• BMB Reports
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• 제43권3호
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• pp.205-211
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• 2010

Sprouty (Spry) proteins have previously been suggested as negative regulators of the MAPK pathway through interaction with Raf-1. However, the molecular basis of this inhibition has not been elucidated. In this study, we used cells expressing FLAGtagged Raf-1 with point mutations at known phosphorylation sites to reveal that activation of Raf-1 mutants does not correlate with their degree of interaction with Spry2. The association of Raf-1 with Spry2 in intact cells was further corroborated by immunofluorescence colocalization. Additionally, there was no significant change observed in the strength of interaction between Raf-1 mutants and Spry2 after paclitaxel treatment despite differences in the activation levels of these mutants. Thus, our study provides the evidence that Spry2 does not directly regulate Raf-1 kinase activity, but instead acts as a scaffolding protein that assists interactions between Raf-1 kinase and its direct regulators.

• 생명과학회지
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• 제8권1호
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• pp.14-26
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• 1998

본 연구에서는 SDS/polyacrylamide 젤 전기영동에 의한 쥐의 성장과정에 따른 소뇌의 단백질양상의 변화양상과 immunocytochemistry를 이용하여 c-raf a-raf kinase의 정상 소뇌에서의 분포에 대해 관찰 하였으며 western blot을 이용하여 소뇌의 단백질들에서 c-raf의 존재에 대해 살펴보았다. 단백질 양상에서 쥐의 성장에 따라 crude에선,ㄴ 49,200 dalton과 169,000 dalton 사이의 bands가 양적 증가를 보였으며 cytosolic fraction 에서는 37,800 dalton의 band가 양적 증가를 보이는데 비해 membrane fraction 에서는 260,600 dalton의 band가 증가하였다. 이러한 결과로 성장 발달에 따라 고분자 량의 물질들이 이들 소뇌 부위에서 기여하였을 것으로 추정할 수 있었다. Immunocytochemistry에 의한 분석에서는 c-raf와 a-raf가 소뇌의 피질주위에서 조롱박 세포(Purkinje cell) 의 세포질 특히 핵 주변부위에서 강하게 검출되었으며 a-raf에 비해 c-raf가 더 강하게 나타났었다. 그리고 그 외에 Nucleus embolifornis의 큰 neuronal cell의 세포질 부위의 나타남을 볼 수 있었다. Immunoblot에 의한 분석에서는 crude와 cytosolic fraction에서 raf protein kinase의 존재를 확인할 수 있었으며, 이상의 결과들을 종합해 보았을 때 소뇌의 정상의 많은 신경세포(neuronal cell)에 raf protein kinase가 분포되어 있으며 이들이 정상의 cell에서 기능을 가질 것으로 추정된다.

• Journal of Microbiology and Biotechnology
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• 제21권5호
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• pp.525-528
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• 2011

The Raf-1 kinase inhibitory protein (RKIP) can regulate multiple key signaling pathways. Specifically, RKIP binds to Raf-1 kinase and inhibits the Ras-Raf-1-MEK1/2- ERK1/2 pathway. Additionally, Raf-1 has been shown to translocate to mitochondria and thereby protect cells from stress-mediated apoptosis. Recently, HBx was found to stimulate the mitochondrial translocation of Raf-1, contributing to the anti-apoptotic effect. We found that RKIP was downregulated during HBx-mediated hepatocarcinogenesis. In this study, we show that RKIP bound to Raf-1 and consequently inhibited the translocation of Raf-1 into mitochondria. This promoted the apoptosis of cells treated with apoptotic stimulus. Thus, the downregulation of RKIP increased the level of free Raf-1 and thereby elevated the mitochondrial translocation of Raf-1 during HBx-mediated hepatocarcinogenesis. The elevated Raf-1 mitochondrial translocation induced the increased anti-apoptotic effect and subsequently promoted HBx-mediated hepatocarcinogenesis.

• 한국독성학회:학술대회논문집
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• 한국독성학회 2002년도 Molecular and Cellular Response to Toxic Substances
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• pp.136-136
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• 2002

Experiments were carried out to determine the role of Raf-1 kinase in the development of drug resistance and apoptosis induced by paclitaxel. In the present study, paclitaxel sensitivity, Raf-1 activity and MAPKs activation were compared in 2 cell lines: parental human breast cancer cells and its drug resistant variant (MCF-7/Adr) cells.(omitted)

• 한국식품과학회지
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• 제52권4호
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• pp.385-395
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• 2020

This study examined the effect of 3,4-dihydroxytoluene (DHT) on UVB-induced photoaging and determined its molecular mechanisms, using HaCaT human keratinocytes and SKH-1 hairless mice. DHT suppressed UVB-induced matrix metalloproteinase-1 (MMP-1) expression in HaCaT cells. In vivo data from mouse skin supported that DHT decreased UVB-induced wrinkle formation, epidermal thickness, and matrix metalloproteinase-13 (MMP-13) expression. DHT appeared to exert its anti-aging effects by suppressing UVB-induced Raf-1 kinase activity and subsequent attenuation of UVB-induced phosphorylation of MEK, ERK, and p90RSK in HaCaT cells. In vitro and in vivo pull-down assays revealed that DHT bound with Raf-1 in ATP-noncompetitive manner. Overall, DHT appears to anti-photoaging effects in vitro and in vivo through the suppression of Raf-1 kinase activity and may have potential as a treatment for the prevention of skin aging.

• Animal cells and systems
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• 제1권4호
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• pp.637-640
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• 1997

Raf-1, a cytoplasmic serine/threonine protein kinase, serves as a central intermediate in many signaling pathways in cell proliferation, differentiation, and development. In this study, we investigated that B-raf, Drosophila homolog of the human c-raf-1, is involved in ultraviolet (UV) responsive events by using hypomorphic mutant $D-raf^{c110}$ and Draf-lacZ transgenic fly. At first, effect of UV damage on the survival of wild-type and $D-raf^{C110}$ strains was examined. In terms of $1/LD_{50}$ value, the relative ratio of UV sensitivities of wild-type versus $D-raf^{C110}$ strain was 1 : 2.2. By using quantitative $\beta$-galactosidase activity analysis, transcriptional activity of the D-raf gene promoter was also examined in UV-irradiated Draf-lacZ transgenic larvae. UV irradiation increased the expression of lacZ reporter gene in Draf-lacZ transgenic fly. However, in $D-raf^{C110}$ strain the transcriptional activity of D-raf gene promoter by UV irradiation was extensively reduced. Results obtained in this study suggest that D-raf plays a role in UV response, leading to better survival of Drosophila to UV damage.

• 한국독성학회:학술대회논문집
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• 한국독성학회 2005년도 춘계 국제심포지엄 및 학술대회
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• pp.121-121
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• 2005

• 한국환경성돌연변이발암원학회:학술대회논문집
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• 한국환경성돌연변이발암원학회 2002년도 Molecular and Cellular Response to Toxic Substances
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• pp.136-136
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• 2002

• 한국식품영양학회지
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• 제28권1호
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• pp.111-118
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• 2015

이전 연구들에서 rutin과 quercetin을 포함한 여러 flavonoids의 암예방 활성이 보고되었으나, rutin의 경우 섭취 시 체내에서 HVA, HPAA, DHT라는 대사체로 변형되어 흡수된다. 그러나, 이들 대사체와 관련한 암예방 효능 및 그 분자생물학적 작용기작에 대한 연구 결과는 보고된 바가 없어, 본 연구에서 이를 규명하였다. DHT는 EGF로 유도된 세포 변형을 억제하였으며, AP-1 전사인자의 활성 또한 억제하였다. DHT는 Raf-1 효소 활성을 효과적으로 저해하므로서 MEK 및 ERK의 인산화를 억제하였으며, Raf-1과 ATP는 비경쟁적으로 직접 결합하여 Raf-1 효소 활성을 저해한다는 사실을 밝혀내었다. 이와는 대조적으로, rutin은 EGF로 유도된 세포 변형, AP-1 활성, ERK 신호전달체계, Raf-1 효소 활성을 억제하지 못하였다. 이상의 연구결과는 DHT의 암예방 활성이 발암과정과 밀접한 연관이 있는 Raf-1 효소 활성을 억제하여 세포 변형을 억제하는 것과 관련되어 있다는 것을 제시한다.

• 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.