TGF-β-signaling with small molecule FKBP12 antagonists that bind myristoylated FKBP12-TGF-β type I receptor fusion proteins Original Research Article

Background: Growth arrest in many cell types is triggered by transforming growth factor beta (TGF-β), which signals through two TGF-β receptors (type I, TGF-βRI, and type II, TGF-βRII). In the signaling pathway, TGF-β binds to the extracellular domain of TGF-βRII, which can then transphosphorylate TGF-βRI in its glycine/serine (GS)-rich box. Activated TGF-βRI phosphorylates two downstream effectors, Smad2 and Smad3, leading to their translocation into the nucleus. Cell growth is arrested and plasminogen activator inhibitor 1 (PAI-1) is upregulated. We investigated the role of the immunophilin FKBP12, which can bind to the GS box of TGF-βRI, in TGF-β signaling. Results: Overexpression of myristoylated TGF-βRI and TGF-βRII cytoplasmic tails caused constitutive nuclear translocation of a green-fluorescent-protein-Smad2 construct in COS-1 cells, and constitutive activation of a PAI-1 reporter plasmid in mink lung cells. Fusing FKBP12 to TGF-βRI resulted in repression of autosignaling that could be alleviated by FK506M or rapamycin (two small molecules that can bind to FKBP12). Mutation of the FKBP12-binding site in the FKBP12-TGF-βRI fusion protein restored constitutive signaling. An acidic mutation in the FKBP12-TGFβRI protein allowed FKBP12 antagonists to activate signaling in the absence of TGF-βRII. Further mutations in the TGF-βRI FKBP12-binding site resulted in TGF-β signaling that was independent of both TGF-βRII and FKBP12 antagonists. Conclusions: Fusing FKBP12 to TGF-βRI results in a novel receptor that is activated by small molecule FKBP12 antagonists. These results suggest that FKBP12 binding to TGF-βRI is inhibitory and that FKBP12 plays a role in inhibiting TGF-β superfamily signals.