• Journal of Microbiology and Biotechnology
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• v.21 no.5
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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.

• Molecules and Cells
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• v.42 no.9
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• pp.646-660
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• 2019

Abscisic acid (ABA) is a phytohormone essential for seed development and seedling growth under unfavorable environmental conditions. The signaling pathway leading to ABA response has been established, but relatively little is known about the functional regulation of the constituent signaling components. Here, we present several lines of evidence that Arabidopsis Raf-like kinase Raf10 modulates the core ABA signaling downstream of signal perception step. In particular, Raf10 phosphorylates subclass III SnRK2s (SnRK2.2, SnRK2.3, and SnRK2.6), which are key positive regulators, and our study focused on SnRK2.3 indicates that Raf10 enhances its kinase activity and may facilitate its release from negative regulators. Raf10 also phosphorylates transcription factors (ABI5, ABF2, and ABI3) critical for ABA-regulted gene expression. Furthermore, Raf10 was found to be essential for the in vivo functions of SnRK2s and ABI5. Collectively, our data demonstrate that Raf10 is a novel regulatory component of core ABA signaling.

• BMB Reports
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• v.43 no.3
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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.

• The Korean Journal of Food And Nutrition
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• v.28 no.1
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• pp.111-118
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• 2015

In summary, the rutin metabolite DHT significantly reduced EGF-induced neoplastic transformation in JB6 P+ cells. This inhibition was mediated mainly by blocking the Raf/MEK/ERK signaling pathway and subsequent suppression of AP-1 activities. DHT attenuated Raf-1 kinase activity by directly binding to Raf-1 in vitro and ex vivo. Taken together, these results suggest that Raf-1 may be a critical molecular target to suppress DHT-induced neoplastic transformation, which is mainly attributable to the chemopreventive potential of several foods containing rutin.

• Journal of Life Science
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• v.19 no.7
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• pp.866-870
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• 2009

Fascin, a cytoskeleton actin binding protein, functions in cell adhesion and cell migration. Fascin is also known as a candidate biomarker for various cancers, however, regulatory mechanisms of fascin expression remains little understood. In this study, we found an abnormal bristle phenotype, which is similar to that of the Drosophila fascin mutant, in Drafmutant flies. Hence, we investigated whether fascin expression is regulated by Raf signaling. RT-PCR and Western blot analysis showed that Drosophila fascin expression was down-regulated in Draf mutant flies and the level was increased in larvae expressing the oncogenic form of Draf (Draf$^{got}$) under the GAL4-UAS system. Immunostaining analysis showed increased fascin in the hemocytes over-expressing Draf$^{got}$. Our results indicate that fascin expression is regulated by Raf signaling and suggest that Raf signaling may play an important role in the actin cytoskeleton-associated developmental process and tumor progression via regulation of fascin gene.

• Biomolecules & Therapeutics
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• v.21 no.2
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• pp.114-120
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• 2013

Most patients with mutant B-Raf melanomas respond to inhibitors of oncogenic B-Raf but resistance eventually emerges. To better understand the mechanisms that determine the long-term responses of mutant B-Raf melanoma cells to B-Raf inhibitor, we used chronic selection to establish B-Raf (V600E) melanoma clones with acquired resistance to the new oncogenic B-Raf inhibitor UI-152. Whereas the parental A375P cells were highly sensitive to UI-152 ($IC_{50}$ < $0.5{\mu}M$), the resistant sub-line (A375P/Mdr) displayed strong resistance to UI-152 ($IC_{50}$ < $20{\mu}M$). Immunofluorescence analysis indicated the absence of an increase in the levels of P-glycoprotein multidrug resistance (MDR) transporter in A375P/Mdr cells, suggesting that resistance was not attributable to P-glycoprotein overexpression. In UI-152-sensitive A375P cells, the anti-proliferative activity of UI-152 appeared to be due to cell-cycle arrest at $G_0/G_1$ with the induction of apoptosis. However, we found that A375P/Mdr cells were resistant to the apoptosis induced by UI-152. Interestingly, UI-152 preferentially induced autophagy in A375P/Mdr cells but not in A375P cells, as determined by GFP-LC3 puncta/cell counts. Further, autophagy inhibition with 3-methyladenine (3-MA) partially augmented growth inhibition of A375P/Mdr cells by UI-152, which implies that a high level of autophagy may protect UI-152-treated cells from undergoing growth inhibition. Together, our data implicate high rates of autophagy as a key mechanism of acquired resistance to the oncogenic B-Raf inhibitor, in support of clinical studies in which combination therapy with autophagy targeted drugs is being designed to overcome resistance.

• Biomolecules & Therapeutics
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• v.19 no.2
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• pp.174-180
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• 2011

Experiments were carried out to determine the role of Raf-1 kinase in the development of drug resistance to paclitaxel in v-H-ras transformed NIH 3T3 fibroblasts (Ras-NIH 3T3). We established a multidrug-resistant cell line (Ras-NIH 3T3/Mdr) from Ras-NIH 3T3 cells by stepwise increases in paclitaxel. Drug sensitivity assays indicated that the $IC_{50}$ value for drug-resistant Ras-NIH 3T3/Mdr cells was more than 1 ${\mu}M$ paclitaxel, 10- or more-fold higher than for the parental Ras-NIH 3T3 cells. Western blot and RT-PCR analysis showed that the drug efflux pump a P-glycoprotein were highly expressed in Ras-NIH 3T3/Mdr cells, while not being detectable in Ras-NIH 3T3 cells. Additionally, verapamil, which appears to inhibit drug efflux by acting as a substrate for P-glycoprotein, completely reversed resistance to paclitaxel in Ras-NIH 3T3/Mdr cell line, indicating that resistance to paclitaxel is associated with overexpression of the multidrug resistance gene. Interestingly, Ras-NIH 3T3/Mdr cells have higher basal Raf-1 activity compared to Ras-NIH 3T3 cells. Unexpectedly, however, the colocalization of Raf-1 and its negative regulator Spry2 was less observed in cytoplasm of Ras-NIH 3T3/Mdr cells due to translocation of Spry2 around the nucleus in the perinuclear zone, implying that Raf-1 may be released from negative feedback inhibition by interacting with Spry2. We also showed that shRNA-mediated knockdown of Raf-1 caused a moderate increase in cell susceptibility to paclitaxel. Thus, the results presented here suggest that a Raf-1-dependent pathway plays an important role in the development of acquired drug-resistance.

• Korean Journal of Food Science and Technology
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• v.52 no.4
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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.

• Korean Journal of Plant Taxonomy
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• v.44 no.3
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• pp.181-187
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• 2014

The taxonomic status of a new forma, Opuntia humifusa (Raf.) Raf. f. jeollaensis E. J. Kim & S. S. Whang, and the taxonomic relationships of three Korean cladodes were studied based on DNA sequences of three genes. The new forma O. humifusa f. jeollaensis (Wanggasi-Chunnyuncho) is simlilar to O. humifusa (Chunnyuncho), but differ in having both flower with the reddish inner center, and strong and many 3 cm long spines. Molecular studies showed that the new forma grouped into Macrocentra series instead of Humifusa series which included Korean O. humifusa. We designated the new taxa firstly studied here as a new forma, because the taxa have been mainly cultivated in farmlands in Jeolla-do area rather than distributed in nature. The taxonomic relationships of three Korean cladodes are discussed in detail.

• Journal of Life Science
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• v.26 no.1
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• pp.101-108
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• 2016

The aim of this study was to evaluate the relationships between the expression of Heat shock protein70 (HSP70), Raf-1 and Bcl-2-associated athanogene-1 (BAG1) protein in diffuse type gastric carcinoma and examine association of HSP70, Raf-1 and BAG1 expression with various clinic-pathological factors and survival. Heat shock protein70 is induced in the cells in response to various stress conditions, including carcinogens. Overexpression of heat shock protein 70 has been observed in many types of cancer. The proto-oncoprotein Raf is pivotal for mitogen-activated protein kinase (MAPK) signaling, and its aberrant activation has been implicated in multiple human cancers. Overexpression of BAG1 protein has been documented in some type of human cancer. BAG1 has been reported to interact with protein involved with a variety of signal pathway, and regulation of cell differentiation, survival and apoptosis. These interaction partners include HSP70 and Raf-1. The percentage of tumors exhibiting HSP70 positivity was significantly in cases of positive lymph node metastasis (64.9%) compared to cases without lymph node metastasis (35.1%, p=0.007). HS70 expression was correlated with pathological N-stage (p=0.006). Expression of BAG1 was detected in the majority of diffuse type gastric carcinoma tissues (71.7%), especially in younger patients (80% vs 52.6%, p=0.035). Furthermore BAG1 expression was correlated with tumor size (p=0.020). Raf-1 expression was found to be significantly associated with tumor size (p=0.005). The result indicate that HSP70 was significantly correlated the progression of diffuse type gastric cancer. Expression of BAG1 and Raf-1 may be used as diagnostic markers for gastric carcinoma.