Inhibition of Transforming Growth Factor-β Signaling by Low Molecular Weight Compounds Interfering with ATP- or Substrate-Binding Sites of the TGFβ Type I Receptor Kinase

Abstract

Transforming growth factor-beta (TGFbeta) is a potent regulator of cell proliferation, differentiation, apoptosis, and migration. TGF-beta type I receptor (TbetaR-I), which has intrinsic serine/threonine kinase activity, is a key component in activation of intracellular TGFbeta signaling. We studied two different classes of TbetaR-I inhibitors, i.e., compounds interfering with the ATP-binding site of the kinase and substrate-mimicking peptides. We found that pyridinylimidazole compounds inhibited TbetaR-I kinase at micromolar concentration. A representative compound, SB203580, inhibited in vivo Smad2 phosphorylation by TbetaR-I and affected TGFbeta-dependent transcriptional activation. Peptides mimicking the TbetaR-I phosphorylation sites at the C-terminus of Smad2 also inhibited the autophosphorylation of TbetaR-I and phosphorylation of Smad2 by TbetaR-I in vitro and in vivo, whereas a similar peptide from Smad5 was without effect. The substrate-mimicking peptide, fused to penetratin, inhibited a TGFbeta1-dependent transcriptional response in a luciferase reporter assay and ligand-dependent growth inhibition of Mv1Lu cells. Thus, the substrate-mimetic peptide is a new type of specific inhibitor of the TGFbeta signaling in vivo.

Mitogen-Activated Protein Kinase 14 Is a Novel Negative Regulatory Switch for the Vascular Smooth Muscle Cell Contractile Gene Program

“…44,46,49,51,62 First, the effects of p38MAPK rely largely on biological context, particularly the intensity and duration of kinase activity. 52 Second, although SB203580 is relatively specific for blocking p38MAPK, it has been reported to target other signaling pathways, such as cyclin G-associated kinase and SMAD 63,64 ; thus, we cannot exclude an off-target effect of SB203580 on VSMC differentiation, particularly at higher concentrations (eg, 10 μmol/L). In this context, we have found that both SB203580 and BIRB (another p38MAPK inhibitor) augment levels of VSMC marker expression at low concentrations, whereas inhibition is seen at higher concentrations ( Figure VII in the online-only Data Supplement).…”

Transforming growth factor-β1 activates interleukin-6 expression in prostate cancer cells through the synergistic collaboration of the Smad2, p38-NF-κB, JNK, and Ras signaling pathways

“…TGF-b1-induced IL-6 expression was significantly suppressed by NF-kB inhibition, and pretreatment of p38 inhibitors SB202190 and SB203580 blocked NF-kB activation. However, it should be noted that SB203580 and SB202190 are also able to suppress TGF-b-specific response by interfering with the ATP-binding site of TbR-I (Yakymovych et al, 2002). Additionally, we identified that inhibitory effect of SB202190 on IL-6 expression is less significant compared to IkBDN or MG132, leading to the conjecture that NF-kB could also be activated by a mechanism(s) distinct from the p38 cascade in TGF-b1-treated prostate cancer cells.…”

Smad2 Phosphorylation by Type I Receptor

“…The peptides used in this study were synthesized using the Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry as described (20). Three lysine residues were added to the N terminus of each peptide to allow peptide binding to the P81 phosphocellulose paper or to the plastic.…”

“…Gels were dried and exposed by phosphorimaging. Phosphorylation of GST-Smad2 proteins in vitro was performed as described earlier (20). After SDS-PAGE, the proteins were transferred onto the nitrocellulose membrane, and phosphorylation of the C terminus was analyzed by two-dimensional phosphopeptide mapping.…”

“…Consensus sequences have been established for more than 50 kinases, suggesting that substrate recognition is mediated by a complementation between amino acid residues in a substrate and in a kinase substrate-binding region in or immediate to the catalytic cleft (19). Previously we showed that the C-terminal Smad2 peptide specifically inhibited T␤R-I kinase, but not other receptor kinases (20). This suggests that the substrate recognition site of T␤R-I kinase has preferences for the C-terminal peptide of Smad2.…”