Cross talk between transforming growth factor b(TGF-b) serine/threonine kinase receptor signaling and tyrosine kinase receptor signaling modulates cell responsiveness to polypeptide growth factors regulating cell proliferation, differentiation, and apoptosis. Here we provide a mechanism through which Smad-dependent TGF-b signaling is modulated by protein kinase C (PKC). PKC, for example, is activated downstream of tyrosine kinase receptors. We show that PKC directly phosphorylates receptor-regulated Smad proteins. This phosphorylation abrogates the ability of Smad3 to bind directly to DNA, which leads to subsequent inability to mediate transcriptional responses dependent on the direct binding of Smad3 to DNA. Interference with PKC regulation of Smad functions increased cell sensitivity to transformation by the tumor promoter phorbol 12-myristate 13-acetate (PMA). PKC-dependent phosphorylation of Smad3 was found also to be a key event in the PMA-dependent inactivation of TGF-b-stimulated cell death. Thus, PKC-dependent phosphorylation of Smad3 leads to down-regulation of the growth inhibitory and apoptotic action of TGF-b.

Transforming growth factor β (TGFβ) family members signal via heterotetrameric complexes of type I (TβRI) and type II (TβRII) dual specificity kinase receptors. The availability of the receptors on the cell surface is controlled by several mechanisms. Newly synthesized TβRI and TβRII are delivered from the Golgi apparatus to the cell surface via separate routes. On the cell surface, TGFβ receptors are distributed between different microdomains of the plasma membrane and can be internalized via clathrin-and caveolae-mediated endocytic mechanisms. Although receptor endocytosis is not essential for TGFβ signaling, localization of the activated receptor complexes on the early endosomes promotes TGFβ-induced Smad activation. Caveolae-mediated endocytosis, which is widely regarded as a mechanism that facilitates the degradation of TGFβ receptors, has been shown to be required for TGFβ signaling via non-Smad pathways. The importance of proper control of TGFβ receptor intracellular trafficking is emphasized by clinical data, as mislocalization of receptors has been described in connection with several human diseases. Thus, control of intracellular trafficking of the TGFβ receptors together with the regulation of their expression, posttranslational modifications and down-regulation, ensure proper regulation of TGFβ signaling.

Transforming growth factor-␤ (TGF␤) signaling involves activation of a number of signaling pathways, several of which are controlled by phosphorylation events. Here, we describe a phosphoproteome profiling of MCF-7 human breast epithelial cells treated with TGF␤1. We identified 32 proteins that change their phosphorylation upon treatment with TGF␤1; 26 of these proteins are novel targets of TGF␤1. We show that Smad2 and Smad3 have different effects on the dynamics of TGF␤1-induced protein phosphorylation. The identified proteins belong to nine functional groups, e.g., proteins regulating RNA processing, cytoskeletal rearrangements, and proteasomal degradation. To evaluate the proteomics findings, we explored the functional importance of TGF␤1-dependent phosphorylation of one of the targets, i.e.,

Phosphorylation of eEF1A1 by TβR-I is a novel regulatory mechanism that provides a direct link to regulation of protein synthesis by TGF-β, as an important component in the TGF-β-dependent regulation of protein synthesis and cell proliferation.

Transforming growth factor-beta (TGFbeta) is a potent regulator of cell proliferation, differentiation, apoptosis, and migration. TGF-beta type I receptor (TbetaR-I), which has intrinsic serine/threonine kinase activity, is a key component in activation of intracellular TGFbeta signaling. We studied two different classes of TbetaR-I inhibitors, i.e., compounds interfering with the ATP-binding site of the kinase and substrate-mimicking peptides. We found that pyridinylimidazole compounds inhibited TbetaR-I kinase at micromolar concentration. A representative compound, SB203580, inhibited in vivo Smad2 phosphorylation by TbetaR-I and affected TGFbeta-dependent transcriptional activation. Peptides mimicking the TbetaR-I phosphorylation sites at the C-terminus of Smad2 also inhibited the autophosphorylation of TbetaR-I and phosphorylation of Smad2 by TbetaR-I in vitro and in vivo, whereas a similar peptide from Smad5 was without effect. The substrate-mimicking peptide, fused to penetratin, inhibited a TGFbeta1-dependent transcriptional response in a luciferase reporter assay and ligand-dependent growth inhibition of Mv1Lu cells. Thus, the substrate-mimetic peptide is a new type of specific inhibitor of the TGFbeta signaling in vivo.

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.

Smad3 is an essential component in the intracellular signaling of transforming growth factor-b (TGFb), which is a potent inhibitor of tumor cell proliferation. BRCA2 is a tumor suppressor involved in early onset of breast, ovarian and prostate cancer. Both Smad3 and BRCA2 possess transcription activation domains. Here, we show that Smad3 and BRCA2 interact functionally and physically. We found that BRCA2 forms a complex with Smad3 in vitro and in vivo, and that both MH1 and MH2 domains of Smad3 contribute to the interaction. TGFb1 stimulates interaction of endogenous Smad3 and BRCA2 in non-transfected cells. BRCA2 co-activates Smad3-dependent transcriptional activation of luciferase reporter and expression of plasminogen activator inhibitor-1 (PAI-1). Smad3 increases the transcriptional activity of BRCA2 fused to the DNA-binding domain (DBD) of Gal4, and reciprocally, BRCA2 co-activates DBD-Gal4-Smad3. Thus, our results show that BRCA2 and Smad3 form a complex and synergize in regulation of transcription.

The adaptor CIN85 enhances TGFβ-induced signaling and cellular responses to TGFβ by promoting the expression of TGFβ receptors on the surface in a Rab11-dependent manner.