title: Transforming growth factor β-mediated site-specific Smad linker region phosphorylation in vascular endothelial cells.
abstract: OBJECTIVE
  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.
  
  
  RESULTS
  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.
authors.affiliation: Discipline of Pharmacy, School of Medical Sciences and Diabetes Complications Group, Health Innovations Research Institute, RMIT University, Bundoora, Vic, Australia.
authors.forename: Danielle
authors.initials: D
authors.lastname: Kamato
authors.forename: Muhamad Ashraf
authors.initials: MA
authors.lastname: Rostam
authors.forename: Terence J
authors.initials: TJ
authors.lastname: Piva
authors.forename: Hossein
authors.initials: H
authors.lastname: Babaahmadi Rezaei
authors.forename: Robel
authors.initials: R
authors.lastname: Getachew
authors.forename: Lyna
authors.initials: L
authors.lastname: Thach
authors.forename: Rebekah
authors.initials: R
authors.lastname: Bernard
authors.forename: Wenhua
authors.initials: W
authors.lastname: Zheng
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Little
authors.forename: Narin
authors.initials: N
authors.lastname: Osman
mesh_terms: D000818:Animals; D001011:Aorta / Q000187:drug effects / Q000378:metabolism; D015153:Blotting, Western; D002417:Cattle; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors; D010766:Phosphorylation; D061251:Primary Cell Culture; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051899:Smad2 Protein / Q000235:genetics / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000378:metabolism / Q000494:pharmacology*
chemical_list: D047428:Protein Kinase Inhibitors; D012333:RNA, Messenger; D051899:Smad2 Protein; D016212:Transforming Growth Factor beta; D020928:Mitogen-Activated Protein Kinases
keywords: Smad linker region; Smads; cell signalling; serine/threonine kinase; transforming growth factor-β