34 results on '"Lei Chen"'
Search Results
2. TET2–BCLAF1 transcription repression complex epigenetically regulates the expression of colorectal cancer gene Ascl2 via methylation of its promoter
- Author
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Yangyang Shang, Tao Jiang, Lijian Ran, Wenjing Hu, Yun Wu, Jun Ye, Zhihong Peng, Lei Chen, and Rongquan Wang
- Subjects
Tumor Suppressor Proteins ,Cell Biology ,DNA Methylation ,Biochemistry ,Dioxygenases ,DNA-Binding Proteins ,Gene Expression Regulation, Neoplastic ,Repressor Proteins ,Tandem Mass Spectrometry ,Cell Line, Tumor ,Basic Helix-Loop-Helix Transcription Factors ,Humans ,Colorectal Neoplasms ,Promoter Regions, Genetic ,Molecular Biology ,Chromatography, Liquid - Abstract
Ascl2 has been shown to be involved in tumorigenesis in colorectal cancer (CRC), although its epigenetic regulatory mechanism is largely unknown. Here, we found that methylation of the Ascl2 promoter (bp -1670 ∼ -1139) was significantly increased compared to the other regions of the Ascl2 locus in CRC cells and was associated with elevated Ascl2 mRNA expression. Furthermore, we found that promoter methylation was predictive of CRC patient survival after analyzing DNA methylation data, RNA-Seq data, and clinical data of 410 CRC patient samples from the MethHC database, the MEXPRESS database, and the Cbioportal website. Using the established TET methylcytosine dioxygenase 2 (TET2) knockdown and ectopic TET2 catalytic domain-expression cell models, we performed glucosylated hydroxymethyl-sensitive quatitative PCR (qPCR), real-time PCR, and Western blot assays to further confirm that hypermethylation of the Ascl2 promoter, and elevated Ascl2 expression in CRC cells was partly due to the decreased expression of TET2. Furthermore, BCLAF1 was identified as a TET2 interactor in CRC cells by LC-MS/MS, coimmunoprecipitation, immunofluorescence colocalization, and proximity ligation assays. Subsequently, we found the TET2-BCLAF1 complex bound to multiple elements around CCGG sites at the Ascl2 promoter and further restrained its hypermethylation by inducing its hydroxymethylation using chromatin immunoprecipitation-qPCR and glucosylated hydroxymethyl-qPCR assays. Finally, we demonstrate that TET2-modulated Ascl2-targeted stem gene expression in CRC cells was independent of Wnt signaling. Taken together, our data suggest an additional option for inhibiting Ascl2 expression in CRC cells through TET2-BCLAF1-mediated promoter methylation, Ascl2-dependent self-renewal of CRC progenitor cells, and TET2-BCLAF1-related CRC progression.
- Published
- 2022
3. Hepatocyte-specific Sirt6 deficiency impairs ketogenesis
- Author
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Aijuan Qu, Rui Li, Shiyun Pu, Qin Tang, Ya Huang, Meng Gong, Yanping Li, Lei Chen, Zijing Zhang, Hong Li, Min Zou, Jinhang Zhang, Qinhui Liu, Jiangying Kuang, Xuping Yang, Hua Wang, Tao Li, Lu Zhang, Jinhan He, Tong Wu, Wen Xie, and Wei Jiang
- Subjects
Male ,0301 basic medicine ,SIRT6 ,medicine.medical_specialty ,Ketone Bodies ,Biochemistry ,Mice ,03 medical and health sciences ,chemistry.chemical_compound ,Internal medicine ,Lipid droplet ,Ketogenesis ,medicine ,Animals ,Sirtuins ,Molecular Biology ,Mice, Knockout ,030102 biochemistry & molecular biology ,Fatty acid metabolism ,Chemistry ,Proteins ,Lipid metabolism ,Cell Biology ,Lipid Metabolism ,medicine.disease ,Mice, Inbred C57BL ,Metabolism ,030104 developmental biology ,Endocrinology ,medicine.anatomical_structure ,Hepatocyte ,Lipogenesis ,Hepatocytes ,Steatosis - Abstract
Sirt6 is an NADH (NAD(+))-dependent deacetylase with a critical role in hepatic lipid metabolism. Ketogenesis is controlled by a signaling network of hepatic lipid metabolism. However, how Sirt6 functions in ketogenesis remains unclear. Here, we demonstrated that Sirt6 functions as a mediator of ketogenesis in response to a fasting and ketogenic diet (KD). The KD-fed hepatocyte-specific Sirt6 deficiency (HKO) mice exhibited impaired ketogenesis, which was due to enhanced Fsp27 (fat-specific induction of protein 27), a protein known to regulate lipid metabolism. In contrast, overexpression of Sirt6 in mouse primary hepatocytes promoted ketogenesis. Mechanistically, Sirt6 repressed Fsp27β expression by interacting with Crebh (cAMP response element–binding protein H) and preventing its recruitment to the Fsp27β gene promoter. The KD-fed HKO mice also showed exacerbated hepatic steatosis and inflammation. Finally, Fsp27 silencing rescued hypoketonemia and other metabolic phenotypes in KD-fed HKO mice. Our data suggest that the Sirt6–Crebh–Fsp27 axis is pivotal for hepatic lipid metabolism and inflammation. Sirt6 may be a pharmacological target to remedy metabolic diseases.
- Published
- 2019
4. Regulatory domains controlling high intestinal vitamin D receptor gene expression are conserved in mouse and human
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James C. Fleet, Dennis Aldea, Lei Chen, Sylvia Christakos, and Michael Verzi
- Subjects
Intestines ,Mice ,Deoxyribonucleases ,Gene Expression Regulation ,Animals ,Gene Expression ,Humans ,Receptors, Calcitriol ,Cell Biology ,Molecular Biology ,Biochemistry ,Transcription Factors - Abstract
Vitamin D receptor (VDR) levels are highest in the intestine where it mediates 1,25 dihydroxyvitamin D-induced gene expression. However, the mechanisms controlling high intestinal VDR gene expression are unknown. Here, we used Assay for Transposase-Accessible Chromatin using Sequencing (ATAC-Seq) to identify the regulatory sites controlling intestine-specific Vdr gene expression in the small intestine (villi and crypts) and colon of developing, adult, and aged mice. We identified 17 ATAC peaks in a 125 kb region from intron 3 to -55.8 kb from exon 1 of the Vdr gene. Interestingly, many of these peaks were missing/reduced in the developing intestine. Chromatin ImmunoPrecipitation-Sequencing (ChIP-Seq) peaks for intestinal transcription factors (TFs) were present within the ATAC peaks and at HiChIP looping attachments that connected the ATAC/TF ChIP peaks to the transcription start site and CCCTF-binding factor sites at the borders of the Vdr gene regulatory domain. Intestine-specific regulatory sites were identified by comparing ATAC peaks to DNAse-Seq data from other tissues that revealed tissue-specific, evolutionary conserved, and species-specific peaks. Bioinformatics analysis of human DNAse-Seq peaks revealed polymorphisms that disrupt TF-binding sites. Our analysis shows that mouse intestinal Vdr gene regulation requires a complex interaction of multiple distal regulatory regions and is controlled by a combination of intestinal TFs. These intestinal regulatory sites are well conserved in humans suggesting that they may be key components of VDR regulation in both mouse and human intestines.
- Published
- 2022
5. OTUB1 protein suppresses mTOR complex 1 (mTORC1) activity by deubiquitinating the mTORC1 inhibitor DEPTOR
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Weijuan Pan, Chenchen Jiao, Lu Deng, Ping Wang, Xiao Tan, Xinbo Wang, Yue Yu, Lei Chen, Xin Ge, Linlin Zhao, Xiaoping Peng, and Guo-li Gao
- Subjects
0301 basic medicine ,mTORC1 ,Mechanistic Target of Rapamycin Complex 1 ,DEPTOR ,Biochemistry ,mTORC2 ,Deubiquitinating enzyme ,03 medical and health sciences ,0302 clinical medicine ,Protein Domains ,Autophagy ,Humans ,Molecular Biology ,Mechanistic target of rapamycin ,PI3K/AKT/mTOR pathway ,Cell Proliferation ,Deubiquitinating Enzymes ,biology ,Protein Stability ,Chemistry ,Intracellular Signaling Peptides and Proteins ,Ubiquitination ,Cell Biology ,Cell biology ,Cysteine Endopeptidases ,030104 developmental biology ,OTUB1 ,030220 oncology & carcinogenesis ,biology.protein ,HeLa Cells ,Deubiquitination - Abstract
Mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) integrates various environmental signals to regulate cell growth and metabolism. DEPTOR, also termed DEPDC6, is an endogenous inhibitor of mTORC1 and mTORC2 activities. The abundance of DEPTOR centrally orchestrates the mTOR signaling network. However, the mechanisms by which DEPTOR stability is regulated are still elusive. Here, we report that OTU domain–containing ubiquitin aldehyde-binding protein 1 (OTUB1) specifically deubiquitinates DEPTOR in a deubiquitination assay. We found that OTUB1 directly interacted with DEPTOR via its N-terminal domain, deubiquitinated DEPTOR, and thereby stabilized DEPTOR in a Cys-91–independent but Asp-88–dependent manner, suggesting that OTUB1 targets DEPTOR for deubiquitination via a deubiquitinase activity–independent non-canonical mechanism. The interaction between OTUB1 and DEPTOR was enhanced when the cells were treated with amino acids. Moreover, OTUB1 suppressed amino acid–induced activation of mTORC1 in a DEPTOR-dependent manner and thereby ultimately controlled cellular autophagy, cell proliferation, and size. Our findings reveal a mechanism that stabilizes the mTORC1 inhibitor DEPTOR via OTUB1's deubiquitinase activity. Our insights may inform research into various mTOR activity–related diseases, such as cancer, and may contribute to the identification of new diagnostic markers and therapeutic strategies for cancer treatments.
- Published
- 2018
6. Structure, Catalysis, and Inhibition of OfChi-h, the Lepidoptera-exclusive Insect Chitinase
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Yong Zhou, Lei Chen, Xi Jiang, Tian Liu, Yanwei Duan, and Qing Yang
- Subjects
0301 basic medicine ,chemistry.chemical_classification ,biology ,fungi ,Mutant ,Substrate (chemistry) ,Cell Biology ,Substrate analog ,biology.organism_classification ,Biochemistry ,03 medical and health sciences ,chemistry.chemical_compound ,030104 developmental biology ,Enzyme ,chemistry ,Chitin ,Hydrolase ,Chitinase ,biology.protein ,Molecular Biology ,Ostrinia furnacalis - Abstract
Chitinase-h (Chi-h) is of special interest among insect chitinases due to its exclusive distribution in lepidopteran insects and high sequence identity with bacterial and baculovirus homologs. Here OfChi-h, a Chi-h from Ostrinia furnacalis, was investigated. Crystal structures of both OfChi-h and its complex with chitoheptaose ((GlcN)7) reveal that OfChi-h possesses a long and asymmetric substrate binding cleft, which is a typical characteristics of a processive exo-chitinase. The structural comparison between OfChi-h and its bacterial homolog SmChiA uncovered two phenylalanine-to-tryptophan site variants in OfChi-h at subsites +2 and possibly −7. The F232W/F396W double mutant endowed SmChiA with higher hydrolytic activities toward insoluble substrates, such as insect cuticle, α-chitin, and chitin nanowhisker. An enzymatic assay demonstrated that OfChi-h outperformed OfChtI, an insect endo-chitinase, toward the insoluble substrates, but showed lower activity toward the soluble substrate ethylene glycol chitin. Furthermore, OfChi-h was found to be inhibited by N,N′,N″-trimethylglucosamine-N,N′,N″,N″′-tetraacetylchitotetraose (TMG-(GlcNAc)4), a substrate analog which can be degraded into TMG-(GlcNAc)1–2. Injection of TMG-(GlcNAc)4 into 5th-instar O. furnacalis larvae led to severe defects in pupation. This work provides insights into a molting-indispensable insect chitinase that is phylogenetically closer to bacterial chitinases than insect chitinases.
- Published
- 2017
7. Citrus-derived DHCP inhibits mitochondrial complex II to enhance TRAIL sensitivity via ROS-induced DR5 upregulation
- Author
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Hairong Cheng, Lin Sun, Jingmin Yan, Guihua Tai, Yifa Zhou, Miao Hao, and Lei Chen
- Subjects
0301 basic medicine ,Mitochondrial ROS ,Citrus ,SQR, succinate ubiquinone reductase ,mitochondrial respiratory chain complex ,OXPHOS, oxidative phosphorylation ,Cell ,Apoptosis ,TRAIL ,Biochemistry ,TNF-Related Apoptosis-Inducing Ligand ,Mice ,Tumor Cells, Cultured ,OCR, oxygen consumption rate ,Cytotoxicity ,Caspase ,Mice, Inbred BALB C ,biology ,PMS, phenazine methosulfate ,Chemistry ,Electron Transport Complex II ,TCA cycle, tricarboxylic acid cycle ,Cell Cycle ,Cell biology ,Gene Expression Regulation, Neoplastic ,cell death ,medicine.anatomical_structure ,colon cancer ,Colonic Neoplasms ,SDH, succinate dehydrogenase ,Female ,Research Article ,Programmed cell death ,GPDH, glycerol-3-phosphate dehydrogenase ,Mice, Nude ,Cyclopentanes ,ETC, electron transfer chain ,reactive oxygen species (ROS) ,MTT, 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide ,MMP, mitochondrial transmembrane potential ,03 medical and health sciences ,ROS, reactive oxygen species ,TMPD, tetramethyl-p-phenylene diamine ,ubiquinone ,Biomarkers, Tumor ,medicine ,Animals ,Humans ,DR5 ,Citrus Pectin ,Molecular Biology ,Cell Proliferation ,TRAIL, TNF-related apoptosis-inducing ligand ,030102 biochemistry & molecular biology ,G-3-P, glycerol-3-phosphate ,Cell Biology ,Xenograft Model Antitumor Assays ,TTFA, 4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedione ,Receptors, TNF-Related Apoptosis-Inducing Ligand ,030104 developmental biology ,3-NPA, 3-nitropropionic acid ,DCPIP, 2,6-diclorophenol-indophenol ,Cancer cell ,biology.protein ,Reactive Oxygen Species - Abstract
Heat-modified citrus pectin, a water-soluble indigestible polysaccharide fiber derived from citrus fruits and modified by temperature treatment, has been reported to exhibit anticancer effects. However, the bioactive fractions and their mechanisms remain unclear. In this current study, we isolated an active compound, trans-4,5-dihydroxy-2-cyclopentene-l-one (DHCP), from heat-treated citrus pectin, and found that is induces cell death in colon cancer cells via induction of mitochondrial ROS. On the molecular level, DHCP triggers ROS production by inhibiting the activity of succinate ubiquinone reductase (SQR) in mitochondrial complex II. Furthermore, cytotoxicity, apoptotic activity, and activation of caspase cascades were determined in HCT116 and HT-29 cell-based systems, the results indicated that DHCP enhances the sensitivity of cancer cells to tumor necrosis factor–related apoptosis-inducing ligand (TRAIL), with DHCP-induced ROS accounting for the synergistic effect between DHCP and TRAIL. Furthermore, the combination of DHCP and TRAIL inhibits the growth of HCT116 and HT-29 xenografts synergistically. ROS significantly increases the expression of TRAIL death receptor 5 (DR5) via the p53 and C/EBP homologous protein pathways. Collectively, our findings indicate that DHCP has a favorable toxicity profile and is a new TRAIL sensitizer that shows promise in the development of pectin-based pharmaceuticals, nutraceuticals, and dietary agents aimed at combating human colon cancer.
- Published
- 2021
8. Citrus-derived DHCP inhibits mitochondrial complex II to enhance TRAIL sensitivity via ROS-induced DR5 upregulation.
- Author
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Lei Chen, Miao Hao, Jingmin Yan, Lin Sun, Guihua Tai, Hairong Cheng, and Yifa Zhou
- Subjects
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PECTINS , *DEATH receptors , *CITRUS fruits , *MITOCHONDRIA , *CASPASES , *COLON cancer , *CELL death - Abstract
Heat-modified citrus pectin, a water-soluble indigestible polysaccharide fiber derived from citrus fruits and modified by temperature treatment, has been reported to exhibit anticancer effects. However, the bioactive fractions and their mechanisms remain unclear. In this current study, we isolated an active compound, trans-4,5-dihydroxy-2-cyclopentene-l-one (DHCP), from heat-treated citrus pectin, and found that is induces cell death in colon cancer cells via induction of mitochondrial ROS. On the molecular level, DHCP triggers ROS production by inhibiting the activity of succinate ubiquinone reductase (SQR) in mitochondrial complex II. Furthermore, cytotoxicity, apoptotic activity, and activation of caspase cascades were determined in HCT116 and HT-29 cell-based systems, the results indicated that DHCP enhances the sensitivity of cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), with DHCP-induced ROS accounting for the synergistic effect between DHCP and TRAIL. Furthermore, the combination of DHCP and TRAIL inhibits the growth of HCT116 and HT-29 xenografts synergistically. ROS significantly increases the expression of TRAIL death receptor 5 (DR5) via the p53 and C/EBP homologous protein pathways. Collectively, our findings indicate that DHCP has a favorable toxicity profile and is a new TRAIL sensitizer that shows promise in the development of pectin-based pharmaceuticals, nutraceuticals, and dietary agents aimed at combating human colon cancer. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
9. MicroRNA-200 (miR-200) Cluster Regulation by Achaete Scute-like 2 (Ascl2)
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Yangyang Shang, Rong Zhu, Lei Chen, Yin Tian, Qiong Pan, Yonghong He, Jun Ye, Yun Liu, Rongquan Wang, Xiaoli Zhong, Zhihong Peng, Wensheng Chen, Shanshan Li, and Jingjing Zhao
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Colorectal cancer ,Wnt signaling pathway ,Cell Biology ,Transfection ,Biology ,medicine.disease ,Biochemistry ,Molecular biology ,HT29 Cells ,microRNA ,medicine ,Cancer research ,Epithelial–mesenchymal transition ,Stem cell ,Progenitor cell ,Molecular Biology - Abstract
Ascl2, a basic helix-loop-helix transcription factor, is a downstream target of WNT signaling that controls the fate of intestinal cryptic stem cells and colon cancer progenitor cells. However, its involvement in colon cancer and downstream molecular events is largely undefined; in particular, the mechanism by which Ascl2 regulates the plasticity of epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) programs in colon cancer cells remains unknown. In this study, we systematically demonstrate that Ascl2 loss of function in colon cancer cells promotes MET by derepressing the expression of microRNA (miR)-200s (i.e. miR-200b, miR-200a, miR-429, miR-200c, and miR-141) and further activating their expression through a transcriptional mechanism that involves direct binding to the most proximal E-box (E-box2) in the miR-200b-a-429 promoter. Activation of miR-200s due to Ascl2 deficiency led to the inhibition of ZEB1/2 expression and the alteration of epithelial and mesenchymal features. Transfection of miR-200b, miR-200a, and miR-429 inhibitors into Ascl2-deficient colon cancer cells promoted the epithelial-mesenchymal transition in a reversible manner. Transfection of miR-200a or miR-429 inhibitors into Ascl2-deficient colon cancer cells increased cellular proliferation and migration. Ascl2 mRNA levels and the miR-200a, miR-200b, miR-200c, miR-141, or miR-429 levels in the colon cancerous samples were inversely correlated. These results provide the first evidence of a link between Ascl2 and miR-200s in the regulation of EMT-MET plasticity in colon cancer.
- Published
- 2014
10. Fully Deacetylated Chitooligosaccharides Act as Efficient Glycoside Hydrolase Family 18 Chitinase Inhibitors
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Qing Yang, Yong Zhou, Mingbo Qu, Lei Chen, and Yong Zhao
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Stereochemistry ,Oligosaccharides ,Chitin ,Moths ,Biology ,Biochemistry ,chemistry.chemical_compound ,Catalytic Domain ,Hydrolase ,Animals ,Humans ,Glycoside hydrolase ,Enzyme Inhibitors ,Molecular Biology ,Glycoside hydrolase family 18 ,chemistry.chemical_classification ,Chitinases ,fungi ,Mutagenesis ,Cell Biology ,Enzyme ,chemistry ,Enzyme inhibitor ,Larva ,Protein Structure and Folding ,Chitinase ,biology.protein ,Insect Proteins - Abstract
Small molecule inhibitors against chitinases have potential applications as pesticides, fungicides, and antiasthmatics. Here, we report that a series of fully deacetylated chitooligosaccharides (GlcN)2-7 can act as inhibitors against the insect chitinase OfChtI, the human chitinase HsCht, and the bacterial chitinases SmChiA and SmChiB with IC50 values at micromolar to millimolar levels. The injection of mixed (GlcN)2-7 into the fifth instar larvae of the insect Ostrinia furnacalis resulted in 85% of the larvae being arrested at the larval stage and death after 10 days, also suggesting that (GlcN)2-7 might inhibit OfChtI in vivo. Crystal structures of the catalytic domain of OfChtI (OfChtI-CAD) complexed with (GlcN)5,6 were obtained at resolutions of 2.0 Å. These structures, together with mutagenesis and thermodynamic analysis, suggested that the inhibition was strongly related to the interaction between the -1 GlcN residue of the inhibitor and the catalytic Glu(148) of the enzyme. Structure-based comparison showed that the fully deacetylated chitooligosaccharides mimic the substrate chitooligosaccharides by binding to the active cleft. This work first reports the inhibitory activity and proposed inhibitory mechanism of fully deacetylated chitooligosaccharides. Because the fully deacetylated chitooligosaccharides can be easily derived from chitin, one of the most abundant materials in nature, this work also provides a platform for developing eco-friendly inhibitors against chitinases.
- Published
- 2014
11. Molecular Mechanism of the Negative Regulation of Smad1/5 Protein by Carboxyl Terminus of Hsc70-interacting Protein (CHIP)
- Author
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Zhijie Chang, Le Wang, Yi-Tong Liu, Lei Chen, Jia-Wei Wu, Rui Hao, Zhi-Xin Wang, and Hong-Rui Wang
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Smad5 Protein ,animal structures ,Ubiquitin-Protein Ligases ,Amino Acid Motifs ,Endoplasmic-reticulum-associated protein degradation ,Protein degradation ,Biochemistry ,Smad1 Protein ,Protein–protein interaction ,Protein structure ,Transforming Growth Factor beta ,Humans ,Molecular Biology ,biology ,HSC70 Heat-Shock Proteins ,Ubiquitination ,Cell Biology ,Protein Structure, Tertiary ,Hsp70 ,Ubiquitin ligase ,Cell biology ,Tetratricopeptide ,HEK293 Cells ,Chaperone (protein) ,embryonic structures ,biology.protein ,biological phenomena, cell phenomena, and immunity ,Peptides ,Hydrophobic and Hydrophilic Interactions ,Protein Binding ,Signal Transduction - Abstract
The transforming growth factor-β (TGF-β) superfamily of ligands signals along two intracellular pathways, Smad2/3-mediated TGF-β/activin pathway and Smad1/5/8-mediated bone morphogenetic protein pathway. The C terminus of Hsc70-interacting protein (CHIP) serves as an E3 ubiquitin ligase to mediate the degradation of Smad proteins and many other signaling proteins. However, the molecular mechanism for CHIP-mediated down-regulation of TGF-β signaling remains unclear. Here we show that the extreme C-terminal sequence of Smad1 plays an indispensable role in its direct association with the tetratricopeptide repeat (TPR) domain of CHIP. Interestingly, Smad1 undergoes CHIP-mediated polyubiquitination in the absence of molecular chaperones, and phosphorylation of the C-terminal SXS motif of Smad1 enhances the interaction and ubiquitination. We also found that CHIP preferentially binds to Smad1/5 and specifically disrupts the core signaling complex of Smad1/5 and Smad4. We determined the crystal structures of CHIP-TPR in complex with the phosphorylated/pseudophosphorylated Smad1 peptides and with an Hsp70/Hsc70 C-terminal peptide. Structural analyses and subsequent biochemical studies revealed that the distinct CHIP binding affinities of Smad1/5 or Smad2/3 result from the nonconservative hydrophobic residues at R-Smad C termini. Unexpectedly, the C-terminal peptides from Smad1 and Hsp70/Hsc70 bind in the same groove of CHIP-TPR, and heat shock proteins compete with Smad1/5 for CHIP interaction and concomitantly suppress, rather than facilitate, CHIP-mediated Smad ubiquitination. Thus, we conclude that CHIP inhibits the signaling activities of Smad1/5 by recruiting Smad1/5 from the functional R-/Co-Smad complex and further promoting the ubiquitination/degradation of Smad1/5 in a chaperone-independent manner.
- Published
- 2011
12. MDM2 Acts Downstream of p53 as an E3 Ligase to Promote FOXO Ubiquitination and Degradation
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Wei Fu, Jiandong Chen, Hengbing Wang, Pengfei Li, Zheng Shen, Yanping Zhang, Yonghua Yang, Lei Chen, Yingtao Zhang, Qiuping Ma, Sivapriya Ramamoorthy, Wenlong Bai, Santo V. Nicosia, Zafar Nawaz, Jack W. Pledger, Xiaohong Zhang, Tsukasa Shimojima, and Mu Zhang
- Subjects
endocrine system ,Small interfering RNA ,Ubiquitin-Protein Ligases ,Apoptosis ,FOXO1 ,Biochemistry ,Cell Line ,Mice ,Ubiquitin ,Animals ,Humans ,Phosphorylation ,Molecular Biology ,Protein kinase B ,Transcription factor ,Cell Nucleus ,biology ,Mechanisms of Signal Transduction ,fungi ,Ubiquitination ,Forkhead Transcription Factors ,Proto-Oncogene Proteins c-mdm2 ,Cell Biology ,Molecular biology ,Ubiquitin ligase ,Protein Transport ,Gene Expression Regulation ,Cytoprotection ,biology.protein ,Mdm2 ,Tumor Suppressor Protein p53 ,Protein Processing, Post-Translational ,Proto-Oncogene Proteins c-akt ,hormones, hormone substitutes, and hormone antagonists ,Protein Binding - Abstract
Members of the FOXO (forkhead O) class of transcription factors are tumor suppressors that also control aging and organismal life span. Mammalian FOXO degradation is proteasome-mediated, although the ubiquitin E3 ligase for FOXO factors remains to be defined. We show that MDM2 binds to FOXO1 and FOXO3A and promotes their ubiquitination and degradation, a process apparently dependent on FOXO phosphorylation at AKT sites and the E3 ligase activity of MDM2. Binding of MDM2 to FOXO occurs through the region of MDM2 that directs its cellular trafficking and the forkhead box of FOXO1. MDM2 promotes the ubiquitination of FOXO1 in a cell-free system, and its knockdown by small interfering RNA causes accumulation of endogenous FOXO3A protein in cells and enhances the expression of FOXO target genes. In cells stably expressing a temperature-sensitive p53 mutant, activation of p53 by shifting to permissive temperatures leads to MDM2 induction and degradation of endogenous FOXO3A. These data suggest that MDM2 acts as an ubiquitin E3 ligase, downstream of p53, to regulate the degradation of mammalian FOXO factors.
- Published
- 2009
13. Bone Morphogenetic Protein 2 Mediates Dentin Sialophosphoprotein Expression and Odontoblast Differentiation via NF-Y Signaling
- Author
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Shuo Chen, Jelica Gluhak-Heinrich, Marcos Martinez, Tong Li, Yimin Wu, Hui-Hsiu Chuang, Lei Chen, Juan Dong, Isabel Gay, and Mary MacDougall
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animal structures ,Transcription, Genetic ,Sialoglycoproteins ,Cellular differentiation ,Amino Acid Motifs ,CAAT box ,Bone Morphogenetic Protein 2 ,Odontoblast differentiation ,Biology ,Response Elements ,Biochemistry ,Bone morphogenetic protein 2 ,Cell Line ,Mice ,Molecular Basis of Cell and Developmental Biology ,stomatognathic system ,Dentin sialophosphoprotein ,Transforming Growth Factor beta ,Gene expression ,Animals ,RNA, Messenger ,Protein Precursors ,Molecular Biology ,Transcription factor ,Sequence Deletion ,Cell Nucleus ,Extracellular Matrix Proteins ,Mice, Inbred ICR ,Base Sequence ,Odontoblasts ,fungi ,Gene Expression Regulation, Developmental ,Cell Differentiation ,Cell Biology ,Phosphoproteins ,Molecular biology ,Up-Regulation ,stomatognathic diseases ,Odontoblast ,CCAAT-Binding Factor ,Bone Morphogenetic Proteins ,embryonic structures ,Odontogenesis ,Carrier Proteins ,Signal Transduction - Abstract
Dentin sialophosphoprotein (DSPP), an important odontoblast differentiation marker, is necessary for tooth development and mineralization. Bone morphogenetic protein 2 (BMP2) plays a vital role in odontoblast function via diverse signal transduction systems. We hypothesize that BMP2 regulates DSPP gene transcription and thus odontoblast differentiation. Here we report that expression of BMP2 and DSPP is detected during mouse odontogenesis by in situ hybridization assay, and BMP2 up-regulates DSPP mRNA and protein expression as well as DSPP-luciferase promoter activity in mouse preodontoblasts. By sequentially deleting fragments of the mouse DSPP promoter, we show that a BMP2-response element is located between nucleotides –97 and –72. By using antibody and oligonucleotide competition assays in electrophoretic mobility shift analysis and chromatin immunoprecipitation experiments, we show that the heterotrimeric transcription factor Y (NF-Y) complex physically interacts with the inverted CCAAT box within the BMP2-response element. BMP2 induces NF-Y accumulation into the nucleus increasing its recruitment to the mouse DSPP promoter in vivo. Furthermore, forced overexpression of NF-Y enhances promoter activity and increases endogenous DSPP protein levels. In contrast, mutations in the NF-Y-binding motif reduce BMP2-induced DSPP transcription. Moreover, inhibiting BMP2 signaling by Noggin, a BMP2 antagonist, results in significant inhibition of DSPP gene expression in preodontoblasts. Taken together, these results indicate that BMP2 mediates DSPP gene expression and odontoblast differentiation via NF-Y signaling during tooth development.
- Published
- 2008
14. Hepatocyte-specific Sirt6 deficiency impairs ketogenesis.
- Author
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Lei Chen, Qinhui Liu, Qin Tang, Jiangying Kuang, Hong Li, Shiyun Pu, Tong Wu, Xuping Yang, Rui Li, Jinhang Zhang, Zijing Zhang, Ya Huang, Yanping Li, Min Zou, Wei Jiang, Tao Li, Meng Gong, Lu Zhang, Hua Wang, and Aijuan Qu
- Subjects
- *
DEACETYLASES , *ENZYMES , *SIRTUINS , *LIPIDS , *BIOMOLECULES , *LIPID metabolism - Abstract
Sirt6 is an NADH (NAD+)-dependent deacetylase with a critical role in hepatic lipid metabolism. Ketogenesis is controlled by a signaling network of hepatic lipid metabolism. However, how Sirt6 functions in ketogenesis remains unclear. Here, we demonstrated that Sirt6 functions as a mediator of ketogenesis in response to a fasting and ketogenic diet (KD). The KD-fed hepatocyte-specific Sirt6 deficiency (HKO) mice exhibited impaired ketogenesis, which was due to enhanced Fsp27 (fatspecific induction of protein 27), a protein known to regulate lipid metabolism. In contrast, overexpression of Sirt6 in mouse primary hepatocytes promoted ketogenesis. Mechanistically, Sirt6 repressed Fsp27β expression by interacting with Crebh (cAMP response element--binding protein H) and preventing its recruitment to the Fsp27βgene promoter. The KD-fed HKO mice also showed exacerbated hepatic steatosis and inflammation. Finally, Fsp27 silencing rescued hypoketonemia and other metabolic phenotypes in KD-fed HKO mice. Our data suggest that the Sirt6--Crebh--Fsp27 axis is pivotal for hepatic lipid metabolism and inflammation. Sirt6 may be a pharmacological target to remedy metabolic diseases. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
15. Activation of Apoptosis Signal-regulating Kinase 1 by Reactive Oxygen Species through Dephosphorylation at Serine 967 and 14-3-3 Dissociation
- Author
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Erinn H. Goldman, Haian Fu, and Lei Chen
- Subjects
Time Factors ,Tyrosine 3-Monooxygenase ,MAP Kinase Kinase 4 ,Blotting, Western ,Biology ,Mitogen-activated protein kinase kinase ,MAP Kinase Kinase Kinase 5 ,Transfection ,medicine.disease_cause ,Models, Biological ,p38 Mitogen-Activated Protein Kinases ,Biochemistry ,Catalysis ,Dephosphorylation ,Serine ,medicine ,Animals ,Humans ,ASK1 ,Phosphorylation ,Molecular Biology ,Mitogen-Activated Protein Kinase Kinases ,chemistry.chemical_classification ,Reactive oxygen species ,MAP kinase kinase kinase ,JNK Mitogen-Activated Protein Kinases ,Hydrogen Peroxide ,Cell Biology ,MAP Kinase Kinase Kinases ,Precipitin Tests ,Cell biology ,Enzyme Activation ,Oxidative Stress ,14-3-3 Proteins ,chemistry ,COS Cells ,Mitogen-Activated Protein Kinases ,Signal transduction ,Reactive Oxygen Species ,Oxidative stress ,Protein Binding ,Signal Transduction - Abstract
Oxidative stress has been indicated in a variety of pathological processes such as atherosclerosis, diabetes, and neurodegenerative diseases. Understanding how intracellular signaling pathways respond to oxidative insults such as hydrogen peroxide (H(2)O(2)) would have significant therapeutic implications. Recent genetic studies have placed apoptosis signal-regulating kinase 1 (ASK1) in a pivotal position in transmitting H(2)O(2)-initiated signals. How ASK1 is activated by H(2)O(2), though, remains a subject of intense investigation. Here we report a mechanism by which H(2)O(2) induces ASK1 activation through dynamic control of its phosphorylation at serine 967. We found that treatment of COS7 cells with H(2)O(2) triggers dephosphorylation of Ser-967 through an okadaic acid-sensitive phosphatase, resulting in dissociation of the ASK1.14-3-3 complex with concomitant increase of ASK1 catalytic activity and ASK1-mediated activation of JNK and p38 pathways.
- Published
- 2004
16. Activation of Protein Kinase C Induces Nuclear Translocation of RFX1 and Down-regulates c-myc via an Intron 1 X Box in Undifferentiated Leukemia HL-60 Cells
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Kangsheng Wang, Martin R. Johnson, Lucinda Smith, Robert B. Diasio, Jeffrey Bingham Smith, and Lei Chen
- Subjects
Indoles ,Bryostatin 1 ,Cellular differentiation ,Active Transport, Cell Nucleus ,Genes, myc ,Down-Regulation ,HL-60 Cells ,Regulatory Factor X Transcription Factors ,Biology ,Response Elements ,Transfection ,Biochemistry ,Maleimides ,Proto-Oncogene Proteins c-myc ,Lactones ,Cytosol ,Genes, Reporter ,Protein biosynthesis ,Humans ,RNA, Messenger ,Nuclear protein ,Molecular Biology ,Protein Kinase C ,Protein kinase C ,Cell Nucleus ,Reporter gene ,Binding Sites ,Nuclear Proteins ,Cell Differentiation ,Cell Biology ,Bryostatins ,Molecular biology ,Introns ,DNA-Binding Proteins ,Enzyme Activation ,Tetradecanoylphorbol Acetate ,RFX1 ,Macrolides ,Regulatory Factor X1 ,Protein Binding ,Transcription Factors - Abstract
Treatment of human promyelocytic leukemia cells (HL-60) with phorbol 12-myristate 13-acetate (PMA) is known to decrease c-myc mRNA by blocking transcription elongation at sites near the first exon/intron border. Treatment of HL-60 cells with either PMA or bryostatin 1, which acutely activates protein kinase C (PKC), decreased the levels of myc mRNA and Myc protein. The inhibition of Myc synthesis accounted for the drop in Myc protein, because PMA treatment had no effect on Myc turnover. Treatment with PMA or bryostatin 1 increased nuclear protein binding to MIE1, a c-myc intron 1 element that defines an RFX1-binding X box. RFX1 antiserum supershifted MIE1-protein complexes. Increased MIE1 binding was independent of protein synthesis and abolished by a selective PKC inhibitor, which also prevented the effect of PMA on myc mRNA and protein levels and Myc synthesis. PMA treatment increased RFX1 in the nuclear fraction and decreased it in the cytosol without affecting total RFX1. Transfection of HL-60 cells with myc reporter gene constructs showed that the RFX1-binding X box was required for the down-regulation of reporter gene expression by PMA. These findings suggest that nuclear translocation and binding of RFX1 to the X box cause the down-regulation of myc expression, which follows acute PKC activation in undifferentiated HL-60 cells.
- Published
- 2000
17. The Activity of Topoisomerase I Is Modulated by Large T Antigen during Unwinding of the SV40 Origin
- Author
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Dahai Gai, Rupa Roy, Daniel T. Simmons, Pamela W. Trowbridge, and Lei Chen
- Subjects
DNA Replication ,viruses ,Replication Origin ,Simian virus 40 ,Biology ,Random hexamer ,Origin of replication ,Biochemistry ,Virus ,chemistry.chemical_compound ,Antigen ,Humans ,Large T-Antigen ,Antigens, Viral, Tumor ,Molecular Biology ,Topoisomerase ,DNA Helicases ,Helicase ,Sequence Analysis, DNA ,Cell Biology ,Molecular biology ,DNA Topoisomerases, Type I ,chemistry ,DNA, Viral ,Mutation ,biology.protein ,Nucleic Acid Conformation ,DNA - Abstract
When simian virus 40 (SV40) large T antigen binds to the virus origin of replication, it forms a double hexamer that functions as a helicase to unwind the DNA bidirectionally. We demonstrate in this report that T antigen can unwind and release an origin DNA single strand of less than full length in the presence of purified human topoisomerase I. The sites nicked by topoisomerase I in the strands released by T antigen during DNA unwinding were localized primarily to the "late" side of the origin, and the template for lagging strand synthesis was preferred significantly over the one for leading strand synthesis. Importantly, these sites were, for the most part, different from the sites nicked by topoisomerase I in the absence of T antigen. These data indicate that T antigen activates topoisomerase I nicking at discrete sites and releases these nicked strands during unwinding. We hypothesize that a single molecule of topoisomerase I can form a functional complex with a double hexamer of T antigen to simultaneously relax and unwind double-stranded origin-containing DNA.
- Published
- 1998
18. Cloning and expression of cyclosporin A- and FK506-sensitive nuclear factor of activated T-cells: NF45 and NF90
- Author
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Peter N. Kao, Julian Ng, Alan Jay Smith, J. Kenny, Lei Chen, G. Brock, and B. Corthesy
- Subjects
chemistry.chemical_classification ,Protein subunit ,RNA ,Cell Biology ,Biology ,Biochemistry ,Jurkat cells ,Molecular biology ,Amino acid ,chemistry ,Bacterial transcription ,Cyclosporin a ,Gene expression ,Molecular Biology ,Transcription factor - Abstract
Nuclear Factor of Activated T-cells (NF-AT) is a crucial transcription factor required for T-cell expression of interleukin 2. Purified NF-AT contains 45-kDa and 90-kDa subunits (Corthesy, B., and Kao, P. N. (1994) J. Biol. Chem. 269, 20682-20690). Partial internal amino acid sequences derived from each subunit indicate that these proteins are novel. The amino acid sequences were used to clone the cDNAs encoding each subunit. The cDNAs predict proteins of novel structures: NF45 has limited similarity to prokaryotic transcription factor sigma-54 and to human DNA topoisomerase II; NF90 has limited similarity to Drosophila Staufen in a domain predicted to bind double-stranded RNA. RNA encoding NF45 and NF90 exists in nonstimulated Jurkat T-cells and in all other cell types examined (HeLa, HepG2, K562). Immunofluorescence microscopy was used to demonstrate that both proteins are located in the nucleus of Jurkat T-cells. Clones NF45 and NF90 with a polyhistidine fusion tag were transiently expressed and processed in the native environment of Jurkat T-cells. Histidine-tagged NF45 and NF90 proteins, affinity-purified on nickel chelate columns, encode a NF-AT DNA-binding activity that is enhanced following T-cell stimulation, and this enhancement is blocked when T-cells are stimulated in the presence of cyclosporin A or FK506.
- Published
- 1994
19. Structure, Catalysis, and Inhibition of OfChi-h, the Lepidoptera-exclusive Insect Chitinase.
- Author
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Tian Liu, Lei Chen, Yong Zhou, Xi Jiang, Yanwei Duan, and Qing Yang
- Subjects
- *
CHITINASE , *TRYPTOPHAN , *LEPIDOPTERA , *GLYCOSIDASES , *PHENYLALANINE , *BIOCHEMICAL substrates - Abstract
Chitinase-h (Chi-h) is of special interest among insect chitinases due to its exclusive distribution in lepidopteran insects and high sequence identity with bacterial and baculovirus homologs. Here OfChi-h, a Chi-h from Ostrinia furnacalis, was investigated. Crystal structures of both OfChi-h and its complex with chitoheptaose ((GlcN)7) reveal that OfChi-h possesses a long and asymmetric substrate binding cleft, which is a typical characteristics of a processive exo-chitinase. The structural comparison between OfChi-h and its bacterial homolog SmChiA uncovered two phenylalanine-to-tryptophan site variants in OfChi-h at subsites +2 and possibly -7. The F232W/F396W double mutant endowedSmChiA with higher hydrolytic activities toward insoluble substrates, such as insect cuticle, α-chitin, and chitin nanowhisker. An enzymatic assay demonstrated that OfChi-h outperformed OfChtI, an insect endochitinase, toward the insoluble substrates, but showed lower activity toward the soluble substrate ethylene glycol chitin. Furthermore, OfChi-h was found to be inhibited by N,N′,N″-trimethylglucosamine-N,N′,N″,N″′-tetraacetylchitotetraose (TMG-(GlcNAc)4), a substrate analog which can be degraded into TMG-(GlcNAc)1-2. Injection of TMG-(GlcNAc)4 into 5th-instar O. furnacalis larvae led to severe defects in pupation. This work provides insights into a molting-indispensable insect chitinase that is phylogenetically closer to bacterial chitinases than insect chitinases. [ABSTRACT FROM AUTHOR]
- Published
- 2017
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20. Fully Deacetylated Chitooligosaccharides Act as Efficient Glycoside Hydrolase Family 18 Chitinase Inhibitors.
- Author
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Lei Chen, Yong Zhou, Mingbo Qu, Yong Zhao, and Qing Yang
- Subjects
- *
OLIGOSACCHARIDES , *GLYCOSIDASE inhibitors , *CHITINASE , *DEACETYLATION , *SMALL molecules - Abstract
Small molecule inhibitors against chitinases have potential applications as pesticides, fungicides, and antiasthmatics. Here, we report that a series of fully deacetylated chitooligosaccharides (GlcN)2-7 can act as inhibitors against the insect chitinase OfChtI, the human chitinase HsCht, and the bacterial chitinases SmChiA and SmChiB with IC50 values at micromolar to millimolar levels. The injection of mixed (GlcN)2-7 into the fifth instar larvae of the insect Ostrinia furnacalis resulted in 85% of the larvae being arrested at the larval stage and death after 10 days, also suggesting that (GlcN)2-7 might inhibit OfChtI in vivo. Crystal structures of the catalytic domain of OfChtI (OfChtI-CAD) complexed with (GlcN)5,6 were obtained at resolutions of 2.0 Å. These structures, together with mutagenesis and thermodynamic analysis, suggested that the inhibition was strongly related to the interaction between the -1 GlcN residue of the inhibitor and the catalytic Glu148 of the enzyme. Structure-based comparison showed that the fully deacetylated chitooligosaccharides mimic the substrate chitooligosaccharides by binding to the active cleft. This work first reports the inhibitory activity and proposed inhibitory mechanism of fully deacetylated chitooligosaccharides. Because the fully deacetylated chitooligosaccharides can be easily derived from chitin, one of the most abundant materials in nature, this work also provides a platform for developing eco-friendly inhibitors against chitinases. [ABSTRACT FROM AUTHOR]
- Published
- 2014
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21. The A-kinase Anchoring Protein Yotiao Facilitates Complex Formation between Adenylyl Cyclase Type 9 and the IKs Potassium Channel in Heart.
- Author
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Yong Li, Lei Chen, Kass, Robert S., and Dessauer, Carmen W.
- Subjects
- *
A-kinase anchoring proteins , *ADENYLATE cyclase , *POTASSIUM channels , *HEART , *REVERSE transcriptase polymerase chain reaction , *TRANSGENIC mice - Abstract
The scaffolding protein Yotiao is a member of a large family of protein A-kinase anchoring proteins with important roles in the organization of spatial and temporal signaling. In heart, Yotiao directly associates with the slow outward potassium ion current (IKs) and recruits both PKA and PP1 to regulate IKs phosphorylation and gating. Human mutations that disrupt IKs-Yotiao interaction result in reduced PKA-dependent phosphorylation of the IKs subunit KCNQ1 and inhibition of sympathetic stimulation of IKs, which can give rise to long-QT syndrome. We have previously identified a subset of adenylyl cyclase (AC) isoforms that interact with Yotiao, including AC1-3 and AC9, but surprisingly, this group did not include the major cardiac isoforms AC5 and AC6. We now show that either AC2 or AC9 can associate with KCNQ1 in a complex mediated by Yotiao. In transgenic mouse heart expressing KCNQ1-KCNE1, AC activity was specifically associated with the IKs-Yotiao complex and could be disrupted by addition of the AC9 N terminus. A survey of all AC isoforms by RT-PCR indicated expression of AC4-6 and AC9 in adult mouse cardiac myocytes. Of these, the only Yotiao-interacting isoform was AC9. Furthermore, the endogenous IKs-Yotiao complex from guinea pig also contained AC9. Finally, AC9 association with the KCNQ1-Yotiao complex sensitized PKA phosphorylation of KCNQ1 to β-adrenergic stimulation. Thus, in heart, Yotiao brings together PKA, PP1, PDE4D3, AC9, and the IKs channel to achieve localized temporal regulation of β-adrenergic stimulation. [ABSTRACT FROM AUTHOR]
- Published
- 2012
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22. Smad6 Inhibits the Transcriptional Activity of Tbx6 by Mediating Its Degradation.
- Author
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Yue-Lei Chen, Bin Liu, Zhen-Ning Zhou, Rui-Ying Hu, Cong Fei, Zhi-Hui Xie, and Xiaoyan Ding
- Subjects
- *
CELL differentiation , *GROWTH factors , *BONE morphogenetic proteins , *PHYSIOLOGICAL control systems , *HOMOLOGY (Biology) , *HEREDITY - Abstract
Members of the bone morphogenetic protein (BMP) and T-box gene families play several critical roles in the early embryonic development and tissue homeostasis. Although BMP proteins are the upstream regulators of T-box genes, few studies have investigated the molecular mechanisms between these two protein families. Here, we report that Tbx6 interacts directly with Smad6, an inhibitory Smad that antagonizes the BMP signal. This interaction is mediated through the Mad homology 2 (MH2) domain of Smad6 and residues 90-180 of Tbx6. We demonstrate that Smad6 facilitates the degradation of Tbx6 protein through recruitment of Smurf1, a ubiquitin E3 ligase. Consequently, Smad6 reduces Tbx6-mediated Myf-5 gene activation. Furthermore, specific knockdown of endogenous Smad6 and Smurf1 by small interfering RNA increases the protein levels of Tbx6 and enhance the expression of Tbx6 target genes. Collectively, these findings reveal that Smad6 serves as a critical mediator of BMP signal via a functional interaction with Tbx6, thus regulating the activation of Tbx6 downstream genes during cell differentiation. [ABSTRACT FROM AUTHOR]
- Published
- 2009
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23. Phosphorylation of the A-kinase-anchoring Protein Yotiao Contributes to Protein Kinase A Regulation of a Heart Potassium Channel.
- Author
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Lei Chen, Kurokawa, Junko, and Kass, Robert S.
- Subjects
- *
PROTEIN kinases , *PROTEINS , *PHOSPHORYLATION , *PROTEIN binding , *CYCLIC adenylic acid , *MEMBRANE proteins , *BIOCHEMISTRY - Abstract
Regulation of the heart by the sympathetic nervous system, fundamental to the physiological response to stress and exercise, requires coordinated phosphorylation of multiple downstream molecular targets, including the IKs (slowly activating potassium current) channel. Sympathetic nervous system stimulation increases intracellular cAMP for which targeted regulation is directed in large part by distinct scaffold or anchoring proteins. Yotiao is an A-kinase-anchoring protein (AKAP) that recruits the cyclic AMP-dependent protein kinase (protein kinase A (PKA)) and protein phosphatase 1 to the carboxyl terminus of the IKs channel to form a molecular complex and control its phosphorylation state, crucial to the cardiac cellular response to sympathetic nervous system stimulation. Here we report that Yotiao itself is a substrate for PKA phosphorylation, and we identify a Yotiao amino-terminal (N-T) residue (Ser-43) that is PKA-phosphorylated in response to β-adrenergic receptor stimulation. The replacement of Ser-43 by Ala ablates the PKA phosphorylation of N-T Yotiao and markedly diminishes the functional response of the wild type and pseudo-phosphorylated IKs channel to cAMP but neither prevents the PKA phosphorylation of KCNQ1 nor its binding to Yotiao. These results suggest, for the first time, a critical role for the PKA phosphorylation of an AKAP in the functional regulation of an ion channel protein and postphosphorylation allosteric modulation of the IKs channel by Yotiao. [ABSTRACT FROM AUTHOR]
- Published
- 2005
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24. Activation of Apoptosis Signal-regulating Kinase 1 by Reactive Oxygen Species through Dephosphorylation at Serine 967 and 14-3-3 Dissociation.
- Author
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Goldman, Erinn H., Lei Chen, and Haian Fu
- Subjects
- *
OXIDATIVE stress , *APOPTOSIS , *PROTEIN kinases , *BIOCHEMISTRY , *BIOLOGY , *CHEMISTRY - Abstract
Oxidative stress has been indicated in a variety of pathological processes such as atherosclerosis, diabetes, and neurodegenerative diseases. Understanding how intracellular signaling pathways respond to oxidative insults such as hydrogen peroxide (H2O2) would have significant therapeutic implications. Recent genetic studies have placed apoptosis signal-regulating kinase 1 (ASK1) in a pivotal position in transmitting H2O2-initiated signals. How ASK1 is activated by H2O2, though, remains a subject of intense investigation. Here we report a mechanism by which H2O2 induces ASK1 activation through dynamic control of its phosphorylation at serine 967. We found that treatment of COS7 cells with H2O2 triggers dephosphorylation of Ser-967 through an okadaic acid-sensitive phosphatase, resulting in dissociation of the ASK1·14-3-3 complex with concomitant increase of ASK1 catalytic activity and ASKl-mediated activation of JNK and p38 pathways. [ABSTRACT FROM AUTHOR]
- Published
- 2004
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25. Regulatory domains controlling high intestinal vitamin D receptor gene expression are conserved in mouse and human.
- Author
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Fleet, James C., Aldea, Dennis, Lei Chen, Christakos, Sylvia, and Verzi, Michael
- Subjects
- *
VITAMIN D receptors , *GENE expression , *INTESTINES , *GENETIC regulation , *REGULATOR genes , *SMALL intestine - Abstract
Vitamin D receptor (VDR) levels are highest in the intestine where it mediates 1,25 dihydroxyvitamin D-induced gene expression. However, the mechanisms controlling high intestinal VDR gene expression are unknown. Here, we used Assay for Transposase-Accessible Chromatin using Sequencing (ATAC-Seq) to identify the regulatory sites controlling intestine-specific Vdr gene expression in the small intestine (villi and crypts) and colon of developing, adult, and aged mice. We identified 17 ATAC peaks in a 125 kb region from intron 3 to -55.8 kb from exon 1 of the Vdr gene. Interestingly, many of these peaks were missing/reduced in the developing intestine. Chromatin ImmunoPrecipitation-Sequencing (ChIP-Seq) peaks for intestinal transcription factors (TFs) were present within the ATAC peaks and at HiChIP looping attachments that connected the ATAC/TF ChIP peaks to the transcription start site and CCCTF-binding factor sites at the borders of the Vdr gene regulatory domain. Intestine-specific regulatory sites were identified by comparing ATAC peaks to DNAse-Seq data from other tissues that revealed tissue-specific, evolutionary conserved, and species-specific peaks. Bioinformatics analysis of human DNAse-Seq peaks revealed polymorphisms that disrupt TF-binding sites. Our analysis shows that mouse intestinal Vdr gene regulation requires a complex interaction of multiple distal regulatory regions and is controlled by a combination of intestinal TFs. These intestinal regulatory sites are well conserved in humans suggesting that they may be key components of VDR regulation in both mouse and human intestines. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
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26. Large Conformational Changes of Insertion 3 in Human Glycyl-tRNA Synthetase (hGlyRS) during Catalysis.
- Author
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Deng, Xiangyu, Xiangjing Qin, Lei Chen, Qian Jia, Yonghui Zhang, Zhiyong Zhang, Dongsheng Lei, Gang Ren, Zhihong Zhou, Zhong Wang, Qing Li, and Wei Xie
- Subjects
- *
TRANSFER RNA synthetases , *GLYCINE , *AMINOACYLATION , *NEUROLOGICAL disorders , *MOLECULAR dynamics - Abstract
Glycyl-tRNA synthetase (GlyRS) is the enzyme that covalently links glycine to cognate tRNA for translation. It is of great research interest because of its nonconserved quaternary structures, unique species-specific aminoacylation properties, and noncanonical functions in neurological diseases, but none of these is fully understood. We report two crystal structures of human GlyRS variants, in the free form and in complex with tRNAGly respectively, and reveal new aspects of the glycylation mechanism.Wediscover that insertion 3 differs considerably in conformation in catalysis and that it acts like a "switch" and fully opens to allow tRNA to bind in a cross-subunit fashion. The flexibility of the protein is supported by molecular dynamics simulation, as well as enzymatic activity assays. The biophysical and biochemical studies suggest that human GlyRS may utilize its flexibility for both the traditional function (regulate tRNA binding) and alternative functions (roles in diseases). [ABSTRACT FROM AUTHOR]
- Published
- 2016
- Full Text
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27. Genomic analysis of 1,25-dihydroxyvitamin D3 action in mouse intestine reveals compartment and segment-specific gene regulatory effects.
- Author
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Aita, Rohit, Aldea, Dennis, Hassan, Sohaib, Hur, Joseph, Pellon-Cardenas, Oscar, Cohen, Evan, Lei Chen, Shroyer, Noah, Christakos, Sylvia, Verzi, Michael P., and Fleet, James C.
- Subjects
- *
GENOMICS , *REGULATOR genes , *VITAMIN D receptors , *INTESTINES , *SMALL intestine - Abstract
1,25-dihydroxyvitamin D (VD) regulates intestinal calcium absorption in the small intestine (SI) and also reduces risk of colonic inflammation and cancer. However, the intestine compartment-specific target genes of VD signaling are unknown. Here, we examined VD action across three functional compartments of the intestine using RNA-seq to measure VDinduced changes in gene expression and Chromatin Immunoprecipitation with next generation sequencing to measure vitamin D receptor (VDR) genomic binding. We found that VD regulated the expression of 55 shared transcripts in the SI crypt, SI villi, and in the colon, including Cyp24a1, S100g, Trpv6, and Slc30a10. Other VD-regulated transcripts were unique to the SI crypt (162 up, 210 down), villi (199 up, 63 down), or colon (102 up, 28 down), but this did not correlate with mRNA levels of the VDR. Furthermore, bioinformatic analysis identified unique VD-regulated biological functions in each compartment. VDR-binding sites were found in 70% of upregulated genes from the colon and SI villi but were less common in upregulated genes from the SI crypt and among downregulated genes, suggesting some transcript-level VD effects are likely indirect. Consistent with this, we show that VD regulated the expression of other transcription factors and their downstream targets. Finally, we demonstrate that compartment-specific VD-mediated gene expression was associated with compartment-specific VDR-binding sites (<30% of targets) and enrichment of intestinal transcription factor-binding motifs within VDR-binding peaks. Taken together, our data reveal unique spatial patterns of VD action in the intestine and suggest novel mechanisms that could account for compartment-specific functions of this hormone. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
28. OTUB1 protein suppresses mTOR complex 1 (mTORC1) activity by deubiquitinating the mTORC1 inhibitor DEPTOR.
- Author
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Linlin Zhao, Xinbo Wang, Yue Yu, Lu Deng, Lei Chen, Xiaoping Peng, Chenchen Jiao, Guoli Gao, Xiao Tan, Weijuan Pan, Xin Ge, and Ping Wang
- Subjects
- *
MTOR inhibitors , *UBIQUITINATION , *ALDEHYDES , *CELL growth , *CELL proliferation - Abstract
Mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) integrates various environmental signals to regulate cell growth and metabolism. DEPTOR, also termed DEPDC6, is an endogenous inhibitor of mTORC1 and mTORC2 activities. The abundance of DEPTOR centrally orchestrates the mTOR signaling network. However, the mechanisms by which DEPTOR stability is regulated are still elusive. Here, we report that OTU domain-containing ubiquitin aldehyde-binding protein 1 (OTUB1) specifically deubiquitinates DEPTOR in a deubiquitination assay. We found that OTUB1 directly interacted with DEPTOR via its N-terminal domain, deubiquitinated DEPTOR,andthereby stabilizedDEPTORin aCys-91-independent but Asp-88-dependent manner, suggesting that OTUB1 targets DEPTOR for deubiquitination via a deubiquitinase activity- independent non-canonical mechanism. The interaction between OTUB1 and DEPTOR was enhanced when the cells were treated with amino acids. Moreover, OTUB1 suppressed amino acid-induced activation of mTORC1 in a DEPTOR-dependent manner and thereby ultimately controlled cellular autophagy, cell proliferation and size. Our findings reveal a mechanism that stabilizes the mTORC1 inhibitor DEPTOR via OTUB1's deubiquitinase activity. Our insights may inform research into various mTOR activity-related diseases, such as cancer and may contribute to the identification of new diagnostic markers and therapeutic strategies for cancer treatments. [ABSTRACT FROM AUTHOR]
- Published
- 2018
- Full Text
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29. MicroRNA-200 (miR-200) Cluster Regulation by Achaete Scute-like 2 (Ascl2).
- Author
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Yin Tian, Qiong Pan, Yangyang Shang, Rong Zhu, Jun Ye, Yun Liu, Xiaoli Zhong, Shanshan Li, Yonghong He, Lei Chen, Peng Jingjing Zhao, Wensheng Chen, Zhihong, and Rongquan Wang
- Subjects
- *
MICRORNA , *GENETICS of colon cancer , *STEM cell research , *MESENCHYMAL stem cells , *EPITHELIAL cells , *CANCER cells - Abstract
Ascl2, a basic helix-loop-helix transcription factor, is a downstream target of WNT signaling that controls the fate of intestinal cryptic stem cells and colon cancer progenitor cells. However, its involvement in colon cancer and downstream molecular events is largely undefined; in particular, the mechanism by which Ascl2 regulates the plasticity of epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) programs in colon cancer cells remains unknown. In this study, we systematically demonstrate that Ascl2 loss of function in colon cancer cells promotes MET by derepressing the expression of microRNA (miR)-200s (i.e. miR-200b, miR- 200a, miR-429, miR-200c, and miR-141) and further activating their expression through a transcriptional mechanism that involves direct binding to the most proximal E-box (E-box2) in the miR-200b-a-429 promoter. Activation of miR-200s due to Ascl2 deficiency led to the inhibition of ZEB1/2 expression and the alteration of epithelial and mesenchymal features. Transfection of miR-200b, miR-200a, and miR-429 inhibitors into Ascl2- deficient colon cancer cells promoted the epithelial-mesenchymal transition in a reversible manner. Transfection of miR-200a or miR-429 inhibitors into Ascl2-deficient colon cancer cells increased cellular proliferation and migration. Ascl2mRNAlevels and the miR-200a, miR-200b, miR-200c, miR-141, or miR- 429 levels in the colon cancerous samples were inversely correlated. These results provide the first evidence of a link between Ascl2 and miR-200s in the regulation of EMT-MET plasticity in colon cancer. [ABSTRACT FROM AUTHOR]
- Published
- 2014
- Full Text
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30. Enhanced Membrane-tethered Mucin 3 (MUC3) Expression by a Tetrameric Branched Peptide with a Conserved TFLK Motif Inhibits Bacteria Adherence.
- Author
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Qiong Pan, Yin Tian, Xiaohuan Li, Jun Ye, Yun Liu, Lili Song, Yongtao Yang, Rong Zhu, Yonghong He, Lei Chen, Wensheng Chen, Xuhu Mao, Zhihong Peng, and Rongquan Wang
- Subjects
- *
PEPTIDES , *MUCINS , *GENE expression , *BACTERIA , *AMYLOID - Abstract
We investigated whether a synthetic tetrameric branched peptide based on the conserved TFLK motif from mammaryassociated serum amyloid A3 (M-SAA3) is more efficient than the monomeric peptide at up-regulating MUC3 expression and examined the possible mechanism(s) and biological significance of this process. We used standard solid-phase methods to synthesize a tetrameric branched peptide (sequence GWLTFLKAAG) containing a trilysine core, termed the TFLK-containing 10-mer BP. The aberrant expression of transcription factors was analyzed using a transcription factor protein/DNA array.MUC3 and relevant transcription factors were detected using real-time PCR and/or Western blots. The luciferase assay, EMSA, and ChIP assays were used to analyze the activity of the human MUC3 promoter. The bacterial adherence assay was used to evaluate the in vitro inhibition of enteropathogenic Escherichia coli or enterohemorrhage E. coli serotype O157:H7 (EHEC O157:H7) adherence to HT-29-Gal cells after treatment with the TFLK-containing 10-mer BP. In HT-29-Gal cells, the TFLKcontaining 10-mer BP induced higher levels of MUC3 expression than the M-SAA3-derived N-terminal 10-mer monomeric peptide, andMUC3expression was activated through transcriptional mechanisms, including the induction of multiple transcription factors and further binding with their cis-elements between nucleotides -242 and -62 within MUC3 promoter. Interestingly, the TFLK-containing 10-mer BP dramatically inhibited enteropathogenic E. coli and EHEC O157:H7 adherence to the HT-29-Gal cells compared with the controls. This finding suggests a potential therapeutic use for this peptide to prevent gastrointestinal infection. [ABSTRACT FROM AUTHOR]
- Published
- 2013
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31. Molecular Mechanism of the Negative Regulation of Smad1/5 Protein by Carboxyl Terminus of Hsc70-interacting Protein (CHIP).
- Author
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Le Wang, Yi-Tong Liu, Rui Hao, Lei Chen, Zhijie Chang, Hong-Rui Wang, Zhi-Xin Wang, and Jia-Wei Wu
- Subjects
- *
PROTEINS , *BIOMOLECULES , *CYTOKINES , *MOLECULAR chaperones , *PEPTIDE hormones , *CHEMICAL reactions , *UBIQUITIN - Abstract
The transforming growth factor-β (TGF-β) superfamily of ligands signals along two intracellular pathways, Smad2/3-mediated TGF-β/activin pathway and Smad1/5/8-mediated bone morphogenetic protein pathway. The C terminus of Hsc70-interacting protein (CHIP) serves as an E3 ubiquitin ligase to mediate the degradation of Smad proteins and many other signaling proteins. However, the molecular mechanism for CHIP-mediated down-regulation of TGF-β signaling remains unclear. Here we show that the extreme C-terminal sequence of Smad1 plays an indispensable role in its direct association with the tetratricopeptide repeat (TPR) domain of CHIP. Interestingly, Smad1 undergoes CHIP-mediated polyubiquitination in the absence of molecular chaperones, and phosphorylation of the C-terminal SXS motif of Smad1 enhances the interaction and ubiquitination. We also found that CHIP preferentially binds to Smad1/5 and specifically disrupts the core signaling complex of Smad1/5 and Smad4. We determined the crystal structures of CHIP-TPR in complex with the phosphorylated/pseudophosphorylated Smad1 peptides and with an Hsp70/Hsc70 C-terminal peptide. Structural analyses and subsequent biochemical studies revealed that the distinct CHIP binding affinities of Smad1/5 or Smad2/3 result from the nonconservative hydrophobic residues at R-Smad C termini. Unexpectedly, the C-terminal peptides from Smad1 and Hsp70/Hsc70 bind in the same groove of CHIP-TPR, and heat shock proteins compete with Smad1/5 for CHIP interaction and concomitantly suppress, rather than facilitate, CHIP-mediated Smad ubiquitination. Thus, we conclude that CHIP inhibits the signaling activities of Smad1/5 by recruiting Smad1/5 from the functional R-/Co-Smad complex and further promoting the ubiquitination/degradation of Smad1/5 in a chaperone-independent manner. [ABSTRACT FROM AUTHOR]
- Published
- 2011
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32. Molecular Basis of Wnt Activation via the DIX Domain Protein Ccd1.
- Author
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Yi-Tong Liu, Qiong-Jie Dan, Jiawei Wang, Yingang Feng, Lei Chen, Juan Liang, Qinxi Li, Sheng-Cai Lin, Zhi-Xin Wang, and Jia-Wei Wu
- Subjects
- *
EMBRYOLOGY , *PROTEIN research , *POLYMERS , *OLIGOMERS , *MUTAGENESIS - Abstract
The Wnt signaling plays pivotal roles in embryogenesis and cancer, and the three DIX domain-containing proteins, Dvl, Axin, and Ccd1, play distinct roles in the initiation and regulation of canonical Wnt signaling. Overexpressed Dvl has a tendency to form large polymers in a cytoplasmic punctate pattern, whereas the biologically active Dvl in fact forms low molecular weight oligomers. The molecular basis for how the polymeric sizes of Dvl proteins are controlled upon Wnt signaling remains unclear. Here we show that Ccd1 up-regulates canonical Wnt signaling via acting synergistically with Dvl. We determined the crystal structures of wild type Ccd1-DIX and mutant Dvl1-DIX(Y17D), which pack into "head-to-tail" helical filaments. Structural analyses reveal two sites crucial for intra-filament homo- and hetero-interaction and a third site for inter-filament homo-assembly. Systematic mutagenesis studies identified critical residues from all three sites required for Dvl homo-oligomerization, puncta formation, and stimulation of Wnt signaling. Remarkably, Ccd1 forms a hetero-complex with Dvl through the "head" of Dvl-DIX and the "tail" of Ccd1-DIX, depolymerizes Dvl homo-assembly, and thereby controls the size of Dvl polymer. These data together suggest a molecular mechanism for Ccd1-mediated Wnt activation in that Ccd1 converts latent polymeric Dvl to a biologically active oligomer(s). [ABSTRACT FROM AUTHOR]
- Published
- 2011
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33. Pyk2 Inhibition of p53 as an Adaptive and Intrinsic Mechanism Facilitating Cell Proliferation and Survival.
- Author
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Ssang-Taek Lim, Miller, Nichol L. G., Nam, Ju-Ock, Xiao Lei Chen, Yangmi Lim, and Schlaepfer, David D.
- Subjects
- *
PROTEIN-tyrosine kinases , *FOCAL adhesion kinase , *CELL proliferation , *CELL cycle , *FIBROBLASTS , *CISPLATIN - Abstract
Pyk2 is a cytoplasmic tyrosine kinase related to focal adhesion kinase (FAK). Compensatory Pyk2 expression occurs upon FAK loss in mice. However, the impact of Pyk2 up-regulation remains unclear. Previous studies showed that nuclear-localized FAK promotes cell proliferation and survival through FAK FERM domain-enhanced p53 tumor suppressor degradation (Lim, S. T., Chen, X. L., Lim, Y., Hanson, D. A., Vo, 1. T., Howerton, K., Larocque, N., Fisher, S. J., Schlaepfer, D. D., and Ilic, D. (2008) Mol. Ce!! 29, 9-22). Here, we show that FAK knockdown triggered p53 activation and G1 cell cycle arrest in human umbilical vein endothelial cells after 4 days. However, by 7 days elevated Pyk2 expression occurred with a reduction in p53 levels and the release of the G1 block under conditions of continued FAK knockdown. To determine whether Pyk2 regulates p53, experiments were performed in FAK-/-p21-/- mouse embryo fibroblasts expressing endogenous Pyk2 and in 1D8 ovarian carcinoma cells expressing both Pyk2 and FAK. In both cell lines, Pyk2 knockdown increased p53 levels and inhibited cell proliferation associated with G1 cell cycle arrest. Pyk2 FERM domain re-expression was sufficient to reduce p53 levels and promote increased BrdUrd incorporation. Pyk2 FERM promoted Mdm2dependent p53 ubiquitination. Pyk2 FERM effects on p53 were blocked by proteasomal inhibition or mutational-inactivation of Pyk2 FERM nuclear localization. Staurosporine stress of 1D8 cells promoted endogenous Pyk2 nuclear accumulation and enhanced Pyk2 binding to p53. Pyk2 knockdown potentiated 1D8 cell death upon staurosporine addition. Moreover, Pyk2 FERM expression in human fibroblasts upon FAK knockdown prevented cisplatin-mediated apoptosis. Our studies demonstrate that nuclear Pyk2 functions to limit p53 levels, thus facilitating cell growth and survival in a kinase-independent manner. [ABSTRACT FROM AUTHOR]
- Published
- 2010
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34. Protein-tyrosine Phosphatase PCP-2 Inhibits β-Catenin Signaling and Increases E-cadherin-dependent Cell Adhesion.
- Author
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He-Xin Yan, Wen Yang, Rui Zhang, Lei Chen, Liang Tang, Bo Zhai, Shu-Qin Liu, Hui-Fang Cao, Xiao-Bo Man, Hong-Ping Wu, Meng-Chao Wu, and Hong-Yang Wang
- Subjects
- *
PHOSPHATASES , *PROTEINS , *TYROSINE , *CADHERINS , *CELL adhesion , *PHOSPHORYLATION - Abstract
β-Catenin is a key molecule involved in both cell adhesion and Wnt signaling pathway. However, the exact relationship between these two roles has not been clearly elucidated. Tyrosine phosphorylation of β-catenin was shown to decrease its binding to E-cadherin, leading to decreased cell adhesion and increased βcatenin signaling. We have previously shown that receptor-like protein-tyrosine phosphatase PCP-2 localizes to the adherens junctions and directly binds and dephosphorylates β-catenin, suggesting that PCP-2 might regulate the balance between signaling and adhesive β-catenin. Here we demonstrate that PCP-2 can inhibit both the wild-type and constitutively active forms of β-catenin in activating target genes such as c-myc. The phosphatase activity of PCP-2 is required for this effect since loss of catalytic activity attenuates its inhibitory effect on β-catenin activation. Expression of PCP-2 in SW480 colon cancer cells can lead to stabilization of cytosolic pools of β-catenin perhaps, by virtue of their physical interaction. PCP-2 expression also leads to increased membrane-bound E-cadherin and greater stabilization of adherens junctions by dephosphorylation of β-catenin, which could further sequester cytosolic β-catenin and thus inhibit β-catenin mediated nuclear signaling. Furthermore, SW480 cells stably expressing PCP-2 have a reduced ability to proliferate and migrate. Thus, PCP-2 may play an important role in the maintenance of epithelial integrity, and a loss of its regulatory function may be an alternative mechanism for activating β-catenin signaling. [ABSTRACT FROM AUTHOR]
- Published
- 2006
- Full Text
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