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

• Published in: Biochemistry (Easton) 2001-02, Vol.40 (5), p.1473-1482
• Main Authors: Correia, John J, Chacko, Benoy M, Lam, Suvana S, Lin, Kai
• Format: Article
• Language: English
• Subjects: Animals; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Humans; Mutagenesis, Insertional; Peptide Fragments - chemistry; Peptide Fragments - genetics; Peptide Fragments - metabolism; Phosphines - chemistry; Phosphorylation; Protein Structure, Tertiary - genetics; Signal Transduction; Smad3 Protein; Smad4 Protein; Solutions; Trans-Activators - chemistry; Trans-Activators - genetics; Trans-Activators - metabolism; Ultracentrifugation - methods
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi0019343

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) × 109 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-β-activated Smad protein signal transduction process.