A novel protein distinguishes between quiescent and activated forms of the type I transforming growth factor beta receptor.
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.
Charng
M J
MJ
Molecular Cardiology Unit, Baylor College of Medicine, Houston, Texas 77030, USA.
Zhang
D
D
Kinnunen
P
P
Schneider
M D
MD
D030201:Activin Receptors, Type I; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002352:Carrier Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D003001:Cloning, Molecular; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism; D015723:Gene Library; D006360:Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008214:Lymphocytes / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000378:metabolism; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis; D000077293:Receptor, Transforming Growth Factor-beta Type I; D018125:Receptors, Transforming Growth Factor beta / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D022021:Tacrolimus Binding Proteins
D002352:Carrier Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D006360:Heat-Shock Proteins; D047908:Intracellular Signaling Peptides and Proteins; D012333:RNA, Messenger; D018125:Receptors, Transforming Growth Factor beta; D011994:Recombinant Proteins; C111893:TGFBRAP1 protein, human; D017346:Protein Serine-Threonine Kinases; D030201:Activin Receptors, Type I; D000077293:Receptor, Transforming Growth Factor-beta Type I; D022021:Tacrolimus Binding Proteins