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A novel protein distinguishes between quiescent and activated forms of the type I transforming growth factor beta
M J Charng1, D Zhang, P Kinnunen
1Molecular Cardiology Unit, Baylor College of Medicine, Houston, Texas 77030, USA.
Abstract:
Transforming growth factor beta (TGFbeta) signal transduction is mediated by two receptor Ser/Thr kinases acting in series, type II TGFbeta receptor (TbetaR-II) phosphorylating type I TGFbeta receptor (TbetaR-I). Because the failure of interaction cloning, thus far, to identify bona fide TbetaR-I substrates might reasonably have been due to the use of inactive TbetaR-I as bait, we sought to identify molecules that interact specifically with active TbetaR-I, employing the triple mutation L193A,P194A,T204D in a yeast two-hybrid system. The Leu-Pro substitutions prevent interaction with FK506-binding protein 12 (FKBP12), whose putative function in TGFbeta signaling we have previously disproved; the charge substitution at Thr204 constitutively activates TbetaR-I. Unlike previous screens using wild-type TbetaR-I, where FKBP12 predominated, none of the resulting colonies encoded FKBP12. A novel protein was identified, TbetaR-I-associated protein-1 (TRAP-1), that interacts in yeast specifically with mutationally activated TbetaR-I, but not wild-type TbetaR-I, TbetaR-II, or irrelevant proteins. In mammalian cells, TRAP-1 was co-precipitated only by mutationally activated TbetaR-I and ligand-activated TbetaR-I, but not wild-type TbetaR-I in the absence of TGFbeta. The partial TRAP-1 protein that specifically binds these mutationally and ligand-activated forms of TbetaR-I can inhibit signaling by the native receptor after stimulation with TGFbeta or by the constitutively activated receptor mutation, as measured by a TGFbeta-dependent reporter gene. Thus, TRAP-1 can distinguish activated forms of the receptor from wild-type receptor in the absence of TGFbeta and may potentially have a functional role in TGFbeta signaling.
Insights
Researchers identified a novel protein, TRAP-1, that specifically binds activated TGFbeta type I receptor (TbetaR-I). This discovery offers new insights into TGFbeta signal transduction pathways and potential therapeutic targets.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Transforming growth factor beta (TGFbeta) signaling is crucial for cellular processes.
- This pathway involves a cascade of receptor kinases, including type II TGFbeta receptor (TbetaR-II) and type I TGFbeta receptor (TbetaR-I).
- Previous attempts to identify TbetaR-I substrates using inactive receptor bait were unsuccessful.
Purpose of the Study:
- To identify novel molecules that specifically interact with the active form of TbetaR-I.
- To investigate the role of these interacting proteins in TGFbeta signal transduction.
Main Methods:
- Utilized a yeast two-hybrid system with a constitutively active mutant of TbetaR-I (L193A,P194A,T204D).
- Screened for interacting proteins, avoiding known binders like FKBP12.
- Validated interactions in mammalian cells using co-precipitation assays.
- Assessed the functional impact of the identified protein on TGFbeta signaling using a reporter gene assay.
Main Results:
- Identified a novel protein, TbetaR-I-associated protein-1 (TRAP-1), which specifically binds to the activated TbetaR-I mutant.
- TRAP-1 did not interact with wild-type TbetaR-I, TbetaR-II, or irrelevant proteins in yeast.
- In mammalian cells, TRAP-1 co-precipitated with both mutationally and ligand-activated TbetaR-I, but not wild-type TbetaR-I without TGFbeta.
- TRAP-1 inhibited TGFbeta signaling mediated by both native and constitutively active TbetaR-I.
Conclusions:
- TRAP-1 can distinguish between activated and wild-type TbetaR-I in the absence of TGFbeta.
- TRAP-1 may play a significant functional role in regulating TGFbeta signal transduction.
- This finding opens new avenues for understanding and potentially modulating TGFbeta signaling pathways.