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SMAD4, also called SMAD family member 4, Mothers against decapentaplegic homolog 4, or DPC4 (Deleted in Pancreatic Cancer-4) is a highly conserved protein present in all metazoans. It belongs to the SMAD family of
transcription factor In molecular biology, a transcription factor (TF) (or sequence-specific DNA-binding factor) is a protein that controls the rate of transcription (genetics), transcription of genetics, genetic information from DNA to messenger RNA, by binding t ...
proteins, which act as mediators of TGF-β signal transduction. The TGFβ family of cytokines regulates critical processes during the lifecycle of metazoans, with important roles during embryo development, tissue homeostasis, regeneration, and immune regulation. SMAD 4 belongs to the co-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 the Dwarfin family of proteins that modulate members of the TGFβ protein superfamily, a family of proteins that all play a role in the regulation of cellular responses. Mammalian SMAD4 is a
homolog In biology, homology is similarity in anatomical structures or genes between organisms of different taxa due to shared ancestry, ''regardless'' of current functional differences. Evolutionary biology explains homologous structures as retained her ...
of the ''
Drosophila ''Drosophila'' (), from Ancient Greek δρόσος (''drósos''), meaning "dew", and φίλος (''phílos''), meaning "loving", is a genus of fly, belonging to the family Drosophilidae, whose members are often called "small fruit flies" or p ...
'' protein " Mothers against decapentaplegic" named Medea. SMAD4 interacts with R-Smads, such as
SMAD2 Mothers against decapentaplegic homolog 2, also known as SMAD family member 2 or SMAD2, is a protein that in humans is encoded by the ''SMAD2'' gene. MAD homolog 2 belongs to the SMAD, a family of proteins similar to the gene products of the ''Dr ...
,
SMAD3 Mothers against decapentaplegic homolog 3 also known as SMAD family member 3 or SMAD3 is a protein that in humans is encoded by the SMAD3 gene. SMAD3 is a member of the SMAD (protein), SMAD family of proteins. It acts as a mediator of the signal ...
,
SMAD1 Mothers against decapentaplegic homolog 1 also known as SMAD family member 1 or SMAD1 is a protein that in humans is encoded by the ''SMAD1'' gene. Nomenclature SMAD1 belongs to the SMAD (protein), SMAD, a family of proteins similar to the gene p ...
, SMAD5 and SMAD9 (also called SMAD8) to form heterotrimeric complexes. Transcriptional coregulators, such as WWTR1 (TAZ) interact with SMADs to promote their function. Once in the nucleus, the complex of SMAD4 and two R-SMADS binds to
DNA Deoxyribonucleic acid (; DNA) is a polymer composed of two polynucleotide chains that coil around each other to form a double helix. The polymer carries genetic instructions for the development, functioning, growth and reproduction of al ...
and regulates the expression of different genes depending on the cellular context. Intracellular reactions involving SMAD4 are triggered by the binding, on the surface of the cells, of growth factors from the TGFβ 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.


Gene

In humans, the ''SMAD4'' 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.


Protein

SMAD4 is a 552 amino-acid
polypeptide Peptides are short chains of amino acids linked by peptide bonds. A polypeptide is a longer, continuous, unbranched peptide chain. Polypeptides that have a molecular mass of 10,000 Da or more are called proteins. Chains of fewer than twenty ...
with a molecular weight of 60.439  Da. SMAD4 has two functional domains known as MH1 and MH2. 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. 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. 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.


Structures


MH1 domain complexes with DNA motifs

The first structure of SMAD4 bound to DNA was the complex with the palindromic GTCTAGAC motif. 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 conserved β-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 the ''
Trichoplax adhaerens ''Trichoplax adhaerens'' is one of the four named species in the phylum Placozoa. The others are ''Hoilungia hongkongensis'', ''Polyplacotoma mediterranea'' and ''Cladtertia collaboinventa''. Placozoa is a basal group of multicellular animals, p ...
'' SMAD4 MH1 domains bound to the GGCGC motif indicates a high conservation of this interaction in metazoans.


MH2 domain complexes

The MH2 domain, corresponding to the
C-terminus The C-terminus (also known as the carboxyl-terminus, carboxy-terminus, C-terminal tail, carboxy tail, C-terminal end, or COOH-terminus) is the end of an amino acid chain (protein Proteins are large biomolecules and macromolecules that comp ...
, is responsible for receptor recognition and association with other SMADs. It interacts with the R-SMADS MH2 domain and forms
heterodimers In biochemistry, a protein dimer is a macromolecular complex or multimer formed by two protein monomers, or single proteins, which are usually non-covalently bound. Many macromolecules, such as proteins or nucleic acids, form dimers. The word ...
and heterotrimers. Some tumor mutations detected in SMAD4 enhance interactions between the MH1 and MH2 domains.


Nomenclature and origin of name

SMADs are highly conserved across species, especially in the
N terminal The N-terminus (also known as the amino-terminus, NH2-terminus, N-terminal end or amine-terminus) is the start of a protein or polypeptide, referring to the free amine group (-NH2) located at the end of a polypeptide. Within a peptide, the amin ...
MH1 domain and the
C terminal The C-terminus (also known as the carboxyl-terminus, carboxy-terminus, C-terminal tail, carboxy tail, C-terminal end, or COOH-terminus) is the end of an amino acid chain (protein or polypeptide), terminated by a free carboxyl group (-COOH). When t ...
MH2 domain. The SMAD proteins are homologs of both the ''Drosophila'' protein MAD and the '' C. elegans'' protein SMA. The name is a combination of the two. During ''Drosophila'' research, it was found that a mutation in the gene ''MAD'' in the mother repressed the gene ''
decapentaplegic ''Decapentaplegic'' (''Dpp'') is a key morphogen involved in the development of the fruit fly ''Drosophila melanogaster'' and is the first validated secreted morphogen. It is known to be necessary for the correct patterning and development of the ...
'' 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 of dpp"; and based on a tradition of unusual naming within the research community. SMAD4 is also known as DPC4, JIP or MADH4.


Function and action mechanism

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 cell nucleus. The abbreviation ''co'' in co-SMAD stands for ''common 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 a
cell surface receptor Cell surface receptors (membrane receptors, transmembrane receptors) are receptors that are embedded in the plasma membrane of cells. They act in cell signaling by receiving (binding to) extracellular molecules. They are specialized integra ...
. 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 a heteromeric 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 the Erk/
MAPK A mitogen-activated protein kinase (MAPK or MAP kinase) is a type of serine/threonine-specific protein kinases involved in directing cellular responses to a diverse array of stimuli, such as mitogens, osmotic stress, heat shock and proinflamm ...
kinase and
GSK3 Glycogen synthase kinase 3 (GSK-3) is a serine/threonine protein kinase that mediates the addition of phosphate molecules onto serine and threonine amino acid residues. First discovered in 1980 as a regulatory kinase for its namesake, glycogen ...
. The FGF (
Fibroblast Growth Factor Fibroblast growth factors (FGF) are a family of cell signalling proteins produced by the macrophages. They are involved in a wide variety of processes, most notably as crucial elements for normal development in animal cells. Any irregularities in ...
) pathway stimulation leads to Smad4
phosphorylation In biochemistry, phosphorylation is described as the "transfer of a phosphate group" from a donor to an acceptor. A common phosphorylating agent (phosphate donor) is ATP and a common family of acceptor are alcohols: : This equation can be writ ...
by Erk of the canonical
MAPK A mitogen-activated protein kinase (MAPK or MAP kinase) is a type of serine/threonine-specific protein kinases involved in directing cellular responses to a diverse array of stimuli, such as mitogens, osmotic stress, heat shock and proinflamm ...
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 a
growth factor A growth factor is a naturally occurring substance capable of stimulating cell proliferation, wound healing, and occasionally cellular differentiation. Usually it is a secreted protein or a steroid hormone. Growth factors are important for ...
-regulated transcription activation domain located in the Smad4 linker region, SAD (Smad-Activation Domain). Second,
MAPK A mitogen-activated protein kinase (MAPK or MAP kinase) is a type of serine/threonine-specific protein kinases involved in directing cellular responses to a diverse array of stimuli, such as mitogens, osmotic stress, heat shock and proinflamm ...
primes Smad4 for
GSK3 Glycogen synthase kinase 3 (GSK-3) is a serine/threonine protein kinase that mediates the addition of phosphate molecules onto serine and threonine amino acid residues. First discovered in 1980 as a regulatory kinase for its namesake, glycogen ...
-mediated phosphorylations that cause transcriptional inhibition and also generate a phosphodegron used as a docking site by the ubiquitin E3 ligase Beta-transducin Repeat Containing ( beta-TrCP) that polyubiquitinates Smad4 and targets it for degradation in the
proteasome Proteasomes are essential protein complexes responsible for the degradation of proteins by proteolysis, a chemical reaction that breaks peptide bonds. Enzymes that help such reactions are called proteases. Proteasomes are found inside all e ...
. Smad4
GSK3 Glycogen synthase kinase 3 (GSK-3) is a serine/threonine protein kinase that mediates the addition of phosphate molecules onto serine and threonine amino acid residues. First discovered in 1980 as a regulatory kinase for its namesake, glycogen ...
phosphorylations have been proposed to regulate the protein stability during pancreatic and
colon cancer Colorectal cancer (CRC), also known as bowel cancer, colon cancer, or rectal cancer, is the development of cancer from the colon or rectum (parts of the large intestine). Signs and symptoms may include blood in the stool, a change in bowel ...
progression. In the nucleus the heteromeric complex binds promoters and interact with transcriptional activators.
SMAD3 Mothers against decapentaplegic homolog 3 also known as SMAD family member 3 or SMAD3 is a protein that in humans is encoded by the SMAD3 gene. SMAD3 is a member of the SMAD (protein), SMAD family of proteins. It acts as a mediator of the signal ...
/SMAD4 complexes can directly bind the SBE. These associations are weak and require additional
transcription factor In molecular biology, a transcription factor (TF) (or sequence-specific DNA-binding factor) is a protein that controls the rate of transcription (genetics), transcription of genetics, genetic information from DNA to messenger RNA, by binding t ...
s such as members of the AP-1 family, TFE3 and
FoxG1 Forkhead box protein G1 is a protein that in humans is encoded by the ''FOXG1'' gene. Function This gene belongs to the forkhead family of transcription factors that is characterized by a distinct forkhead domain. The complete function of thi ...
to regulate
gene expression Gene expression is the process (including its Regulation of gene expression, regulation) by which information from a gene is used in the synthesis of a functional gene product that enables it to produce end products, proteins or non-coding RNA, ...
. Many TGFβ ligands use this pathway and subsequently SMAD4 is involved in many cell functions such as differentiation,
apoptosis Apoptosis (from ) is a form of programmed cell death that occurs in multicellular organisms and in some eukaryotic, single-celled microorganisms such as yeast. Biochemistry, Biochemical events lead to characteristic cell changes (Morphology (biol ...
,
gastrulation Gastrulation is the stage in the early embryonic development of most animals, during which the blastula (a single-layered hollow sphere of cells), or in mammals, the blastocyst, is reorganized into a two-layered or three-layered embryo known as ...
,
embryonic development In developmental biology, animal embryonic development, also known as animal embryogenesis, is the developmental stage of an animal embryo. Embryonic development starts with the fertilization of an egg cell (ovum) by a sperm, sperm cell (spermat ...
and the
cell cycle The cell cycle, or cell-division cycle, is the sequential series of events that take place in a cell (biology), cell that causes it to divide into two daughter cells. These events include the growth of the cell, duplication of its DNA (DNA re ...
.


Clinical significance

Genetic experiments such as
gene knockout Gene knockouts (also known as gene deletion or gene inactivation) are a widely used genetic engineering technique that involves the gene targeting, targeted removal or inactivation of a specific gene within an organism's genome. This can be done t ...
(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 The house mouse (''Mus musculus'') is a small mammal of the rodent family Muridae, characteristically having a pointed snout, large rounded ears, and a long and almost hairless tail. It is one of the most abundant species of the genus ''Mus (genu ...
''). It has been shown that, in mouse KO of ''SMAD4'', the
granulosa cells A granulosa cell or follicular cell is a somatic cell of the sex cord that is closely associated with the developing female gamete (called an oocyte or egg) in the ovary of mammals. Structure and function In the primordial ovarian follicle, and ...
, which secrete hormones and growth factors during the
oocyte An oocyte (, oöcyte, or ovocyte) is a female gametocyte or germ cell involved in reproduction. In other words, it is an immature ovum, or egg cell. An oocyte is produced in a female fetus in the ovary during female gametogenesis. The female ger ...
development, undergo premature luteinization and express lower levels of follicle-stimulating hormone receptors (FSHR) and higher levels of luteinizing hormone receptors (LHR). This may be due in part to impairment of bone morphogenetic protein-7 effects as BMP-7 uses the SMAD4 signaling pathway. Deletions in the genes coding for SMAD1 and SMAD5 have also been linked to metastasic granulosa cell tumors in mice. 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 both somatic cells and cells of the reproductive organs. If the ''SMAD 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 by
fibroblasts A fibroblast is a type of biological cell typically with a spindle shape that synthesizes the extracellular matrix and collagen, produces the structural framework ( stroma) for animal tissues, and plays a critical role in wound healing. Fibrobla ...
. The functional SMAD 4 participates in the regulation of the TGF-β signal transduction pathway, which negatively regulates growth of epithelial cells and the
extracellular matrix In biology, the extracellular matrix (ECM), also called intercellular matrix (ICM), is a network consisting of extracellular macromolecules and minerals, such as collagen, enzymes, glycoproteins and hydroxyapatite that provide structural and bio ...
(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 multiploid
colorectal cancer Colorectal cancer (CRC), also known as bowel cancer, colon cancer, or rectal cancer, is the development of cancer from the Colon (anatomy), colon or rectum (parts of the large intestine). Signs and symptoms may include Lower gastrointestinal ...
and pancreatic carcinoma. It is found inactivated in at least 50% of pancreatic cancers. 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 the
autosomal dominant In genetics, dominance is the phenomenon of one variant (allele) of a gene on a chromosome masking or overriding the Phenotype, effect of a different variant of the same gene on Homologous chromosome, the other copy of the chromosome. The firs ...
disease juvenile 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 developing
gastrointestinal cancer Gastrointestinal cancer refers to malignant conditions of the Human gastrointestinal tract, gastrointestinal tract (GI tract) and accessory organs of digestion, including the esophagus, stomach, biliary system, pancreas, small intestine, large in ...
s, in particular
colon cancer Colorectal cancer (CRC), also known as bowel cancer, colon cancer, or rectal cancer, is the development of cancer from the colon or rectum (parts of the large intestine). Signs and symptoms may include blood in the stool, a change in bowel ...
. 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 forming heteromeric complexes. Mutations in ''SMAD4'' (mostly substitutions) can cause Myhre syndrome, a rare inherited disorder characterized by mental disabilities, short stature, unusual facial features, and various bone abnormalities.


References


Further reading

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External links


GeneReviews/NCBI/NIH/UW entry on Hereditary Hemorrhagic Telangiectasia

GeneReviews/NCBI/NIH/UW entry on Juvenile Polyposis Syndrome

SMAD4 gene variant database
{{DEFAULTSORT:Mothers Against Decapentaplegic Homolog 4 Developmental genes and proteins MH1 domain MH2 domain Transcription factors Human proteins Genes on human chromosome 18