Kinetic characterization of novel pyrazole TGF-β receptor I kinase inhibitors and their blockade of the epithelial-mesenchymal transition

Transforming growth factor β (TGF-β) signaling pathways regulate a wide variety of cellular processes including cell proliferation, differentiation, extracellular matrix deposition, development, and apoptosis. TGF-β type-I receptor (TβRI) is the major receptor that triggers several signaling events by activating downstream targets such as the Smad proteins. The intracellular kinase domain of TβRI is essential for its function. In this study, we have identified a short phospho-Smad peptide, pSmad3(−3), KVLTQMGSPSIRCSS(PO4)VS as a substrate of TβRI kinase for in vitro kinase assays. This peptide is uniquely phosphorylated by TβRI kinase at the C-terminal serine residue, the phosphorylation site of its parent Smad protein in vivo. Specificity analysis demonstrated that the peptide is phosphorylated by only TβRI and not TGF-β type-II receptor kinase, indicating that the peptide is a physiologically relevant substrate suitable for kinetic analysis and screening of TβRI kinase inhibitors. Utilizing pSmad3(−3) a...