Phosphorylation of Smad7 at Ser-249 does not interfere with its inhibitory role in transforming growth factor-beta-dependent signaling but affects Smad7-dependent transcriptional activation

• Smad proteins are major components in the intracellular signaling pathway of transforming growth factor-beta (TGF-beta), and phosphorylation is an important mechanism in regulation of their functions. Smad7 was identified as a potent inhibitor of TGF-beta-dependent signaling. We have identified serine 249 in Smad7 as a major phosphorylation site, the phosphorylation of which was not affected by TGF-beta1. Abrogation of the phosphorylation by substitution of Ser-249 with alanine or aspartic acid residues did not affect the ability of Smad7 to inhibit TGF-beta1 and BMP7 signaling. No differences were found in the stability or in the intracellular distribution of Smad7 mutants compared with the wild-type molecule. However, Smad7 fused to the DNA-binding domain of GAL4 induced transcription from a reporter with mutated TATA minimal promoter in a Ser-249-dependent manner. Moreover, a reporter with the SV40 minimal promoter was inhibited by GAL4-Smad7, and this effect was also dependent on Ser-249 phosphorylation. The amplitude of effects on transcriptional regulation was dependent on cell type. Our results suggest that phosphorylation of Smad7, unlike phosphorylation of the receptor-regulated Smads, does not regulate TGF-beta signaling but rather affects TGF-beta-independent effects of Smad7 on transcriptional regulation.

Nyckelord

3T3 Cells

Amino Acid Sequence

Animals

Aspartic Acid/chemistry

Bone Morphogenetic Proteins/metabolism

COS Cells

Cell Nucleus/metabolism

DNA/metabolism

DNA-Binding Proteins/genetics/*metabolism/*physiology

Genes; Reporter

Ligands

Luciferases/metabolism

Mice

Microscopy; Fluorescence

Molecular Sequence Data

Mutation

Phosphorylation

Promoter Regions (Genetics)

Protein Structure; Tertiary

Research Support; Non-U.S. Gov't

Serine/chemistry

Signal Transduction

Time Factors

Trans-Activation (Genetics)

Trans-Activators/genetics/*metabolism/*physiology

Transcription; Genetic

Transfection

Transforming Growth Factor beta/*metabolism

Publikations- och innehållstyp

ref (ämneskategori)

art (ämneskategori)