Transforming growth factor β-mediated site-specific Smad linker region phosphorylation in vascular endothelial cells

J Pharm Pharmacol. 2014 Dec;66(12):1722-33. doi: 10.1111/jphp.12298. Epub 2014 Oct 14.


Objectives: Transforming growth factor (TGF)-β regulates the function of vascular endothelial cells and may be involved in endothelial dysfunction. The canonical TGF-β pathway involves TGF-β receptor-mediated carboxy-terminal phosphorylation of Smad2; however, TGF-β signalling also activates numerous serine/threonine kinases that phosphorylate Smad2 in its linker region. The expression of phosphorylated Smad linker proteins were determined following TGF-β stimulation in the absence and presence of different serine/threonine kinase inhibitors in vascular endothelial cells.

Methods: Proteins were quantified by Western blotting using specific antibodies to individual phosphorylated Smad2 linker region residues.

Key findings: TGF-β mediated the phosphorylation of all four Smad2 linker region residues of interest. Erk and Jnk specifically phosphorylate Ser245 while all mitogen-activated protein kinases phosphorylate Ser250 and Ser255. Thr220 and Ser245 are phosphorylated by phosphoinositide 3 kinase (PI3K), while Ser255 was phosphorylated by the PI3K/Akt pathway. CDK and GSK-3 were shown to phosphorylate Thr220 and Ser245. TGF-β also mediated plasminogen activator inhibitor-1 gene expression that was attenuated by p38 and CDK inhibitors.

Conclusions: TGF-β-mediated phosphorylation of individual serine/threonine sites in the linker region of Smad2 occurs in a highly specific manner by kinases. These phosphorylations provide an opportunity to further understand a therapeutically targeted and very specific signalling pathway in vascular endothelial cells.

Keywords: Smad linker region; Smads; cell signalling; serine/threonine kinase; transforming growth factor-β.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Aorta / drug effects
  • Aorta / metabolism
  • Blotting, Western
  • Cattle
  • Endothelial Cells / drug effects*
  • Endothelial Cells / metabolism
  • Endothelium, Vascular / drug effects
  • Endothelium, Vascular / metabolism
  • Mitogen-Activated Protein Kinases / antagonists & inhibitors
  • Phosphorylation
  • Primary Cell Culture
  • Protein Kinase Inhibitors / pharmacology
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Smad2 Protein / genetics
  • Smad2 Protein / metabolism*
  • Transforming Growth Factor beta / metabolism
  • Transforming Growth Factor beta / pharmacology*


  • Protein Kinase Inhibitors
  • RNA, Messenger
  • Smad2 Protein
  • Transforming Growth Factor beta
  • Mitogen-Activated Protein Kinases