Sedimentation Studies Reveal a Direct Role of Phosphorylation in Smad3:Smad4 Homo- and Hetero-Trimerization

Abstract

SMAD proteins are known to oligomerize and hetero-associate during their activation and translocation to the nucleus for transcriptional control. Analytical ultracentrifuge studies on Smad3 and Smad4 protein constructs are presented to clarify the model of homo- and hetero-oligomerization and the role of phosphorylation in the activation process. These constructs all exhibit a tendency to form disulfide cross-linked aggregates, primarily dimers, and a strong reducing agent, TCEP, was found to be required to determine the best estimates for reversible association models and equilibrium constants. A Smad4 construct, S4AF, consisting of the middle linker (L) domain and the C-terminal (C) domain, is shown to be a monomer, while a Smad3 construct, S3LC, consisting of the LC domains, is shown to form a trimer with an affinity K(3) = (1.2-3.1) x 10(9) M(-2). A Smad3 construct that mimics phosphorylation at the C-terminal target sequence, S3LC(3E), has 17--35-fold enhanced ability to form trimer over that of the wild-type construct, S3LC. S4AF associates with either S3LC or S3LC(3E) to form a hetero-trimer. In each case, the hetero-trimer is favored over the formation of the homo-trimer. Despite high sequence homology between Smad3 and Smad4, a chimeric Smad4 construct with an engineered Smad3 C-terminal pseudo-phosphorylation sequence, S4AF(3E), shows no tendency to form trimer. This suggests a Smad4-specific sequence insert inhibits homo-trimer formation, or other domains or sequences in S3LC are required in addition to the target sequence to mediate the formation of trimer. These results represent a direct molecular measure of the importance of hetero-trimerization and phosphorylation in the TGF-beta-activated Smad protein signal transduction process.

Both Max and TFE3 Cooperate with Smad Proteins to Bind the Plasminogen Activator Inhibitor-1 Promoter, but They Have Opposite Effects on Transcriptional Activity

“…8). This stoichiometry is consistent with the differential DNA sequence requirements for Smad3 and Smad4 binding to the PE2.1 element and the predominantly trimeric state of Smads in solution (52,53) as well as in the x-ray crystal structure of the MH2 domain (51). Complexes formed by Smad2 and Smad4 with either FAST-1 or FAST-3 at the Mix.2 promoter contain one FAST, two Smad2s, and one A, specificity of Smad4 binding to the PE2.1 oligonucleotide in the surface plasmon resonance assay.…”

Differential regulation of TGF-β signaling through Smad2, Smad3 and Smad4

“…For example, HO-1 induction was dependent on the presence of all three Smads (Figure 2), whereas other genes appeared to be selectively regulated by one Smad (Figures 2 and 4 and Table 1). Biochemical and crystallographic studies have shown that Smads can exist in a multitude of signaling complexes representing multiple oligomeric states that range from heterodimers to high molecular weight assemblies (Nakao et al, 1997;Jayaraman and Massague, 2000;Chacko et al, 2001;Correia et al, 2001;Wu et al, 2001;Qin et al, 2002). Still, the exact stoichiometry of Smad complexes remains controversial (Inman and Hill, 2002;Moustakas and Heldin, 2002).…”

Stoichiometry of Active Smad-Transcription Factor Complexes on DNA

“…Similar analyses with Smad3 indicate that endogenous Smad3 exists in multiple oligomeric states in unstimulated cells (8), while purified C-terminal fragments of Smad3 can be either monomeric or trimeric (11,12). Similarly, in the uninduced state, Smad4 can exist as monomers (7), dimers, or trimers (8), and crystallographic studies have shown that purified C-terminal fragments of Smad4 are trimers (13,14).…”