The C-terminal domain of Mad-like signal transducers is sufficient for biological activity in the Xenopus embryo and transcriptional activation

Smad proteins are essential intracellular signal transducers of the transforming growth factor-␤ (TGF-␤) superfamily. The TGF-␤ superfamily signals through phosphorylation and activation of R-Smad proteins, receptor-regulated Smads, by heteromeric complexes of ligand-specific type I and type II serine/threonine kinase receptors. R-Smads receive a signal from the activated receptor complex and transmit it to the nucleus. A cDNA was isolated that encodes a 649-amino acid protein found to be homologous to members of R-Smad subfamily with highest homology scored to clawed African frog and human Smad2. The Schistosoma mansoni homologue (SmSmad2) was overexpressed in bacteria as a Sj26-GST fusion protein and used to raise specific antibodies. The IgG fraction of the immunized rabbit serum identified 70-and 72-kDa protein bands in Western analysis of schistosome extracts. Treatment with alkaline phosphatase removed the 72-kDa band, which indicates that this band represents the phosphorylated form of schistosome Smad2. SmSmad2 was localized in the subtegument, parenchymal cells, and sex organs in both male and female worm cryosections. Similar results were also obtained from the analysis of the Smad2 mRNA distribution pattern revealed by in situ hybridization of adult worm pair paraffin sections. SmSmad2 mRNA levels were determined by reverse transcriptasepolymerase chain reaction in different mammalian host developmental stages and found to be constitutively expressed. SmSmad2 was also found to interact with a previously identified SmT␤R-I, a serine/threonine type I kinase receptor. Furthermore, SmSmad2 was shown to undergo phosphorylation by constitutively active forms of SmT␤R-I in vitro. In addition, SmSmad2 localized in the nuclei of mink lung epithelial cells upon treatment with TGF-␤ 1 . These data indicate that the SmSmad2 responds to the TGF-␤ signals by interaction with receptor I, which phosphorylates it, whereupon it translocates into the nucleus presumably to regulate target gene transcription and consequently elicit a specific TGF-␤ effect.

Section: Discussionsupporting

confidence: 89%

mentioning

confidence: 99%

Identification and Characterization of a Smad2 Homologue fromSchistosoma mansoni, a Transforming Growth Factor-β Signal Transducer

Osman

Niles

LoVerde

2001

“…Fusing Smad7 with a heterologous DNA-binding domain revealed that the Smad7 MH2 domain has a potential transcriptional activation domain (data not shown), although less efficient than that of the MH2 domains of Smad1 and Smad4 (21,22). The N-terminal region of Smad7 shows little similarity to MH1 domains, which are capable of binding DNA directly.…”

Section: Discussionmentioning

confidence: 98%

“…Smad proteins share two regions of high sequence similarity, termed Mad homology (MH) 1 and MH2 domains, at the N-and C-terminal regions, respectively. The N-terminal domain has been shown to have direct sequencespecific DNA-binding activity (17)(18)(19)(20), and the C-terminal domain can act as a transcriptional activator (21,22).…”

mentioning

confidence: 99%

Transforming Growth Factor β1 Induces Nuclear Export of Inhibitory Smad7

Itoh

Landström

Hermansson

et al. 1998

234

190

Transforming growth factor ␤ (TGF-␤) signals from membrane to nucleus through serine/threonine kinase receptors and their downstream effector molecules, termed Smad proteins. Recently, Smad6 and Smad7 were identified, which antagonize TGF-␤ family signaling by preventing the activation of signal-transducing Smad complexes. Here we report that Smad7, but not Smad6, inhibits TGF-␤1-induced growth inhibition and the expression of immediate early response genes, including Smad7. Interestingly, in the absence of ligand, Smad7 was found to be predominantly localized in the nucleus, whereas Smad7 accumulated in the cytoplasm upon TGF-␤ receptor activation. The latter is in accordance with the physical association of Smad7 with the ligand-activated TGF-␤ receptor complex in the cell membrane. Whereas the ectopically expressed C-terminal domain of Smad7 was also exported from the nucleus to the cytoplasm upon TGF-␤ challenge, a Smad7 mutant with a small deletion at the C terminus or only the N-terminal domain of Smad7 was localized mainly in the cytoplasm in the absence or presence of ligand. This suggests that an intact Mad homology 2 domain is important for nuclear localization of Smad7. The nuclear localization of Smad7 suggests a functional role distinct from its antagonistic effect in receptor-mediated Smad activation.

“…Smad proteins share two regions of high similarity, termed MH1 and MH2 domains, connected with a variable proline-rich sequence (18,19). The C-terminal part of Smad2 when fused to a heterologous DNA-binding domain, was found to have transcriptional activity (20,21), whereas the intact Smad2 protein fused to a DNA-binding domain was latent, but transcriptional activity was unmasked after stimulation with ligand (20).…”

mentioning

confidence: 99%

Phosphorylation of Ser465 and Ser467 in the C Terminus of Smad2 Mediates Interaction with Smad4 and Is Required for Transforming Growth Factor-β Signaling

Souchelnytskyi

Tamaki²,

Engström

et al. 1997

369

273

Members of the Smad family of intracellular signal transducers are essential for transforming growth factor-␤ (TGF-␤) to exert its multifunctional effects. After activation of TGF-␤ receptors, Smad2 and Smad3 become phosphorylated and form heteromeric complexes with Smad4. Thereafter, these activated Smad complexes translocate to the nucleus, where they may direct transcriptional responses. Here we report that TGF-␤ mediates phosphorylation of Smad2 at two serine residues in the C terminus, i.e. Ser 465 and Ser 467 , which are phosphorylated in an obligate order; phosphorylation of Ser 465 requires that Ser 467 be phosphorylated. Transfection of Smad2 with mutation of Ser 465 and/or Ser 467 to alanine residues into Mv1Lu cells resulted in dominantnegative inhibition of TGF-␤ signaling. These Smad2 mutants were found to stably interact with an activated TGF-␤ receptor complex, in contrast to wild-type Smad2, which interacts only transiently. Mutation of Ser 465 and Ser 467 in Smad2 abrogated complex formation of this mutant with Smad4 and blocked the nuclear accumulation not only of Smad2, but also of Smad4. Thus, heteromeric complex formation of Smad2 with Smad4 is required for nuclear translocation of Smad4. Moreover, peptides from the C terminus of Smad2 containing phosphorylated Ser 465 and Ser 467 were found to bind Smad4 in vitro, whereas the corresponding unphosphorylated peptides were less effective. Thus, phosphorylated Ser 465 and Ser 467 in Smad2 may provide a recognition site for interaction with Smad4, and phosphorylation of these sites is a key event in Smad2 activation.