Heteromeric and Homomeric Interactions Correlate with Signaling Activity and Functional Cooperativity of Smad3 and Smad4/DPC4

Abstract

Homologs of Drosophila Mad function as downstream mediators of the receptors for transforming growth factor ␤ (TGF-␤)-related factors. Two homologs, the receptor-associated Smad3 and the tumor suppressor Smad4/DPC4, synergize to induce ligand-independent TGF-␤ activities and are essential mediators of the natural TGF-␤ response. We now show that Smad3 and Smad4 associate in homomeric and heteromeric interactions, as assessed by yeast two-hybrid and coimmunoprecipitation analyses. Heteromeric interactions are mediated through the conserved C-terminal domains of Smad3 and Smad4. In Smad3, the homomeric interaction is mediated by the same domain. In contrast, the homomeric association of Smad4 requires both the N-terminal domain and the C-terminal domain, which by itself does not homomerize. Mutations that have been associated with impaired Mad activity in Drosophila or decreased tumor suppressor activity of Smad4/ DPC4 in pancreas cancer, including a short C-terminal truncation and two point mutations in the conserved C-terminal domains, impair the ability of Smad3 and Smad4 to undergo homo-and heteromeric associations. Analyses of the biological activity of Smad3 and Smad4 and their mutants show that full signaling activity correlates with their ability to undergo efficient homo-and heteromeric interactions. Mutations that interfere with these interactions result in decreased signaling activity. Finally, we evaluated the ability of Smad3 or Smad4 to induce transcriptional activation in yeast. These results correlate the ability of individual Smads to homomerize with transcriptional activation and additionally with their biological activity in mammalian cells.Transforming growth factor ␤ (TGF-␤)-related factors play important regulatory roles in cell differentiation and proliferation and in tissue morphogenesis in animals ranging from flies and nematodes to mammals (12,16). Among the many members of the TGF-␤ superfamily, which include activins and bone morphogenetic proteins (BMPs) (12, 16), TGF-␤1 is considered the prototype factor to characterize the various cellular responses and the mechanism of receptor signaling. The growth-inhibitory effect of TGF-␤ in a variety of cell types, such as epithelial cells, and its ability to induce gene expression that leads to increased extracellular matrix deposition have been best characterized (5). Similarly to BMP-2/4 and activin, TGF-␤ exerts its activities through two types of cell surface serine/threonine kinase receptors, type II and type I receptors. The type II and type I receptors have the ability to form a heterotetrameric complex, consisting of two type II and two type I receptors, and this complex is thought to represent the fully functional receptor complex that mediates TGF-␤ signaling following ligand binding. In this tetrameric complex, the constitutively active type II receptors phosphorylate the type I receptor cytoplasmic domains, and this phosphorylation is required for activation of the signaling pathways leading to the TGF-␤ response (4,18,22).The down...

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

“…In addition, mutant forms of Smad2 or Smad3 with short deletions in their C termini were found not to interact with Smad4 in this assay (45,46). Our observation that C-terminal peptides of Smad2 bind GST-Smad4 is consistent with these data.…”

“…Recent studies using the yeast two-hybrid interaction screen indicated that the C-terminal domains of Smad2 and Smad3 are important for the heteromeric interactions with Smad4 (45,46). In addition, mutant forms of Smad2 or Smad3 with short deletions in their C termini were found not to interact with Smad4 in this assay (45,46).…”

Smad3 is acetylated by p300/CBP to regulate its transactivation activity

“…As shown in Figure 6a, TGF-b augmented the activity of GAL4-Smad3 by 7.0-fold. GAL4-Smad3C, which has only the MH2 domain, had stronger activity (16.7-fold) than the wild type, consistent with a previous report (Wu et al, 1997). However, mutation of K378 (GAL4-Smad3C(K378R)) and mutation of all four lysines (GAL4-Smad3C(4KR)) significantly decreased the transcriptional activity (85 and 97% reduction, respectively).…”

“…K378 is fully conserved among all Smads and among all members of this family in various species. The MH2 domain of the Smad proteins exhibits a transactivation function through heterologous fusion with the GAL4 DNA-binding domain (Wu et al, 1997). We therefore evaluated the effect of the Smad3 acetylation of Smad3 on its function as a transactivator (Figure 6).…”

Inhibition of the TGF-Beta Tumor Suppression Pathway Through Repression of Smad Dependent Transcription

“…lA). It is known that overexpressed Smad2 and SmadS can partially bypass the requirement for ligand and localize in the nucleus, oligomerize, and activate downstream events (Wu et al 1997;Zhang et al 1997). Activation of TGF-p signaling can further enhance these events.…”