Identification and characterization of ERK MAP kinase phosphorylation sites in Smad3

Biochemistry. 2005 Sep 20;44(37):12546-53. doi: 10.1021/bi050560g.

Abstract

Smad3 is phosphorylated by ERK MAP kinase upon EGF treatment. We have mapped the ERK phosphorylation sites to Ser 207, Ser 203, and Thr 178 in Smad3. We show that, upon EGF treatment, Smad3 is rapidly phosphorylated in these sites, peaking at approximately 15-30 min and that MEK1 inhibitors PD98059 and U0216 inhibit Smad3 phosphorylation induced by EGF. Ser 207 is the best ERK site in Smad3. Its phosphorylation shows the highest EGF induction in Smad3. It is also a very sensitive site to EGF treatment, significantly responding to low concentrations of EGF. These three sites are also phosphorylated by recombinant ERK2 in vitro. We have compared the kinetic parameters of Smad3 with those of ELK1 and MBP for ERK2. We further show that mutation of the ERK phosphorylation sites increases the ability of Smad3 to stimulate a Smad target gene, suggesting that ERK phosphorylation inhibits Smad3 activity.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Binding Sites
  • Cell Line
  • DNA-Binding Proteins / chemistry
  • DNA-Binding Proteins / metabolism*
  • Epidermal Growth Factor / pharmacology
  • Extracellular Signal-Regulated MAP Kinases / chemistry*
  • Extracellular Signal-Regulated MAP Kinases / metabolism*
  • Kinetics
  • Peptide Fragments / chemistry
  • Peptide Fragments / isolation & purification
  • Phosphopeptides / chemistry
  • Phosphopeptides / isolation & purification
  • Phosphorylation
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Smad3 Protein
  • Trans-Activators / chemistry
  • Trans-Activators / metabolism*

Substances

  • DNA-Binding Proteins
  • Peptide Fragments
  • Phosphopeptides
  • Recombinant Proteins
  • Smad3 Protein
  • Trans-Activators
  • Epidermal Growth Factor
  • Extracellular Signal-Regulated MAP Kinases