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

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

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Discipline of Pharmacy, School of Medical Sciences and Diabetes Complications Group, Health Innovations Research Institute, RMIT University, Bundoora, Vic, Australia.



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.


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


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.


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.


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

[Indexed for MEDLINE]

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