B R Stockwell1, S L Schreiber. 1. Howard Hughes Medical Institute Department of Chemistry and Chemical Biology Harvard University 12 Oxford Street, Cambridge, Massachusetts, 02138, USA.
Abstract
BACKGROUND: Growth arrest in many cell types is triggered by transforming growth factor beta (TGF-beta), which signals through two TGF-beta receptors (type I, TGF-beta RI, and type II, TGF-beta). In the signaling pathway, TGF-beta binds to the extracellular domain of TGF-betaRII, which can then transphosphorylate TGF-betaRI in its glycine/serine (GS)-rich box. Activated TGF-betaRI 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-betaRI, in TGF-beta signaling. RESULTS: Overexpression of myristoylated TGF-betaRI and TGF-betaRII 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-betaRI 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 FKBP1-TGF-betaRI fusion protein restored constitutive signaling. An acidic mutation in the FKBP12-TGF-betaRI protein allowed FKBP12 antagonists to activate signaling in the absence of TGF-betaRII. Further mutations in the TGF-betaRI FKBP12-binding site resulted in TGF-beta signaling that was independent of both TGF-betaRII and FKBP12 antagonists. CONCLUSIONS: Fusing FKBP12 to TGF-betaRI results in a novel receptor that is activated by small molecule FKBP12 antagonists. These results suggest that FKBP12 binding to TGF-betaRI is inhibitory and that FKBP12 plays a role in inhibiting TGF-beta superfamily signals.
BACKGROUND:Growth arrest in many cell types is triggered by transforming growth factor beta (TGF-beta), which signals through two TGF-beta receptors (type I, TGF-beta RI, and type II, TGF-beta). In the signaling pathway, TGF-beta binds to the extracellular domain of TGF-betaRII, which can then transphosphorylate TGF-betaRI in its glycine/serine (GS)-rich box. Activated TGF-betaRI 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-betaRI, in TGF-beta signaling. RESULTS: Overexpression of myristoylated TGF-betaRI and TGF-betaRII 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-betaRI 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 FKBP1-TGF-betaRI fusion protein restored constitutive signaling. An acidic mutation in the FKBP12-TGF-betaRI protein allowed FKBP12 antagonists to activate signaling in the absence of TGF-betaRII. Further mutations in the TGF-betaRI FKBP12-binding site resulted in TGF-beta signaling that was independent of both TGF-betaRII and FKBP12 antagonists. CONCLUSIONS: Fusing FKBP12 to TGF-betaRI results in a novel receptor that is activated by small molecule FKBP12 antagonists. These results suggest that FKBP12 binding to TGF-betaRI is inhibitory and that FKBP12 plays a role in inhibiting TGF-beta superfamily signals.
Authors: Brian K Law; Anna Chytil; Nancy Dumont; Elizabeth G Hamilton; Mary E Waltner-Law; Mary E Aakre; Cassondra Covington; Harold L Moses Journal: Mol Cell Biol Date: 2002-12 Impact factor: 4.272