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The phosphorylation of the Smad2/3 linker region by Nemo-like kinase regulates TGF-β signaling

ABSTRACT: Smad2 and Smad3 (Smad2/3) primarily mediates the transforming growth factor-β (TGF-β) signaling that drives cell proliferation, differentiation, and migration. The dynamics of the Smad2/3 phosphorylation provides the key mechanism for regulating the TGF-β signaling pathway. Here we identified NLK as a novel regulator of TGF-β signaling pathway via modulating the phosphorylation of Smad2/3 in the linker region.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Yanchi Zhou

LAB HEAD: Wei Song

PROVIDER: PXD023957 | Pride | 2021-09-10

REPOSITORIES: Pride

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			Action	DRS
	MS120755-2.msf	Msf
	MS120755-2.raw	Raw
	MS120755-3.msf	Msf
	MS120755-3.raw	Raw
	checksum.txt	Txt

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Publications

The phosphorylation of the Smad2/3 linker region by nemo-like kinase regulates TGF-β signaling.

Liang Junbo J Zhou Yanchi Y Zhang Ning N Wang Dingding D Cheng Xiaowen X Li Kai K Huang Rong R Lu Yan Y Wang Hailong H Han Deqiang D Wu Wei W Han Meng M Miao Shiying S Wang Linfang L Zhao Hong H Song Wei W

The Journal of biological chemistry 20210101

Smad2 and Smad3 (Smad2/3) are structurally similar proteins that primarily mediate the transforming growth factor-β (TGF-β) signaling responsible for driving cell proliferation, differentiation, and migration. The dynamics of the Smad2/3 phosphorylation provide the key mechanism for regulating the TGF-β signaling pathway, but the details surrounding this phosphorylation remain unclear. Here, using in vitro kinase assay coupled with mass spectrometry, we identified for the first time that nemo-li ...[more]

PMID: 33676893

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Smad2 and Smad3 binding sites in H345-TbRII cells

Project description:Transforming growth factor (TGF)-beta induces apoptosis of many types of cancer cells and acts as a tumor suppressor. We found lower expression of TGF-beta type II receptor (TbRII) in most of SCLC cells and tissues than in normal lung epithelial cells and normal lung tissues, respectively. In vitro cell growth and in vivo tumor formation were suppressed by TGF-beta-mediated apoptosis when the wild-type TbRII was overexpressed in SCLC cells. We therefore determined Smad2 and Smad3 (Smad2/3) binding sites in a SCLC cell line H345 stably expressing exogenous TbRII (H345-TbRII) to identify target genes of TGF-beta. Smad2 and Smad3 binding sites in H345-TbRII cells were determined by ChIP-seq (one sample analysis, without replicates).

2015-09-15 | E-GEOD-63871 | biostudies-arrayexpress

HNF4A-binding sites in HepG2 hepatoblastoma cells treated with TGF-beta

Project description:Specific regulation of target genes by transforming growth factor-β (TGF-β) in a given cellular context is determined in part by transcription factors and cofactors that interact with the Smad complex. In the present study, we determined Smad2 and Smad3 (Smad2/3) binding regions in the promoters of known genes in HepG2 hepatoblastoma cells, and compared them to those in HaCaT epidermal keratinocytes to elucidate the mechanisms of cell type- and context-dependent regulation of transcription induced by TGF-β. Our results show that 81% of the Smad2/3 binding regions in HepG2 cells were not shared with those found in HaCaT cells. Hepatocyte nuclear factor 4α (HNF4α) is expressed in HepG2 cells, but not in HaCaT cells, and the HNF4α binding motif was identified as an enriched motif in the HepG2-specific Smad2/3 binding regions. ChIP-sequencing analysis of HNF4A binding regions under TGF-β stimulation revealed that 32.5% of the Smad2/3 binding regions overlapped HNF4A bindings. MIXL1 was identified as a new combinatorial target of HNF4A and Smad2/3, and both the HNF4A protein and its binding motif were required for the induction of MIXL1 by TGF-β in HepG2 cells. These findings generalize the importance of binding of HNF4A on Smad2/3 binding genomic regions for HepG2-specific regulation of transcription by TGF-β, and suggest that certain transcription factors expressed in a cell-type-specific manner play important roles in the transcription regulated by the TGF-β-Smad signaling pathway. HepG2 cells were treated with TGF-beta for 1.5 h or left untreated. anti-HNF4A ChIP-seq was performed. One lane was used for each sample.

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Invasion control by a positive feedback loop mechanism Smad4-null cells

Project description:New findings demonstrate that transcriptional factors alternative to Smad4 can bind to Smad2/3 and mediate different transcriptional effects. In this study, we detected constitutively phosphorylation of Smad2/3 in Smad4-null pancreatic cancer cell line BxPC-3. Both pan-specific TGF-β-neutralizing antibody and specific TGF-β type I receptor (TβR-I) inhibitor, SB-431542, can decrease steady-state p-Smad2/3 levels. Moreover, exogenous TGF-β strongly stimulated translocation of phosphorylated Smad2/3 (p-Smad2/3) into the nucleus. Therefore, we identified TGF-β-responsive genes using genome-wide oligonucleotide microarrays and confirmed their dependency on Smad2/3 by the combination of RNA interference (RNAi) and microarray assay. The major finding from our microarray analysis was that of the 262 target genes seen to be regulated via TGF-β induction, 87 were differentially transcriptionally controlled by Smad2/3 signaling and 175 were Smad2/3-independent. Our results showed that integrin β6 was transcriptionally up-regulated via TGF-β induction in a Smad3-dependent manner, which was validated by real-time RT-PCR and western blot. We also provide evidence that αVβ6 integrin can activate TGF-β-Smad2/3 signaling. Thus, we for the first time suggest the positive feedback loop compose of TGF-β-Smad3 signaling and integrin β6. Functional analysis revealed that exogenous TGF-β can amplify the invasive property of Smad4-deficient pancreatic cancer cells; however, TGF-β-neutralizing antibody, specific TβR-I inhibitor, and anti-αVβ6 integrin antibody can reduce it. Therefore, integrin β6 mediated the invasion of BxPC-3 cells induced by TGF-β signaling. Keywords: cell type comparison

2007-09-01 | GSE8464 | GEO

Stress-induced tyrosine phosphorylation of RtcB modulates IRE1 activity and signaling outputs.

Project description:Endoplasmic Reticulum (ER) stress is a hallmark of various diseases, which is dealt with through the activation of an adaptive signaling pathway named the Unfolded Protein Response (UPR). This response is mediated by three ER-resident sensors and the most evolutionary conserved, IRE1α signals through its cytosolic kinase and endoribonuclease (RNase) activities. IRE1α RNase activity can either catalyze the initial step of XBP1 mRNA unconventional splicing or degrade a number of RNAs through Regulated IRE1-Dependent Decay (RIDD). The balance between these two activities plays an instrumental role in cells’ life and death decisions upon ER stress. Until now, the biochemical and biological outputs of IRE1α RNase activity have been well documented, however, the precise mechanisms controlling whether IRE1 signaling is adaptive or pro-death (terminal) remain unclear. This prompted us to further investigate those mechanisms and we hypothesized that XBP1 mRNA splicing and RIDD activity could be co-regulated by the IRE1α RNase regulatory network. We showed that a key nexus in this pathway is the tRNA ligase RtcB which, together with IRE1α, is responsible for XBP1 mRNA splicing. We demonstrated that RtcB is tyrosine phosphorylated by c-Abl and dephosphorylated by PTP1B. Moreover, we identified RtcB Y306 as a key residue which, when phosphorylated, perturbs RtcB interaction with IRE1α, thereby attenuating XBP1 mRNA splicing and favoring RIDD. Our results demonstrate that the IRE1α RNase regulatory network is dynamically fine-tuned by tyrosine kinases and phosphatases upon various stresses and that the nature of the stress determines cell adaptive or death outputs.

2022-02-21 | PXD023433 | Pride

TGF-beta-induced gene expression data and Smad2/3 binding sites of HaCaT keratinocytes

Project description:Smad2/3 are transcription factors that engage in TGF-beta-induced transcription. Here we analyzed the effect of identified Smad2/3 binding sites to transcription. We used expression microarrays to compare the Smad2/3 binding sites identified by ChIP-chip to TGF-beta-induced gene expressions. Keywords: time course We also examined the effect of either ETS1/TFAP2A/SMAD2/SMAD3 siRNAs on TGF-beta-induced gene expression change.

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Expression data of the human hepatoblastoma cell line HepG2 treated with TGF-beta

Project description:Smad2/3 are transcription factors that engage in TGF-beta-induced transcription. We determined and analyzed HepG2 and Hep3B-specific Smad2/3 binding sites by ChIP-chip. We used expression microarrays to compare the Smad2/3 and HNF4alpha binding sites identified by ChIP-chip or ChIP-seq, respectively, to TGF-beta-induced gene expressions. HepG2 cells were transfected with control or HNF4A siRNAs and treated with 3 ng/ml TGF-beta for 0, 1.5 and 24 h (6 samples in total, no replicates). Total RNA was extracted and expression microarray analysis was performed as described in the protocols.

2011-06-08 | E-GEOD-28590 | biostudies-arrayexpress

Identification of the gene transcription mediated by TGF-β-Smad2/3-Smad4 signaling

Project description:TGF-β signaling is known to be very much dependent on the formation of Smad2/3-Smad4 transcription regulatory complexes. However, the signaling functions of Smad2/3-Smad4 in TGF-β-induced responses are obscure as TGF-β also initiates a number of other signaling pathways. In this study, we systematically assessed the contribution of TGF-β-Smad2/3-Smad4 signaling to target gene transcription. Individual Smads were selectively knocked down in Hep3B cells by stable RNA interference (RNAi). We identified TGF-β-responsive genes using genome-wide oligonucleotide microarrays and confirmed their dependency on Smad2, Smad3 or Smad4 by the combination of RNAi and microarray assay. The major finding from our microarray analysis was that of the 2039 target genes seen to be regulated via TGF-β induction, 190 were differentially transcriptionally controlled by Smad2-Smad4 and Smad3-Smad4 signaling and the latter control mechanism appeared to be functionally more important. We also found evidence of competition between Smad2 and Smad3 for their activation when controlling the transcription of target genes. Keywords: cell type comparison

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Expression data of the human hepatoblastoma cell line HepG2 treated with TGF-beta

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