Roles for lysine residues of the MH2 domain of Smad3 in transforming growth factor-beta signaling.
Sma and MAD-related protein 3 (Smad3) plays a key role in the intracellular signaling of the transforming growth factor-beta (TGF-beta) family of growth factors, which exhibits a diverse set of cellular responses, including cell proliferation and differentiation. Smad3 has the N-terminal Mad homology (MH) 1 and the C-terminal MH2 domains. MH2 domain is essential for the TGF-beta-induced transcriptional activation, because the MH2 domain of Smad3 is involved in the interactions with several transcriptional cofactors as well as the type I TGF-beta receptor (TbetaR-I). In this study, we examined the roles for four lysine residues (Lys-333, Lys-341, Lys-378, and Lys-409) in the Smad3 MH2 domain. Mutation of the lysine (K)-378 to arginine (R) (K378R) abolished the interaction with TbetaR-I, phosphorylation, transcriptional activation by an active TbetaR-I. The K341R mutant also failed to stimulate TGF-beta-induced transcription by resting in the cytoplasm. However, the K409R mutant showed a higher transcriptional activity by stronger interactions with co-activators, such as p300/CBP. Furthermore, both the K341R and K378R mutants act as dominant-negative inhibitors in the TGF-beta-induced target genes of endogenous TGF-beta signal. Thus, the lysine residues of Smad3 MH2 domain play important roles in the transcriptional regulation of TGF-beta signals through TbetaR-I.
Imoto
Seiyu
S
Department of Immunology, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
Sugiyama
Kenji
K
Sekine
Yuichi
Y
Matsuda
Tadashi
T
D000595:Amino Acid Sequence; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D008239:Lysine / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D051900:Smad3 Protein; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism*; D014158:Transcription, Genetic / Q000502:physiology; D016212:Transforming Growth Factor beta / Q000378:metabolism* / Q000502:physiology
D004268:DNA-Binding Proteins; C497790:SMAD3 protein, human; D051900:Smad3 Protein; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D008239:Lysine