title: Interactome of transforming growth factor-beta type I receptor (TbetaRI): inhibition of TGFbeta signaling by Epac1.
abstract: Transforming growth factor-beta (TGFbeta) is a potent regulator of cell growth, differentiation, and apoptosis. Type I TGFbeta receptor (TbetaRI) is the key receptor for initiation of intracellular signaling by TGFbeta. Here we report proteomics-based identification of proteins that form a complex with TbetaRI. Using 2D-GE and MALDI TOF mass spectrometry, we identified 16 proteins that specifically interacted with a GST-fused TbetaRI Thr204Asp construct with constitutively active serine/threonine kinase. We confirmed interactions of the receptor with cAMP regulated guanine nucleotide exchange factor 1 (Epac1), beta-spectrin, PIASy, and beta-catenin proteins using immunoblotting. Interaction of the receptor with Epac1 required intact kinase activity of TbetaRI but was not affected by deletion of cAMP-binding domain of Epac1. TGFbeta1-induced C-terminal phosphorylation of Smad2 was inhibited in vivo and in vitro in the presence of Epac1. Epac1 inhibited also TGFbeta1/TbetaRI-dependent transcriptional activation, as evaluated by luciferase reporter assays. We observed that expression of Epac1 counteracted TGFbeta/TbetaRI-dependent decrease of cell adhesion and TGFbeta/TbetaRI-induced stimulation of cell migration. Thus, we have reported novel TRI-interacting proteins and have shown that Epac1 inhibited TGFbeta-dependent regulation of cell migration and adhesion.
authors.affiliation: Ludwig Institute for Cancer Research, Uppsala University, Box 595, BMC, SE-751 24, Uppsala, Sweden.
authors.forename: Paolo
authors.initials: P
authors.lastname: Conrotto
authors.forename: Ihor
authors.initials: I
authors.lastname: Yakymovych
authors.forename: Mariya
authors.initials: M
authors.lastname: Yakymovych
authors.forename: Serhiy
authors.initials: S
authors.lastname: Souchelnytskyi
mesh_terms: D000818:Animals; D002448:Cell Adhesion; D045744:Cell Line, Tumor; D002465:Cell Movement; D005982:Glutathione Transferase / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D040901:Proteomics / Q000379:methods*; D018125:Receptors, Transforming Growth Factor beta / Q000378:metabolism*; D015398:Signal Transduction; D015533:Transcriptional Activation; D016212:Transforming Growth Factor beta / Q000378:metabolism*
chemical_list: D020662:Guanine Nucleotide Exchange Factors; C116122:RAPGEF3 protein, human; D018125:Receptors, Transforming Growth Factor beta; D016212:Transforming Growth Factor beta; D005982:Glutathione Transferase