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

J Biol Chem. 2001 Apr 27;276(17):14344-9. doi: 10.1074/jbc.M011019200. Epub 2001 Jan 19.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3T3 Cells
  • Amino Acid Sequence
  • Animals
  • Aspartic Acid / chemistry
  • Bone Morphogenetic Proteins / metabolism
  • COS Cells
  • Cell Nucleus / metabolism
  • DNA / metabolism
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • DNA-Binding Proteins / physiology*
  • Genes, Reporter
  • Ligands
  • Luciferases / metabolism
  • Mice
  • Microscopy, Fluorescence
  • Molecular Sequence Data
  • Mutation
  • Phosphorylation
  • Promoter Regions, Genetic
  • Protein Structure, Tertiary
  • Serine / chemistry
  • Signal Transduction*
  • Smad7 Protein
  • Time Factors
  • Trans-Activators / genetics
  • Trans-Activators / metabolism*
  • Trans-Activators / physiology*
  • Transcription, Genetic
  • Transcriptional Activation*
  • Transfection
  • Transforming Growth Factor beta / metabolism*

Substances

  • Bone Morphogenetic Proteins
  • DNA-Binding Proteins
  • Ligands
  • Smad7 Protein
  • Smad7 protein, mouse
  • Trans-Activators
  • Transforming Growth Factor beta
  • Aspartic Acid
  • Serine
  • DNA
  • Luciferases