Interactome of Transforming Growth Factor-β Type I Receptor (TβRI):  Inhibition of TGFβ 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.

EPAC1 activation by cAMP stabilizes CFTR at the membrane by promoting its interaction with NHERF1

“…Interestingly, EPAC1, the expression of which in the adult lung is dominant over that of EPAC2 (Ulucan et al, 2007), also interacts with one of the receptors for TGF-β -a pro-inflammatory cytokine, the levels of which are increased in individuals with cystic fibrosis. This interaction leads to the subsequent inhibition of Smaddependent TGF-β signaling (Conrotto et al, 2007). These observations further strengthen the relevance of EPAC1 in the context of CFTR trafficking and in the overall context of the normal versus cystic fibrosis cells.…”

cAMP-induced Epac-Rap activation inhibits epithelial cell migration by modulating focal adhesion and leading edge dynamics

“…Furthermore, neither exogenous Epac1 expression nor 8-CPT-2OMe-cAMP stimulation suppressed Smad2 phosphorylation after TGFβ addition (Fig. 3E), as was reported in 293T cells [42]. Finally, Rap activation did not affect the down regulation of E-cadherin protein levels that is observed in the presence of TGFβ (Fig.…”

“…Surprisingly, neither the stabilization of cell-cell junctions nor the activation of integrins could account for this effect on migration. Major receptor signaling pathways like ERK and Smad (unlike previously reported [42]), are also not affected, indicating that signaling through Epac/Rap does not interfere in HGF or TGFβ signaling, but rather acts downstream to block the induction of cell migration by these transforming growth factors.…”

The cyclic AMP effector Epac integrates pro- and anti-fibrotic signals

“…Smad3 is required for TGF␤-induced gene expression in fibroblasts (19); its loss prevents interstitial fibrosis in the heart and attenuates cardiac remodeling (20), suggesting a role for Smad3 in the decrease of Epac1 by TGF␤. Epac1 protein can interact with type I TGF␤ receptor and inhibit transcriptional activation and phosphorylation of Smad2 (21). Functionally, the decrease in Epac expression would facilitate localization of fibroblasts at sites where fibrogenesis is required and contribute to this process by blunting the negative impact of Epac activation on collagen synthesis.…”