,SMAD3,SMAD1,SMAD5 andSMAD9 (also called SMAD8) to form heterotrimeric complexes. Transcriptional coregulators, such asWWTR1 (TAZ) interact with SMADs to promote their function. Once in the nucleus, the complex of SMAD4 and two R-SMADS binds toDNA and regulates the expression of different genes depending on the cellular context.[6] Intracellular reactions involving SMAD4 are triggered by the binding, on the surface of the cells, of growth factors from theTGFβ family. The sequence of intracellular reactions involving SMADS is called the SMAD pathway or the transforming growth factor beta (TGF-β) pathway since the sequence starts with the recognition of TGF-β by cells.
In humans, theSMAD4 gene contains 54 829 base pairs and is located from pair n° 51,030,212 to pair 51,085,041 in the region 21.1 of the chromosome 18.[7][8]
Pattern of the chromosome 18 inHomo sapiens. TheSMAD 4 gene is located on the long arm of the chromosome, at locus 21.1. This locus corresponds to the black stripe between the regions 12.3 and 21.2.
SMAD4 is a 552 amino-acidpolypeptide with a molecular weight of 60.439 Da. SMAD4 has two functional domains known asMH1 andMH2.
SMAD 4 is composed of three major domains, including MH1 (up), MH2 (down) and a linking domain (right).
The complex of two SMAD3 (or of two SMAD2) and one SMAD4 binds directly to DNA though interactions of their MH1 domains. These complexes are recruited to sites throughout the genome by cell lineage-defining transcription factors (LDTFs) that determine the context-dependent nature of TGF-β action. Early insights into the DNA binding specificity of Smad proteins came from oligonucleotide binding screens, which identified the palindromic duplex 5'–GTCTAGAC–3' as a high affinity binding sequence for SMAD3 and SMAD4 MH1 domains.[9] Other motifs have also been identified in promoters and enhancers. These additional sites contain the CAGCC motif and the GGC(GC)|(CG) consensus sequences, the latter also known as 5GC sites.[10] The 5GC-motifs are highly represented as clusters of sites, in SMAD-bound regions genome-wide. These clusters can also contain CAG(AC)|(CC) sites. SMAD3/SMAD4 complex also binds to the TPA-responsive gene promoter elements, which have the sequence motif TGAGTCAG.[11]
The first structure of SMAD4 bound to DNA was the complex with the palindromic GTCTAGAC motif.[12] Recently, the structures of SMAD4 MH1 domain bound to several 5GC motifs have also been determined. In all complexes, the interaction with the DNA involves a conservedβ-hairpin present in the MH1 domain. The hairpin is partially flexible in solution and its high degree of conformational flexibility allows recognition of the different 5-bp sequences. Efficient interactions with GC-sites occur only if a G nucleotide is located deep in the major grove, and establishes hydrogen bonds with the guanidinium group of Arg81. This interaction facilitates a complementary surface contact between the Smad DNA-binding hairpin and the major groove of the DNA. Other direct interactions involve Lys88 and Gln83. The X-ray crystal structure of theTrichoplax adhaerens SMAD4 MH1 domains bound to the GGCGC motif indicates a high conservation of this interaction in metazoans.[10]
Smad4 MH1 domain bound to the GGCT DNA motif, fromPDB:5MEZClose-up view of the Smad4 MH1 domain bound to the GGCGC DNA motif, fromPDB:5MEYSmad4 MH1 domain bound to the GGCGC DNA motif, fromPDB:5MEY
The MH2 domain, corresponding to theC-terminus, is responsible for receptor recognition and association with other SMADs. It interacts with the R-SMADS MH2 domain and formsheterodimers andheterotrimers. Some tumor mutations detected in SMAD4 enhance interactions between the MH1 and MH2 domains.[13]
SMADs are highly conserved across species, especially in theN terminalMH1 domain and theC terminalMH2 domain.The SMAD proteins are homologs of both theDrosophila protein MAD and theC. elegans protein SMA. The name is a combination of the two. DuringDrosophila research, it was found that a mutation in the geneMAD in the mother repressed the genedecapentaplegic in the embryo. The phrase "Mothers against" was added, since mothers often form organizations opposing various issues, e.g.Mothers Against Drunk Driving (MADD), reflecting "the maternal-effect enhancement ofdpp";[14] and based on a tradition of unusual naming within the research community.[15] SMAD4 is also known as DPC4, JIP or MADH4.
SMAD4 is a protein defined as an essential effector in the SMAD pathway. SMAD4 serves as a mediator between extracellular growth factors from the TGFβ family and genes inside the cellnucleus. The abbreviationco in co-SMAD stands forcommon mediator. SMAD4 is also defined as a signal transducer.
In the TGF-β pathway, TGF-β dimers are recognized by a transmembrane receptor, known as type II receptor. Once the type II receptor is activated by the binding of TGF-β, it phosphorylates a type I receptor. Type I receptor is also acell surface receptor. This receptor then phosphorylates intracellular receptor regulated SMADS (R-SMADS) such as SMAD2 or SMAD3. The phosphorylated R-SMADS then bind to SMAD4. The R-SMADs-SMAD4 association is aheteromeric complex. This complex is going to move from the cytoplasm to the nucleus: it is the translocation. SMAD4 may form heterotrimeric, heterohexameric or heterodimeric complexes with R-SMADS.
SMAD4 is a substrate of theErk/MAPK kinase[16] andGSK3.[17] The FGF (Fibroblast Growth Factor) pathway stimulation leads to Smad4phosphorylation byErk of the canonicalMAPK site located at Threonine 277. This phosphorylation event has a dual effect on Smad4 activity. First, it allows Smad4 to reach its peak of transcriptional activity by activating agrowth factor-regulated transcription activation domain located in the Smad4 linker region, SAD (Smad-Activation Domain).[18] Second,MAPK primes Smad4 forGSK3-mediated phosphorylations that cause transcriptional inhibition and also generate a phosphodegron used as a docking site by the ubiquitinE3 ligase Beta-transducin Repeat Containing (beta-TrCP) that polyubiquitinates Smad4 and targets it for degradation in theproteasome.[19] Smad4GSK3 phosphorylations have been proposed to regulate the protein stability during pancreatic andcolon cancer progression.[20]
In the nucleus the heteromeric complex binds promoters and interact with transcriptional activators.SMAD3/SMAD4 complexes can directly bind the SBE. These associations are weak and require additionaltranscription factors such as members of theAP-1 family,TFE3 andFoxG1 to regulategene expression.[21]
Genetic experiments such asgene knockout (KO), which consist in modifying or inactivating a gene, can be carried out in order to see the effects of a dysfunctional SMAD 4 on the study organism. Experiments are often conducted in the house mouse (Mus musculus).
Deletions in the genes coding for SMAD1 andSMAD5 have also been linked to metastasic granulosa cell tumors in mice.[24]
SMAD4, is often found mutated in many cancers. The mutation can be inherited or acquired during an individual's lifetime. If inherited, the mutation affects bothsomatic cells and cells of the reproductive organs. If theSMAD 4 mutation is acquired, it will only exist in certain somatic cells. Indeed, SMAD 4 is not synthesized by all cells. The protein is present in skin, pancreatic, colon, uterus and epithelial cells. It is also produced byfibroblasts. The functional SMAD 4 participates in the regulation of the TGF-β signal transduction pathway, which negatively regulates growth of epithelial cells and theextracellular matrix (ECM). When the structure of SMAD 4 is altered, expression of the genes involved in cell growth is no longer regulated and cell proliferation can go on without any inhibition. The important number of cell divisions leads to the forming of tumors and then to multiploidcolorectal cancer andpancreatic carcinoma. It is found inactivated in at least 50% of pancreatic cancers.[25]
Somatic mutations found in human cancers of the MH1 domain of SMAD 4 have been shown to inhibit the DNA-binding function of this domain.
SMAD 4 is also found mutated in theautosomal dominant diseasejuvenile polyposis syndrome (JPS). JPS is characterized by hamartomatous polyps in the gastrointestinal (GI) tract. These polyps are usually benign, however they are at greater risk of developinggastrointestinal cancers, in particularcolon cancer.Around 60 mutations causing JPS have been identified. They have been linked to the production of a smaller SMAD 4, with missing domains that prevent the protein from binding to R-SMADS and formingheteromeric complexes.[8]
Mutations inSMAD4 (mostly substitutions) can causeMyhre syndrome, a rare inherited disorder characterized by mental disabilities, short stature, unusual facial features, and various bone abnormalities.[26][27]
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^Caputo V, Bocchinfuso G, Castori M, Traversa A, Pizzuti A, Stella L, et al. (July 2014). "Novel SMAD4 mutation causing Myhre syndrome".American Journal of Medical Genetics. Part A.164A (7):1835–1840.doi:10.1002/ajmg.a.36544.PMID24715504.S2CID5294309.
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