Sedimentation studies reveal a direct role of phosphorylation in Smad3:Smad4 homo- and hetero-trimerization.
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.
Correia
J J
JJ
Department of Biochemistry, University of Mississippi Medical Center, Jackson, Mississippi 39216, USA. jcorreia@biochem.umsmed.edu
Chacko
B M
BM
Lam
S S
SS
Lin
K
K
D000818:Animals; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D016254:Mutagenesis, Insertional; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010720:Phosphines / Q000737:chemistry; D010766:Phosphorylation; D017434:Protein Structure, Tertiary / Q000235:genetics; D015398:Signal Transduction; D051900:Smad3 Protein; D051901:Smad4 Protein; D012996:Solutions; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014461:Ultracentrifugation / Q000379:methods
D004268:DNA-Binding Proteins; D010446:Peptide Fragments; D010720:Phosphines; D051900:Smad3 Protein; D051901:Smad4 Protein; D012996:Solutions; D015534:Trans-Activators; C497790:SMAD3 protein, human; C497796:SMAD4 protein, human; C080938:tris(2-carboxyethyl)phosphine