Phosphorylation of threonine 276 in Smad4 is involved in transforming growth factor-beta-induced nuclear accumulation

Am J Physiol Cell Physiol. 2003 Oct;285(4):C823-30. doi: 10.1152/ajpcell.00053.2003. Epub 2003 Jun 11.


Smad4, the common Smad, is central for transforming growth factor (TGF)-beta superfamily ligand signaling. Smad4 has been shown to be constitutively phosphorylated (Nakao A, Imamura T, Souchelnytskyi S, Kawabata M, Ishisaki A, Oeda E, Tamaki K, Hanai J, Heldin C-H, Miyazono K, and ten Dijke P. EMBO J 16: 5353-5362, 1997), but the site(s) of phosphorylation, the kinase(s) that performs this phosphorylation, and the significance of the phosphorylation of Smad4 are currently unknown. This report describes the identification of a consensus ERK phosphorylation site in the linker region of Smad4 at Thr276. Our data show that ERK can phosphorylate Smad4 in vitro but not Smad4 with mutated Thr276. Flag-tagged Smad4-T276A mutant protein accumulates less efficiently in the nucleus after stimulation by TGF-beta and is less efficient in generating a transcriptional response than Smad4 wild-type protein. Tryptic phosphopeptide mapping identified a phosphopeptide in Smad4 wild-type protein that was absent in phosphorylated Smad4-T276A mutant protein. Our results suggest that MAP kinase can phosphorylate Thr276 of Smad4 and that phosphorylation can lead to enhanced TGF-beta-induced nuclear accumulation and, as a consequence, enhanced transcriptional activity of Smad4.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence / genetics
  • Animals
  • Cell Line
  • Cell Nucleus / metabolism*
  • Consensus Sequence
  • DNA-Binding Proteins / genetics*
  • DNA-Binding Proteins / metabolism*
  • Genetic Linkage
  • LLC-PK1 Cells
  • Mitogen-Activated Protein Kinases / metabolism
  • Molecular Sequence Data
  • Mutation
  • Phosphoproteins / metabolism
  • Phosphorylation
  • Protein Structure, Tertiary / genetics
  • Swine
  • Threonine / metabolism
  • Trans-Activators / genetics*
  • Trans-Activators / metabolism*
  • Transcription, Genetic / physiology
  • Transforming Growth Factor beta / pharmacology*


  • DNA-Binding Proteins
  • Phosphoproteins
  • Trans-Activators
  • Transforming Growth Factor beta
  • Threonine
  • Mitogen-Activated Protein Kinases