• BMB Reports
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• 제47권12호
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• pp.685-690
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• 2014

The Ras/Raf/MEK/Erk signaling pathway is important for regulation of cell growth, proliferation, differentiation, survival, and apoptosis in response to a variety of extracellular stimuli. Lack of Erk MAPK activation is observed in several cancer cells despite active activation of Ras. However, little is known about the modulation of Erk1/2 activity by active Ras. Here, we show that overexpression of active H-Ras (H-RasG12R) in NIH3T3 fibroblasts impaired FGF2-induced Erk1/2 phosphorylation, as compared to wild-type cells. Northern blot analysis revealed that prolonged expression of active Ras increased MAP kinase phosphatase 3 (MKP3) mRNA expression, a negative regulator of Erk MAPK. Inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway abrogated active Ras-induced up-regulation of MKP3 expression, leading to the rescue of Erk1/2 phosphorylation. Our results demonstrated that the Ras/Raf/MEK/Erk signaling cascade is negatively regulated by the PI3K/Aktdependent transcriptional activation of the MKP3 gene.

• Biomolecules & Therapeutics
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• 제8권2호
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• pp.153-159
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• 2000

A novel serine/threonine protein phosphatase with EF-hand motif, which belongs to PPEF family was partially cloned from rat brain cDNA by employing RT-PCR method. The size of the amplified clone was 1.6kbp. The amplified DNA was subcloned into pGEM-T-Easy vector and the resulting plasmid was maned as pGEM-rPPEF2. The nucleuotide sequence is shared by 88% with that of mouse PPEF-2 cDNA, and the deduced amino acid sequence reveal 92% homology with that of mouse PPEF-2 cDNA. The N-terminal region of the cloned rat brain PPEF contains a putative phosphatase catalytic domain (PP domain) and the C-terminal region contains multiple $Ca^{2+}$ binding sites (EF region). The putative catalytic domin (PP) and the EF-hand motif (EF) regions were subcloned into pGEX4T-1 and were overexpressed in E. coli DH5 as glutathione-S-transferase (GST) fusion proteins. Expression of the desired fusion protein was identified by SDS-PAGE and also by immunoblot analysis using monoclonal antibody against GST. The recombinant proteins were purified by glutathione-agarose chromatography. This report is first to demonstrate the cloning of PPEF family from rat brain tissues. The clone reported here would be invaluable for the investigation of the role of this new type-phosphatase in rat brain.

• BMB Reports
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• 제49권7호
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• pp.370-375
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• 2016

Ras oncoproteins are small molecular weight GTPases known for their involvement in oncogenesis, which operate in a complex signaling network with multiple effectors. Approximately 25% of human tumors possess mutations in a member of this family. The Raf1/MEK/Erk1/2 pathway is one of the most intensively studied signaling mechanisms. Different levels of regulation account for the inactivation of MAP kinases by MAPK phosphatases in a cell type- and stimuli-dependent manner. In the present study, using three inducible Ras-expressing NIH/3T3 cell lines, we demonstrated that MKP3 upregulation requires the activation of the Erk1/2 pathway, which correlates with the shutdown of this pathway. We also demonstrated, by applying pharmacological inhibitors and effector mutants of Ras, that induction of MKP3 at the protein level is positively regulated by the oncogenic Ras/Raf/MEK/Erk1/2 signaling pathway.

• Bulletin of the Korean Chemical Society
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• 제33권5호
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• pp.1505-1508
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• 2012

Protein tyrosine phosphatase 1B (PTP1B) is a key factor in negative regulation of the insulin pathway, and is a promising target for the treatment of type-II diabetes, obesity and cancer. Herein, compound ($\mathbf{4}$) was first observed to have moderate inhibitory activity against PTP1B with an $IC_{50}$ value of $13.72{\pm}1.53{\mu}M$. To obtain more potent PTP1B inhibitors, we synthesized a series of chalcone derivatives using compound ($\mathbf{4}$) as the lead compound. Compound $\mathbf{4l}$ ($IC_{50}=3.12{\pm}0.18{\mu}M$) was 4.4-fold more potent than the lead compound $\mathbf{4}$ ($IC_{50}=13.72{\pm}1.53{\mu}M$), and more potent than the positive control, ursolic acid ($IC_{50}=3.40{\pm}0.21{\mu}M$). These results may help to provide suitable drug-like lead compounds for the design of inhibitors of PTP1B as well as other PTPs.

• 대한한의학회지
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• 제24권4호
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• pp.43-53
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• 2003

Drynariae Rhizorna (DR), an herbal medicine known for its effect to purify blood quality and improve circulation, frequently appears as the main ingredient in prescriptions for bone injuries. Currently, how pharmacologically it contributes to the reformation of bone is unclear. In the present study, the effect of the aqueous extract of DR on bone cells was investigated in vitro for the first time. The human osteoprecursor cells (OPC-I) were incubated in the medium with different concentrations of the aqueous extract of DR and the cell proliferation was studied. When the concentration of DR aqueous extract was $<120{\;}\mu\textrm{g}/ml$, the proliferation of OPC-I was enhanced. However, the proliferation of OPC-I was inhibited by DR extract with the concentrations $>250{\;}\mu\textrm{g}/ml$. Under most treatments, the cells presented very pale expression for cyclooxygenase-2 (Cox 2) protein; a slightly intensified band showed at the highest DR concentration, 1.0 mg/ml during the course of culture. From the results, it was concluded that the aqueous extract of DR was found to directly stimulate the proliferation, alkaline phosphatase (ALP) activity, protein secretion and particularly type I collagen synthesis of OPC-I at dose-dependent manner.

• 한국미생물·생명공학회지
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• 제33권3호
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• pp.189-193
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• 2005

RBL-2H3 cell lysates에 anti-protein phosphatase(PP) 1, 2A, 2B 항체를 첨가한 후 immunoprecipitation을 실시한 결과 PP2B를 가해준 샘플에서만 HRF를 확인하였다. 역으로 monoclonal anti-HRF 항체를 가한 후 immunoprecipitation을 실시한 결과 PP1, 2A는 검출되지 않았으나 PP2B의 경우는 regulatory subunit(19 kDa), catalyic subunit(60 kDa) 모두 확인할 수 있었다. Affinity chromatography를 통해서도 PP2B가 HRF의 탈인산화에 관여함을 확인하였다 즉 19kDa의 PP2B regulatory subunit과 60kDa의 catalytic subunit 모두가 확인되었으며 외부 $Ca^{2+}$이온 첨가 여부에 따른 차이는 관찰할 수 없었다. 결론적으로 RBL-2H3 cell에서 PP2B는 PP1이나 PP2A에 비해 상대적으로 그 존재량은 적으나 HRF와 상호작용하는 phosphatase로서 검출된 반면 PP1이나 PP2A는 검출되지 않았다.

• International Journal of Industrial Entomology and Biomaterials
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• 제35권1호
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• pp.7-13
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• 2017

In this study, changes in gene expression after administration of silk fibroin fragments ($size{\approx}30kDa$) were evaluated in MG63 cells using a cDNA microarray assay. In addition, the level of alkaline phosphatase (ALP) activity and cellular proliferation in the group administered moderately sized silk fibroin fragments ($size{\approx}30kDa$) (MSF) were compared to those in the group administered smaller silk fibroin fragments (size < 1 kDa) (SSF). The results of the cDNA microarray assay show increased expression of genes that are related to the cell cycle and inflammation. ALP, bone morphogenetic protein-7, bone morphogenetic protein receptor type IA, and runt-related transcription factor 2 exhibited significantly lower expression compared to control cells (fold ratio < 0.5). Relative ALP activity of the $100{\mu}g/mL$ MSF group was significantly lower than that of the SSF group (P < 0.05). Thus, the MSF group showed increased expression of genes associated with cellular proliferation and inflammation but decreased expression of genes associated with osteogenesis.

• 식품보건융합연구
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• 제5권3호
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• pp.21-33
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• 2019

The incidence of both obesity and Type 2 Diabetes Mellitus( DM) is increasing proportionately so that causes of deaths from these has overtaken from that of malnourishment. Hence it has been recommended to treat the 2 in parallel considering the role of diabesity on health. Important causes of T2DM are insulin resistance (IR) and /or inadequate insulin secretion. Protein tyrosine phosphatase 1B(PTPIB) has a negative impact in insulin signaling pathways and hence plays crucial role inT2DM,since its overexpression might induce IR. Thus PTPIB is considered a therapeutic target for both obesity and T2DM, there has been a search for novel ,promising natural inhibitors. We conducted a pubmed search for articles related to PTPIB inhibitors from natural causes be it marine sources or other natural sources. Out of 988 articles we selected 100 articles for review. Thus various bioactive molecules isolated from marine organisms that can acts as PTPIB Inhibitors and thus possess antidiabetic activity both in vitro/ in vivo studies ,besides products from fruits like Chinese raspberry or curcumin used as routine spices are described with their chemical classes, structure-activity relationships and potency as assessed by IC 50 values are discussed. More work is required to make this a reality.

• 대한의생명과학회지
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• 제18권1호
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• pp.1-9
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• 2012

Differentially expressed genes by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were identified in order to evaluate them as dioxin-sensitive markers and crucial signaling molecules to understand dioxin-induced toxic mechanisms in human bronchial cells. Gene expression profiling was analyzed by cDNA microarray and ten genes were selected for further study. They were cytochrome P450, family 1, subfamily B, polypeptide 1 (CYP1B1), S100 calcium binding protein A8 (calgranulin A), S100 calcium binding protein A9 (calgranulin B), aldehyde dehydrogenase 1 family, member A3 (ALDH6) and peroxiredoxin 5 (PRDX5) in up-regulated group. Among them, CYP1B1 was used as a hallmark for dioxin and sharply increased by TCDD exposure. Down-regulated genes were IK cytokine, interferon-induced protein with tetratricopeptide repeats 1 (IFIT1), nuclease sensitive element binding protein 1 (NSEP1), protein tyrosine phosphatase type VI A, member 1 (PTP4A1), ras oncogene family 32 (RAB32). Although up-regulated 4 genes in microarray were coincided with northern hybridization, down-regulated 5 genes showed U-shaped expression pattern which is sharply decreased at lower doses and gradually increased at higher doses. These results introduce some of TCDD-responsive genes can be sensitive markers against TCDD exposure and used as signaling cues to understand toxicity initiated by TCDD inhalation in pulmonary tissues.

• BMB Reports
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• 제47권10호
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• pp.552-557
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• 2014

The main purpose of this study was to investigate whether type 3 muscarinic acetylcholine receptor (M3R) dysfunction induced vascular hyperpermeability. Transwell system analysis showed that M3R inhibition by selective antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) and small interfering RNA both increased endothelial permeability. Using coimmunoprecipitation and Western blot assay, we found that M3R inhibition increased VE-cadherin and ${\beta}$-catenin tyrosine phosphorylation without affecting their expression. Using PTP1B siRNA, we found that PTP1B was required for maintaining VE-cadherin and ${\beta}$-catenin protein dephosphorylation. In addition, 4-DAMP suppressed PTP1B activity by reducing cyclic adenosine monophosphate (cAMP), but not protein kinase $C{\alpha}$ ($PKC{\alpha}$). These data indicate that M3R preserves the endothelial barrier function through a mechanism potentially maintaining PTP1B activity, keeping the adherens junction proteins (AJPs) dephosphorylation.