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

Danielle Kamato, Muhamad Ashraf Rostam, Terrence Jerald Piva, Hossein Babaahmadi Rezaei, Robel Getachew, Lyna Thach, Rebekah Bernard, Wenhua Zheng, Peter J Little, Narin Osman

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21 Citations (Scopus)

Abstract

Objectives Transforming 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. Methods Proteins were quantified by Western blotting using specific antibodies to individual phosphorylated Smad2 linker region residues. Key findings TGF-? 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. Conclusions TGF-?-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.
Original languageEnglish
Pages (from-to)1722 - 1733
Number of pages12
JournalJournal of Pharmacy and Pharmacology
Volume66
Issue number12
DOIs
Publication statusPublished - 2014

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