Small C-terminal domain phosphatases dephosphorylate the regulatory linker regions of Smad2 and Smad3 to enhance transforming growth factor-β signaling (original) (raw)
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Cellular and Molecular Life Sciences, 2019
Transforming growth factor (TGF)-β signalling pathways are intensively investigated because of their diverse association with physiological and pathophysiological states. Smad transcription factors are the key mediators of TGF-β signalling. Smads can be directly phosphorylated in the carboxy terminal by the TGF-β receptor or in the linker region via multiple intermediate serine/threonine kinases. Growth factors in addition to hormones and TGF-β can activate many of the same kinases which can phosphorylate the Smad linker region. Historically, Smad linker region phosphorylation was shown to prevent nuclear translocation of Smads and inhibit TGF-β signalling pathways; however, it was subsequently shown that Smad linker region phosphorylation can be a driver of gene expression. This review will cover the signalling pathways of Smad linker region phosphorylation that drive the expression of genes involved in pathology and pathophysiology. The role of Smad signalling in cell biology is expanding rapidly beyond its role in TGF-β signalling and many signalling paradigms need to be re-evaluated in terms of Smad involvement.
Smad regulation in TGF-β signal transduction
Journal of Cell Science, 2001
Smad proteins transduce signals from transforming growth factor-β (TGF-β) superfamily ligands that regulate cell proliferation, differentiation and death through activation of receptor serine/threonine kinases. Phosphorylation of receptor-activated Smads (R-Smads) leads to formation of complexes with the common mediator Smad (Co-Smad), which are imported to the nucleus. Nuclear Smad oligomers bind to DNA and associate with transcription factors to regulate expression of target genes. Alternatively, nuclear R-Smads associate with ubiquitin ligases and promote degradation of transcriptional repressors, thus facilitating target gene regulation by TGF-β. Smads themselves can also become ubiquitinated and are degraded by proteasomes. Finally, the inhibitory Smads (I-Smads) block phosphorylation of R-Smads by the receptors and promote ubiquitination and degradation of receptor complexes, thus inhibiting signalling.
SMAD regulation in TGF-beta signal transduction
Journal of Cell Science
Smad proteins transduce signals from transforming growth factor-beta (TGF-beta) superfamily ligands that regulate cell proliferation, differentiation and death through activation of receptor serine/threonine kinases. Phosphorylation of receptor-activated Smads (R-Smads) leads to formation of complexes with the common mediator Smad (Co-Smad), which are imported to the nucleus. Nuclear Smad oligomers bind to DNA and associate with transcription factors to regulate expression of target genes. Alternatively, nuclear R-Smads associate with ubiquitin ligases and promote degradation of transcriptional repressors, thus facilitating target gene regulation by TGF-beta. Smads themselves can also become ubiquitinated and are degraded by proteasomes. Finally, the inhibitory Smads (I-Smads) block phosphorylation of R-Smads by the receptors and promote ubiquitination and degradation of receptor complexes, thus inhibiting signalling.
Identification of novel Smad2 and Smad3 associated proteins in response to TGF-β1
Journal of Cellular Biochemistry, 2008
Transforming growth factor-beta 1 (TGF-β1) is an important growth inhibitor of epithelial cells and insensitivity to this cytokine results in uncontrolled cell proliferation and can contribute to tumorigenesis. TGF-β1 signals through the TGF-β type I and type II receptors, and activates the Smad pathway via phosphorylation of Smad2 and Smad3. Since little is known about the selective activation of Smad2 versus Smad3, we set out to identify novel Smad2 and Smad3 interacting proteins in epithelial cells. A nontransformed human cell line was transduced with Myc-His 6 -Smad2 or Myc-His 6 -Smad3-expressing retrovirus and was treated with TGF-β1. Myc-His 6 -Smad2 or Myc-His 6 -Smad3 was purified by tandem affinity purification, eluates were subject to SDS-PAGE and Colloidal Blue staining, and select protein bands were digested with trypsin. The resulting tryptic peptides were analyzed by liquid chromatography and tandem mass spectrometry and the SEQUEST algorithm was employed to identify proteins in the bands. A number of proteins that are known to interact with Smad2 or Smad3 were detected in the eluates. In addition, a number of putative novel Smad2 and Smad3 associated proteins were identified that have functions in cell proliferation, apoptosis, Actin cytoskeleton regulation, cell motility, transcription, and Ras or insulin signaling. Specifically, the interaction between Smad2/3 and the Cdc42 guanine nucleotide exchange factor, Zizimin1, was validated by co-immunoprecipitation. The discovery of these novel Smad2 and/or Smad3 associated proteins may reveal how Smad2 and Smad3 are regulated and/or uncover new functions of Smad2 and Smad3 in TGF-β1 signaling.
Tgf-beta induced Erk phosphorylation of smad linker region regulates smad signaling
PloS one, 2012
The Transforming Growth Factor-Beta (TGF-β) family is involved in regulating a variety of cellular processes such as apoptosis, differentiation, and proliferation. TGF-β binding to a Serine/Threonine kinase receptor complex causes the recruitment and subsequent activation of transcription factors known as smad2 and smad3. These proteins subsequently translocate into the nucleus to negatively or positively regulate gene expression. In this study, we define a second signaling pathway leading to TGF-β receptor activation of Extracellular Signal Regulated Kinase (Erk) in a cell-type dependent manner. TGF-β induced Erk activation was found in phenotypically normal mesenchymal cells, but not normal epithelial cells. By activating phosphotidylinositol 3-kinase (PI3K), TGF-β stimulates p21-activated kinase2 (Pak2) to phosphorylate c-Raf, ultimately resulting in Erk activation. Activation of Erk was necessary for TGF-β induced fibroblast replication. In addition, Erk phosphorylated the linke...
Evidence for a Role of Smad3 and Smad2 in Stabilization of the Tumor-derived Mutant Smad2.Q407R
Journal of Biological Chemistry, 2003
Transforming growth factor  (TGF-) is a potent inhibitor of cell proliferation and the loss of responsiveness to TGF- may contribute to the development of human cancers. In hepatocellular carcinomas, the potential role of TGF- signaling as a tumor suppressor pathway can be illustrated by the presence of mutations in genes encoding TGF- receptors or downstream components of this signaling such as Smad2. Although Smad2 is mutated in hepatocellular carcinomas, the alteration of TGF- signaling with respect to tumor progression remains to be established. Using the HepG2 hepatoma cells, we showed here that expression of Smad2.Q407R, a missense mutation found in human hepatocellular carcinoma, was less effective than expression of wild-type Smad2 in enhancing the ability of TGF- to induce transcription from the Mix.2 promoter. This effect was specifically associated with a decrease in the steady-state level of Smad2.Q407R, presumably because of an enhancement of its ubiquitination and degradation through the proteasome machinery. More importantly, we found that the unstability of Smad2.Q407R was reversed when this mutant undergoes homo-oligomerization with wild-type Smad2 or hetero-oligomerization with Smad3 within the cells. Therefore, our findings allowed us to propose a novel mechanism for suppression of the deleterious effect of a tumor-derived mutation of Smad2, which loss may lead to dysregulated cell proliferation during tumorigenesis. Transforming growth factor- (TGF-) 1 regulates a broad range of cellular functions (1), including cell cycle arrest, regulation of extracellular matrix production, and induction of programmed cell death. TGF- signals through a heteromeric complex of two types of transmembrane serine/threonine kinases, the type I (TRI) and type II (TRII) receptors. TGF- binding to TRII induces recruitment and phosphorylation of TRI that transduces signals to downstream intracellular substrates, the Smad proteins (2, 3). The interaction of receptor complex and receptor-regulated Smads (R-Smad), Smad2 and Smad3, is facilitated by SARA (Smad anchor for receptor acti
Signaling of transforming growth factor-β family members through Smad proteins
European Journal of Biochemistry, 2000
Smads are pivotal intracellular nuclear effectors of transforming growth factor-b (TGF-b) family members. Ligand-induced activation of TGF-b family receptors with intrinsic serine/threonine kinase activity trigger phosphorylation of receptor-regulated Smads (R-Smads), whereas Smad2 and Smad3 are phosphorylated by TGF-b, and activin type I receptors, Smad1, Smad5 and Smad8, act downstream of BMP type I receptors. Activated R-Smads form heteromeric complexes with common-partner Smads (Co-Smads), e.g. Smad4, which translocate efficiently to the nucleus, where they regulate, in co-operation with other transcription factors, coactivators and corepressors, the transcription of target genes. Inhibitory Smads act in most cases in an opposite manner from R-and Co-Smads. Like other components in the TGF-b family signaling cascade, Smad activity is intricately regulated. The multifunctional and context dependency of TGF-b family responses are reflected in the function of Smads as signal integrators. Certain Smads are somatically mutated at high frequency in particular types of human cancers. Gene ablation of Smads in the mouse has revealed their critical roles during embryonic development. Here we review the latest advances in our understanding of the Smad mechanism of action and their in vivo functions.
2023
Purpose: The dual modality of TGFb, both as a potent tumor suppressor and a stimulator of tumor progression, invasion, and metastasis, make it a critical target for therapeutic intervention in human cancers. The ability to carry out real-time, noninvasive imaging of TGFb-activated Smad signaling in live cells and animal models would significantly improve our understanding of the regulation of this unique signaling cascade. To advance these efforts, we developed a highly sensitive molecular imaging tool that repetitively, noninvasively, and dynamically reports on TGFBR1 kinase activity. Experimental Design: The bioluminescent TGFbR1 reporter construct was developed using a split firefly luciferase gene containing a functional sensor of Smad2 phosphorylation, wherein inhibition of TGFb receptor1 kinase activity leads to an increase in reporter signaling. The reporter was stably transfected into mammalian cells and used to image in vivo and in vitro bioluminescent activity as a surrogate for monitoring TGFBR1 kinase activity. Results: The reporter was successfully used to monitor direct and indirect inhibition of TGFb-induced Smad2 and SMAD3 phosphorylation in live cells and tumor xenografts and adapted for high-throughput screening, to identify a role for receptor tyrosine kinase inhibitors as modulators of TGFb signaling. Conclusion: The reporter is a dynamic, noninvasive imaging modality for monitoring TGFb-induced Smad2 signaling in live cells and tumor xenografts. It has immense potential for identifying novel effectors of R-Smad phosphorylation, for validating drug-target interaction, and for studying TGFb signaling in different metastasis models. Clin Cancer Res; 17(23); 7424-39. Ó2011 AACR.