The type II TGF-ß receptor phosphorylates Tyr182 in the type I receptor to activate downstream Src signaling.

Transforming growth factor-ß (TGF-ß) signaling has important roles during embryonic development and in tissue homeostasis. TGF-ß ligands exert cellular effects by binding to type I (TßRI) and type II (TßRII) receptors and inducing both SMAD-dependent and SMAD-independent intracellular signaling pathways, the latter of which includes the activation of the tyrosine kinase Src. We investigated the mechanism by which TGF-ß stimulation activates Src in human and mouse cells. Before TGF-ß stimulation, inactive Src was complexed with TßRII. Upon TGF-ß1 stimulation, TßRII associated with and phosphorylated TßRI at Tyr182. Binding of Src to TßRI involved the interaction of the Src SH2 domain with phosphorylated Tyr182 and the interaction of the Src SH3 domain with a proline-rich region in TßRI and led to the activation of Src kinase activity and Src autophosphorylation. TGF-ß1-induced Src activation required the kinase activities of TßRII and Src but not that of TßRI. Activated Src also phosphorylated TßRI on several tyrosine residues, which may stabilize the binding of Src to the receptor. Src activation was required for the ability of TGF-ß to induce fibronectin production and migration in human breast carcinoma cells and to induce α-smooth muscle actin and actin reorganization in mouse fibroblasts. Thus, TGF-ß induces Src activation by stimulating a direct interaction with TßRI that depends on tyrosine phosphorylation of TßRI by TßRII.

Intracellular trafficking of transforming growth factor ß receptors.

Transforming growth factor ß (TGFß) family members signal via heterotetrameric complexes of type I (TßRI) and type II (TßRII) dual specificity kinase receptors. The availability of the receptors on the cell surface is controlled by several mechanisms. Newly synthesized TßRI and TßRII are delivered from the Golgi apparatus to the cell surface via separate routes. On the cell surface, TGFß 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 TGFß signaling, localization of the activated receptor complexes on the early endosomes promotes TGFß-induced Smad activation. Caveolae-mediated endocytosis, which is widely regarded as a mechanism that facilitates the degradation of TGFß receptors, has been shown to be required for TGFß signaling via non-Smad pathways. The importance of proper control of TGFß 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 TGFß receptors together with the regulation of their expression, posttranslational modifications and down-regulation, ensure proper regulation of TGFß signaling.

CIN85 modulates TGFß signaling by promoting the presentation of TGFß receptors on the cell surface.

Members of the transforming growth factor ß (TGFß) family initiate cellular responses by binding to TGFß receptor type II (TßRII) and type I (TßRI) serine/threonine kinases, whereby Smad2 and Smad3 are phosphorylated and activated, promoting their association with Smad4. We report here that TßRI interacts with the SH3 domains of the adaptor protein CIN85 in response to TGFß stimulation in a TRAF6-dependent manner. Small interfering RNA-mediated knockdown of CIN85 resulted in accumulation of TßRI in intracellular compartments and diminished TGFß-stimulated Smad2 phosphorylation. Overexpression of CIN85 instead increased the amount of TßRI at the cell surface. This effect was inhibited by a dominant-negative mutant of Rab11, suggesting that CIN85 promoted recycling of TGFß receptors. CIN85 enhanced TGFß-stimulated Smad2 phosphorylation, transcriptional responses, and cell migration. CIN85 expression correlated with the degree of malignancy of prostate cancers. Collectively, our results reveal that CIN85 promotes recycling of TGFß receptors and thereby positively regulates TGFß signaling.

TRAF6 ubiquitinates TGFß type I receptor to promote its cleavage and nuclear translocation in cancer.

Transforming growth factor ß (TGFß) is a pluripotent cytokine promoting epithelial cell plasticity during morphogenesis and tumour progression. TGFß binding to type II and type I serine/threonine kinase receptors (TßRII and TßRI) causes activation of different intracellular signaling pathways. TßRI is associated with the ubiquitin ligase tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6). Here we show that TGFß, via TRAF6, causes Lys63-linked polyubiquitination of TßRI, promoting cleavage of TßRI by TNF-alpha converting enzyme (TACE), in a PKCζ-dependent manner. The liberated intracellular domain (ICD) of TßRI associates with the transcriptional regulator p300 to activate genes involved in tumour cell invasiveness, such as Snail and MMP2. Moreover, TGFß-induced invasion of cancer cells is TACE- and PKCζ- dependent and the TßRI ICD is localized in the nuclei of different kinds of tumour cells in tissue sections. Thus, our data reveal a specific role for TßRI in TGFß mediated tumour invasion.

Phosphorylation of eEF1A1 at Ser300 by TßR-I results in inhibition of mRNA translation.

BACKGROUND: Transforming growth factor ß (TGF-ß) is a potent inhibitor of cell proliferation that regulates cell functions by activating specific serine/threonine kinase receptors on the cell surface. Type I TGF-ß receptor (TßR-I) is essential for TGF-ß signaling, and substrates of TßR-I provide insights into molecular mechanisms of TGF-ß signaling. RESULTS: Here we identify eukaryotic elongation factor 1A1 (eEF1A1) as a novel substrate of TßR-I. We show that TßR-I phosphorylates eEF1A1 at Ser300 in vitro and in vivo. Ser300 was found to be important for aminoacyl-tRNA (aa-tRNA) binding to eEF1A1. Ser300 phosphorylation or mutations of Ser300 correlate with inhibition of protein synthesis in vitro and in vivo. We show that mimicking eEF1A1 phosphorylation at Ser300 results in inhibition of cell proliferation, and that mutations of Ser300 affect TGF-ß dependency in inhibition of protein synthesis and cell proliferation. Increased expression of eEF1A has been reported to enhance carcinogenesis. An analysis of human breast cancer cases revealed a decrease of eEF1A1 phosphorylation at Ser300 in malignant tumor cells as compared to epithelial cells in noncancerous tissues. CONCLUSIONS: Phosphorylation of eEF1A1 by TßR-I is a novel regulatory mechanism that provides a direct link to regulation of protein synthesis by TGF-ß, as an important component in the TGF-ß-dependent regulation of protein synthesis and cell proliferation.

Interactome of transforming growth factor-beta type I receptor (TbetaRI): inhibition of TGFbeta signaling by Epac1.

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.

Interaction and functional cooperation between the serine/threonine kinase bone morphogenetic protein type II receptor with the tyrosine kinase stem cell factor receptor.

Transmembrane receptors with intrinsic serine/threonine or tyrosine kinase domains regulate vital functions of cells in multicellular eukaryotes, e.g., differentiation, apoptosis, and proliferation. Here, we show that bone morphogenetic protein type II receptor (BMPR-II) which has a serine/threonine kinase domain, and stem cell factor receptor (c-kit) which contains a tyrosine kinase domain form a complex in vitro and in vivo; the interaction is induced upon treatment of cells with BMP2 and SCF. Stem cell factor (SCF) modulated BMP2-dependent activation of Smad1/5/8 and phosphorylation of Erk kinase. SCF also enhanced BMP2-dependent differentiation of C2C12 cells. We found that BMPR-II was phosphorylated at Ser757 upon co-expression with and activation of c-kit. BMPR-II phosphorylation required intact kinase activity of BMPR-II. Abrogation of the c-kit/SCF-dependent phosphorylation of BMPR-II at the Ser757 interfered with the cooperative effect of BMP2 and SCF. Our data suggest that the complex formation between c-kit and BMPR-II leads to phosphorylation of BMPR-II at Ser757, which modulates BMPR-II-dependent signaling.

Proteins associated with type II bone morphogenetic protein receptor (BMPR-II) and identified by two-dimensional gel electrophoresis and mass spectrometry.

Bone morphogenetic proteins (BMP) are polypeptide growth factors that regulate cell differentiation and proliferation. BMPs bind to type I and type II serine/threonine kinase receptors to initiate intracellular signalling. BMPR-II is the type II receptor, its mutations lead to hereditary pulmonary hypertension, and knockout of Bmpr-II results in early embryonic lethality. To identify novel interacting proteins and explore signalling pathways that can be initiated by BMPR-II, we performed glutathione-S-transferase (GST) pull-down assays with BMPR-II protein constructs fused to GST and extracts of mouse myoblast C2C12 cells. We generated three constructs which contain different parts of the cytoplasmic region of BMPR-II: full-length cytoplasmic part of BMPR-II, only the kinase domain, or only the C-terminal tail of BMPR-II. Proteins which formed complexes with these BMPR-II constructs were analyzed by two-dimensional gel electrophoresis (2-D GE), and specifically interacting proteins were identified by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). We identified 33 interacting proteins; 11 proteins interacted with the C-terminal tail of BMPR-II, 4 with full-length BMPR-II, and 18 with a short form of the receptor with a deleted tail. Fourteen proteins have assigned functions in various signalling processes, suggesting links of BMP signalling to regulation of MAP kinase pathway, apoptosis, transcription, PKCss, and PKA. Five of the identified proteins are components of the cytoskeleton, and four are enzymes involved in metabolism, e.g., processing of estrogens or lipids. We confirmed interaction of PKC beta and CtBP with BMPR-II using immunodetection. We showed that the C-terminal tail of BMPR-II provides binding sites for a number of regulatory proteins that may initiate Smad-independent signalling.

Potential role of transforming growth factor beta1 in drug resistance of tumor cells.

Acquired drug resistance of tumor cells is frequently observed in cancer patients undergoing chemotherapy. We studied murine leukemia L1210 cells sensitive and resistant to the cytotoxic action of cisplatin and showed that cisplatin-resistant leukemia cells were also refractory to TGF beta1-dependent growth inhibition and apoptosis. Addressing the question about the mechanisms responsible for the cross-resistance to cisplatin and TGF beta1, we found that cisplatin- and TGF beta1-resistant L1210 cells possessed a decreased expression of type I TGF beta1 receptor, while the expression of type II TGF beta1 receptor was not affected. Western blot analysis of Smad proteins 2, 3, 4, 6, and 7, which participate in signal transduction pathway down-stream of the TGF beta1 receptors, revealed an increased expression of Smad 6, inhibiting TGF beta1 action, only in cisplatin- and TGF beta1-resistant L1210 cells. TGF beta1 and especially the cytotoxic mistletoe agglutinin increased Smad 6 expression in TGF beta1-sensitive but not in TGF beta1-resistant L1210 cells. TGF beta1-resistant L1210 cells also differed from TGF beta1-sensitive cells by the lack of expression of the pro-apoptotic p53 protein and higher level of expression of the anti-apoptotic Bcl-2 protein. Thus, the described co-expression of tumor cell refractoriness to an anti-cancer drug and to the inhibitory cytokine TGF beta1 is accompanied by multiple changes in the TGF beta1 signal transduction pathway and in other regulatory systems of the target cells. Besides, we found that various anti-tumor drugs and cytotoxic plant lectins increased the level of TGF beta1 expression in both TGFbeta1-sensitive and -resistant L1210 cells. A hypothesis is proposed that TGFbeta1 can at least partly mediate the effect of cell-stressing agents and, thus, the development of TGF beta1 resistance may be responsible for the appearance of tumor cell refractoriness to the action of some anti-cancer drugs.

BRCA2 and Smad3 synergize in regulation of gene transcription.

Smad3 is an essential component in the intracellular signaling of transforming growth factor-beta (TGFbeta), 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. TGFbeta1 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.