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

ObjectivesTransforming growth factor (TGF)- regulates the function of vascular endothelial cells and may be involved in endothelial dysfunction. The canonical TGF- pathway involves TGF- receptor-mediated carboxy-terminal phosphorylation of Smad2; however, TGF- 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- stimulation in the absence and presence of different serine/threonine kinase inhibitors in vascular endothelial cells. MethodsProteins were quantified by Western blotting using specific antibodies to individual phosphorylated Smad2 linker region residues. Key findingsTGF- 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- also mediated plasminogen activator inhibitor-1 gene expression that was attenuated by p38 and CDK inhibitors. ConclusionsTGF--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.