| 1. |
- Conrotto, Paolo, et al.
(författare)
-
Interactome of transforming growth factor-beta type I receptor (T beta RI) : Inhibition of TGF beta signaling by Epac1
- 2007
-
Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 6:1, s. 287-297
-
Tidskriftsartikel (refereegranskat)abstract
- Transforming growth factor-beta (TGF beta) is a potent regulator of cell growth, differentiation, and apoptosis. Type I TGF beta receptor (T beta RI) is the key receptor for initiation of intracellular signaling by TGF beta. Here we report proteomics-based identification of proteins that form a complex with T beta RI. Using 2D-GE and MALDI TOF mass spectrometry, we identified 16 proteins that specifically interacted with a GST-fused T beta RI Thr204Asp construct with constitutively active serine/threonine kinase. We confirmed interactions of the receptor with cAMP regulated guanine nucleotide exchange factor 1 (Epac1), alpha-spectrin, PIASy, and alpha-catenin proteins using immunoblotting. Interaction of the receptor with Epac1 required intact kinase activity of T beta RI but was not affected by deletion of cAMP-binding domain of Epac1. TGF beta 1-induced C-terminal phosphorylation of Smad2 was inhibited in vivo and in vitro in the presence of Epac1. Epac1 inhibited also TGF beta 1/T beta RI-dependent transcriptional activation, as evaluated by luciferase reporter assays. We observed that expression of Epac1 counteracted TGF beta/T beta RI-dependent decrease of cell adhesion and TGF beta/T beta RI-induced stimulation of cell migration. Thus, we have reported novel T beta RI-interacting proteins and have shown that Epac1 inhibited TGF beta-dependent regulation of cell migration and adhesion.
|
|
| 2. |
- Lin, Kah Wai, et al.
(författare)
-
Phosphorylation of eEF1A1 at Ser300 by T beta R-I Results in Inhibition of mRNA Translation
- 2010
-
Ingår i: Current Biology. - : Elsevier BV. - 0960-9822 .- 1879-0445. ; 20:18, s. 1615-1625
-
Tidskriftsartikel (refereegranskat)abstract
- Background: Transforming growth factor beta (TGF-beta) 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-beta receptor (T beta R-I) is essential for TGF-beta signaling, and substrates of T beta R-I provide insights into molecular mechanisms of TGF-beta signaling. Results: Here we identify eukaryotic elongation factor 1A1 (eEF1A1) as a novel substrate of T beta R-I. We show that T beta R-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-beta 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 T beta R-I is a novel regulatory mechanism that provides a direct link to regulation of protein synthesis by TGF-beta, as an important component in the TGF-beta-dependent regulation of protein synthesis and cell proliferation.
|
|
| 3. |
- Preobrazhenska, Olena, et al.
(författare)
-
BRCA2 and Smad3 synergize in regulation of gene transcription
- 2002
-
Ingår i: Oncogene. - : Springer Science and Business Media LLC. - 0950-9232 .- 1476-5594. ; 21:36, s. 5660-5664
-
Tidskriftsartikel (refereegranskat)abstract
- 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.
|
|
| 4. |
- Stoika, Rostyslav, et al.
(författare)
-
Potential role of transforming growth factor beta1 in drug resistance of tumor cells
- 2003
-
Ingår i: Acta Biochimica Polonica. - 0001-527X .- 1734-154X. ; 50:2, s. 497-508
-
Tidskriftsartikel (refereegranskat)abstract
- 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.
|
|
| 5. |
- Yakymovych, Ihor, et al.
(författare)
-
CIN85 modulates TGF beta signaling by promoting the presentation of TGF beta receptors on the cell surface
- 2015
-
Ingår i: Journal of Cell Biology. - : Rockefeller University Press. - 0021-9525 .- 1540-8140. ; 210:2, s. 319-332
-
Tidskriftsartikel (refereegranskat)abstract
- Members of the transforming growth factor beta (TGF beta) family initiate cellular responses by binding to TGF beta receptor type II (Tf3R11) and type I (TpRI) serine/threonine kinases, whereby Srnad2 and Smad3 are phosphorylated and activated, promoting their association with Smadzi. We report here that T beta RI interacts with the SH3 domains of the adaptor protein CIN85 in response to TGF beta stimulation in a TRAF6-dependent manner. Small interfering RNA mediated knockdown of CIN85 resulted in accumulation of T beta RI in intracellular compartments and diminished TGF beta-stimulated Sniad2 phosphorylation. Overexpression of CIN85 instead increased the amount of T beta RI at the cell surface. This effect was inhibited by a dominant-negative mutant of Rab11, suggesting that CIN85 promoted recycling of TGF beta receptors. CIN85 enhanced TGF beta-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 TGF beta receptors and thereby positively regulates TGF beta signaling.
|
|
| 6. |
- Yakymovych, Ihor, et al.
(författare)
-
Intracellular trafficking of transforming growth factor beta receptors
- 2018
-
Ingår i: Acta biochimica et biophysica Sinica. - : Oxford University Press. - 1672-9145 .- 1745-7270. ; 50:1, s. 3-11
-
Forskningsöversikt (refereegranskat)abstract
- Transforming growth factor beta (TGF beta) family members signal via heterotetrameric complexes of type I (T beta RI) and type II (T beta RII) dual specificity kinase receptors. The availability of the receptors on the cell surface is controlled by several mechanisms. Newly synthesized T beta RI and T beta RII are delivered from the Golgi apparatus to the cell surface via separate routes. On the cell surface, TGF beta 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 TGF beta signaling, localization of the activated receptor complexes on the early endosomes promotes TGF beta-induced Smad activation. Caveolae-mediated endocytosis, which is widely regarded as a mechanism that facilitates the degradation of TGF beta receptors, has been shown to be required for TGF beta signaling via non-Smad pathways. The importance of proper control of TGF beta 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 TGF beta receptors together with the regulation of their expression, posttranslational modifications and down-regulation, ensure proper regulation of TGF beta signaling.
|
|
| 7. |
|
|
| 8. |
- Yakymovych, Ihor, et al.
(författare)
-
The type II TGF-β receptor phosphorylates Tyr 182 in the type I receptor to activate downstream Src signaling
- 2022
-
Ingår i: Science Signaling. - : American Association for the Advancement of Science (AAAS). - 1945-0877 .- 1937-9145. ; 15:760
-
Tidskriftsartikel (refereegranskat)abstract
- Transforming growth factor–β (TGF-β) signaling has important roles during embryonic development and in tissue homeostasis. TGF-β ligands exert cellular effects by binding to type I (TβRI) and type II (TβRII) receptors and induce both SMAD-dependent as well as SMAD-independent intracellular signaling pathways. Activation of the tyrosine kinase Src is one such SMAD-independent consequence of TGF-β signaling. We investigated the mechanism by which TGF-β stimulation activates Src in human and mouse cells. Before TGF-β stimulation, inactive Src was present in a complex with TβRII. Upon TGF-β1 stimulation, which induces the formation of a complex of TβRI and TβRII, TβRII phosphorylated TβRI on serine and threonine residues, which promotes TβRI kinase activity, and on Tyr182. The SH2 domain of Src bound to phosphporylated Tyr182, leading to activation of Src kinase activity. Interaction of the Src SH3 domain with a proline-rich region in TβRI also contributed to binding. TGF-β1–induced Src activation depended on the kinase activity of TβRII but not on that of TβRI, indicating that binding to TβRI activated Src through a non-enzymatic mechanism. Activated Src then phosphorylated TβRI on several tyrosine residues, which may stabilize Src binding to the receptor. In functional assays, Src activation was required for the TGF-β–induced production of fibronectin and for migration in human breast carcinoma cells and for the induction of α-smooth muscle actin (α-SMA) and actin reorganization in mouse fibroblasts. Thus, TGF-β induces Src activation by stimulating a direct interaction with TβRI that depends on tyrosine phosphorylation of TβRI by TβRII.
|
|
| 9. |
- Hassel, Sylke, et al.
(författare)
-
Interaction and functional cooperation between the serine/threonine kinase bone morphogenetic protein type II receptor with the tyrosine kinase stem cell factor receptor
- 2006
-
Ingår i: Journal of Cellular Physiology. - : Wiley. - 0021-9541 .- 1097-4652. ; 206:2, s. 457-467
-
Tidskriftsartikel (refereegranskat)abstract
- 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.
|
|
| 10. |
- Hassel, Sylke, et al.
(författare)
-
Proteins associated with type II bone morphogenetic protein receptor (BMPR-II) and identified by two-dimensional gel electrophoresis and mass spectrometry
- 2004
-
Ingår i: Proteomics. - : Wiley. - 1615-9853 .- 1615-9861. ; 4:5, s. 1346-1358
-
Tidskriftsartikel (refereegranskat)abstract
- 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.
|
|
