Role of Smad4 in TGF-β superfamily signal transduction pathways
TGF-β superfamily members have more than 35 species in vertebrates, including TGF-βs, activins, bone forming proteins, inhibins, Muller's inhibitors, fruit fly dpp, etc., which can regulate the proliferation, differentiation, adhesion and apoptosis of a variety of cells, and play an important role in the development of mammals and the maintenance of tissue homeostasis.
TGF-β superfamily signaling molecules are mediated by membrane receptors to transmit signals into cells, and the most important downstream regulatory protein Smads regulates the expression of various genes in different ways in cells.
Human diseases, such as cancer, osteoporosis, cardiovascular disease, autoimmune disease, fibrosis, and growth disorders, show alterations in the TGF-β-Smad signaling pathway.
First, the reception of signals
TGF-β superfamily signaling molecules have two classes of transmembrane receptors, namely TβRI and TβRII., both of which have Ser/Thr kinase activity.
At present, 7 TBR I and 5 TBRIIs have been found in vertebrates, and these transmembrane receptors are also known as ALKs.
ALK1, ALK2, ALK3 and ALK6 can phosphorylate Smad1, 5, 8, ALK4, ALK5 and ALK7 can phosphorylate Smad2, 3, and the many different reactions caused by a large number of TGF-β superfamily members are in stark contrast to only 2 Smad signaling pathways, so the mechanism of signal specificity and diversity has been raised as an important issue.
When TGF-β superfamily signaling molecules bind to the corresponding TβRII.-type receptor, TBRII undergoes autophosphorylation and binds to TBRI, and the three form trimers, and the GS domain of TBR I (rich in Gly and Ser) is phosphorylated, which is required in the TGF-β signaling pathway.
Second, the transmission of signals
During signaling, the relative levels of R-Smads, Co-Smads, and I-Smads in the cell are important determinants in regulating signal transduction.
Smads binds to SARA proteins via the MH2 domain and is then delivered to phosphorylated receptors.
The activated TBRI briefly binds to SSXS in the R-Smads MH2 domain, activating R-Smads, which were previously inactive due to intramolecular interactions between MH1 and MH2.
Smad4 binds to activated R-Smads to form heterologous complexes under the action of chaperone TRAP1.
c-Ski enhances the binding of the inactivated complex R-Smad-Smad4 to the Smad-binding element, thereby inhibiting the TGF-β signaling pathway. Through the analysis of the MH2 domain crystal structure of Smad4, the presence of trimers was found, which suggests that the heterologous complex formed by R-Smads and Smad4 may be a heterotrimer formed by 2 R-Smads and 1 Smad4.
In addition, I-Smads counteracts the effects of R-Smads.
Smad7 can recruit the complex of GADD34 and the catalytic subunit formation of protein phosphatase I to the activated TBRI, thereby dephosphorylating and inactivating TBRI, hindering the phosphorylation of R-Smads.
Smad6 prevents R-Smads from forming heterologous complexes with Smad4 by competitive binding to R-Smads.
Smad7 mainly inhibits TGF-βs/activator signaling pathway, Smurfl can enhance the inhibitory activity of Smad7, and Smad6 can recruit co-inhibitor CtBP to inhibit BMPs signaling pathway.
Activated R-Smads, which form a complex with Smad4, are released from TβRI and SARA and then enter the nucleus to activate specific target genes.
SNIP1 is a nuclear protein, and its relative level with intranuclear Smad4 determines the background transcription level of the target gene, and also determines the maximum transcriptional activation of the target gene after pathway activation.
It is still unclear which target genes are regulated by the Smad4 protein.
Once displaced to the nucleus, the transcriptional regulatory activity of Smad4 is no longer affected by TGF-β signaling.
R-Smads in the nucleus are continuously dephosphorylated at a very low rate, and this dephosphorylation allows R-Smads to separate from Smad4 and then be retransported into the cytoplasm.
The nucleation of Smad4 requires a nuclear output factor CRM1, which can be combined with the nuclear output signal of the intermediate junction region of Smad4.
3. Activation or inhibition of transcription of target genes
After the heterologous complex enters the nucleus, it acts with the target gene in three ways.
The first way is that the R-Smads/Smad4 complex binds directly to DNA.
Inside the nucleus, Smad4 regulates the transcription of the target gene by recognizing and binding to the 5' AGAC3' sequence on the target gene, the SBE sequence, while the sequence around the "GAC" affects the binding efficiency of SmadsSBE.
The second way is for the Smads protein complex to interact with other DNA-binding factors.
Because the ability of Smad3 and Smad4 to bind DNA is relatively weak, and although Smad2 has 91% sequence consistency with Smad3, Smad2 does not bind to DNA due to the loss of insertion sequences of 30 amino acids in the MH1 domain of naturally spliced Smad2.
These transcription factors are cell-specific and therefore determine the specificity of the response, such as the interaction of members of the FoxH1 family with the Smad2/Smad4 complex in early vertebrate development.
These transcription factors are also regulated by other signaling pathways, such as p53 and IRF7.
The third way is that the Smads protein complex interacts with non-DNA-binding factors.
CBP and p300 are linker proteins closely related to transcription, have histone acyltransferase activity, interact with Smad2, 3, 4, and are required for transcriptional activation of many TGF-β-dependent promoters.
