TGF-beta-signaling with small molecule FKBP12 antagonists that bind myristoylated FKBP12-TGF-beta type I receptor fusion proteins.
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.
Stockwell
B R
BR
Howard Hughes Medical Institute Department of Chemistry and Chemical Biology Harvard University 12 Oxford Street, Cambridge, Massachusetts, 02138, USA.
Schreiber
S L
SL
D030201:Activin Receptors, Type I; D000818:Animals; D019556:COS Cells / Q000187:drug effects; D002352:Carrier Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics; D005455:Fluorescent Antibody Technique; D006360:Heat-Shock Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics; D007166:Immunosuppressive Agents / Q000494:pharmacology; D008168:Lung / Q000187:drug effects / Q000378:metabolism; D008907:Mink; D011090:Polyenes / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000187:drug effects / Q000378:metabolism*; D000077293:Receptor, Transforming Growth Factor-beta Type I; D018125:Receptors, Transforming Growth Factor beta / Q000187:drug effects / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000187:drug effects / Q000378:metabolism*; D012097:Repressor Proteins; D015398:Signal Transduction; D020123:Sirolimus; D016559:Tacrolimus / Q000494:pharmacology; D022021:Tacrolimus Binding Proteins; D016212:Transforming Growth Factor beta / Q000187:drug effects / Q000502:physiology*
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D006360:Heat-Shock Proteins; D007166:Immunosuppressive Agents; D011090:Polyenes; D018125:Receptors, Transforming Growth Factor beta; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D016212:Transforming Growth Factor beta; D017346:Protein Serine-Threonine Kinases; D030201:Activin Receptors, Type I; D000077293:Receptor, Transforming Growth Factor-beta Type I; D022021:Tacrolimus Binding Proteins; D020123:Sirolimus; D016559:Tacrolimus