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TGF-beta-signaling with small molecule FKBP12 antagonists that bind myristoylated FKBP12-TGF-beta type I receptor
1Howard Hughes Medical Institute Department of Chemistry and Chemical Biology Harvard University 12 Oxford Street, Cambridge, Massachusetts, 02138, USA.
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.
Insights
FKBP12 binding to TGF-betaRI inhibits transforming growth factor beta (TGF-beta) signaling. Small molecule antagonists targeting FKBP12 activate this pathway, suggesting FKBP12
Area of Science:
- Cellular signaling pathways
- Molecular biology
- Immunology
Background:
- Transforming growth factor beta (TGF-beta) triggers cell growth arrest via TGF-beta receptors (type I and II).
- TGF-beta signaling involves receptor transphosphorylation, Smad protein activation, and nuclear translocation.
- The immunophilin FKBP12 is known to bind the TGF-beta receptor I (TGF-betaRI) GS box.
Purpose of the Study:
- To investigate the role of FKBP12 in TGF-beta signaling.
- To determine if FKBP12 binding to TGF-betaRI affects receptor activity.
- To explore the potential of FKBP12 antagonists in modulating TGF-beta signaling.
Main Methods:
- Constructing fusion proteins of FKBP12 and TGF-betaRI.
- Utilizing COS-1 and mink lung cell lines for signaling assays.
- Employing reporter plasmids and green fluorescent protein (GFP)-Smad2 constructs to monitor pathway activation.
- Introducing specific mutations in the FKBP12-binding site of TGF-betaRI.
Main Results:
- Fusing FKBP12 to TGF-betaRI repressed receptor autosignaling.
- FKBP12-mediated repression was alleviated by FKBP12-binding small molecules (FK506M, rapamycin).
- Mutations in the FKBP12-binding site restored signaling, and some mutations rendered signaling independent of TGF-betaRII and FKBP12 antagonists.
Conclusions:
- FKBP12 binding to TGF-betaRI acts as an inhibitory mechanism.
- A novel TGF-betaRI construct fused with FKBP12 can be activated by small molecule FKBP12 antagonists.
- These findings indicate FKBP12's role in negatively regulating TGF-beta superfamily signals.