• Asian Pacific Journal of Cancer Prevention
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• v.14 no.7
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• pp.4279-4282
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• 2013

Background: The activation and inactivation of receptor tyrosine kinases are tightly regulated to ensure faithful replication of cells. After having transduced extracellular growth activating signals, activated EGFR is subjected to downregulation either by clathrin mediated endocytosis or c-Cbl mediated proteasome degradation depending on the ligand concentration. c-Cbl is an ubiquitin ligase which requires a phosphorylated tyrosine residue at position 1045 in the cytoplasmic domain of EGFR to interact and add ubiquitin molecules. While activating mutations in exons 19 and 21 have been associated with the development of several cancers, the status of mutations at tyrosine 1045 coding exon 27 of EGFR remain to be investigated. Consistently, defective phosphorylation at 1045 has been associated with sustained phosphorylation of EGFR in non-small lung carcinomas. Hence in the present study we investigated the genetic status of the tyrosine 1045 coding site within exon 27 of EGFR gene to explore for possible occurrence of mutations in this region, especially since no studies have addressed this issue so far. Materials and Methods: Tumor chromosomal DNA isolated from thirty five surgically excised oral squamous cell carcinoma tissues was subjected to PCR amplification with intronic primers flanking the tyrosine 1045 coding exon 27 of EGFR gene. The PCR amplicons were subsequently subjected to direct sequencing to elucidate the mutation status. Results: Sequence analysis identified no mutations in the tyrosine 1045 codon of EGFR in any of the thirty five samples that were analyzed. Conclusions: The lack of identification of mutation in the tyrosine 1045 codon of EGFR suggests that mutations in this region may be relatively rare in oral squamous cell carcinomas. To the best of our knowledge, this study is the first to have explored the genetic status of exon 27 of EGFR in oral squamous cell carcinoma tissue samples.

• Molecules and Cells
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• v.41 no.8
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• pp.799-807
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• 2018

Emerging evidence has suggested that cellular crosstalk between RNF168 and poly(ADP-ribose) polymerase 1 (PARP1) contributes to the precise control of the DNA damage response (DDR). However, the direct and reciprocal functional link between them remains unclear. In this report, we identified that RNF168 ubiquitinates PARP1 via direct interaction and accelerates PARP1 degradation in the presence of poly (ADP-ribose) (PAR) chains, metabolites of activated PARP1. Through mass spectrometric analysis, we revealed that RNF168 ubiquitinated multiple lysine residues on PARP1 via K48-linked ubiquitin chain formation. Consistent with this, micro-irradiation-induced PARP1 accumulation at damaged chromatin was significantly increased by knockdown of endogenous RNF168. In addition, it was confirmed that abnormal changes of HR and HNEJ due to knockdown of RNF168 were restored by overexpression of WT RNF168 but not by reintroduction of mutants lacking E3 ligase activity or PAR binding ability. The comet assay also revealed that both PAR-binding and ubiquitin-conjugation activities are indispensable for the RNF168-mediated DNA repair process. Taken together, our results suggest that RNF168 acts as a counterpart of PARP1 in DDR and regulates the HR/NHEJ repair processes through the ubiquitination of PARP1.

• Journal of Life Science
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• v.28 no.4
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• pp.435-443
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• 2018

AMP-activated protein kinase (AMPK) functions as a metabolic master through regulating and restoring cellular energy balance. In skeletal muscle, AMPK increases myofibril protein degradation through the expression of muscle-specific ubiquitin ligases. Mori Folium, the leaf of Morus alba, is a traditional medicinal herb with various pharmacological functions; however, the effects associated with muscle atrophy have not been fully identified. In this study, we confirmed the effects of AMPK activation by examining the effects of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), an activator of AMPK, on the induction of atrophy and expression of atrophy-related genes in C2C12 myotubes. We also investigated the effects of the ethanol extract of Mori Folium (EEMF) on the recovery of AICAR-induced muscle atrophy in C2C12 myotubes. It was found that exposure to AICAR resulted in the stimulation of Forkhead box O3a (FOXO3a); an up-regulation of muscle-specific ubiquitin ligases such as Muscle Atrophy F-box (MAFbx)/atrogin-1 and muscle RING finger-1 (MuRF1), and a down-regulation of muscle-specific transcription factors, such as MyoD and myogenin; with the activation of AMPK. In addition, AICAR without cytotoxicity indicated a decrease in diameter of C2C12 myotubes. However, treatment with EEMF significantly suppressed AICAR-induced muscle atrophy of C2C12 myotubes in a dose-dependent manner as confirmed by a decrease in myotube diameter, which is associated with a reversed stimulation of FOXO3a by the inhibition of AMPK activation. These results indicate that the activation of AMPK by AICAR induces muscle atrophy, and EEMF has preeminent effects on the inhibition of AICAR-induced muscle atrophy through the AMPK signaling pathway.

• Journal of Life Science
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• v.27 no.12
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• pp.1479-1485
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• 2017

Muscle atrophy due to aging, starvation, and various chronic diseases leads to a decrease in muscle fiber area and density due to reduced muscle protein synthesis and increased protein breakdown. This study investigated the effect of dexamethasone and hydrogen peroxide on the induction of muscle atrophy and expression of atrophy-related genes in differentiated C2C12 myotubes. C2C12 myoblasts were differentiated into myotubes in differentiation medium. During myoblast differentiation, muscle-specific transcription factors, such as myogenin, and MyoD expression increased. Differentiated C2C12 myotubes exposed to noncytotoxic levels of dexamethasone and hydrogen peroxide showed a decrease in myotube diameter, which was associated with up-regulation of muscle-specific ubiquitin ligases, such as muscle atrophy F-box (MAFbx)/atrogin-1 and muscle RING finger-1 (MuRF1), and down-regulation of myogenin and MyoD. These results demonstrated that dexamethasone and hydrogen peroxide induced atrophy through regulation of muscle-specific ubiquitin ligases and muscle-specific transcription factors in C2C12 myotubes. In this study, we confirmed the process of differentiation of C2C12 myoblasts into myotubes in in vitro experiments in the presence of atrophy. This muscle atrophy model of C2C12 cells induced by dexamethasone or hydrogen peroxide seems suited to studies of the mechanism of muscle atrophy suppression and to exploit the experiment for excavating new muscle atrophy.

• BMB Reports
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• v.45 no.9
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• pp.515-520
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• 2012

High expression of osmotically responsive genes1 (HOS1), a key regulator of low temperature response and flowering time, encodes an E3 ubiquitin ligase in Arabidopsis. Here, we report characterization of a newly identified splice variant (HOS1-L) of HOS1. Comparative analyses revealed that HOS1-L has a longer 5' nucleotide sequence than that of the previously identified HOS1 (HOS1-S) and that its protein sequence was more conserved than that of HOS1-S in plants. HOS1-L transcripts were spatio-temporally more abundant than those of HOS1-S. The recovery rate of HOS1-S expression was faster than that of HOS1-L after cold treatment. Diurnal oscillation patterns of HOS1-L revealed that HOS1-L expression was affected by photoperiod. An in vitro pull-down assay revealed that the HOS1-L protein interacted with the ICE1 protein. HOS1-L overexpression caused delayed flowering in wild-type plants. Collectively, these results suggest regulation of HOS1 expression at the post-transcriptional level.

• The Korean Journal of Physiology and Pharmacology
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• v.13 no.6
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• pp.483-489
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• 2009

Despite the potential importance of the human regulator of calcineurin 1 (RCAN-1) gene in the modulation of cell survival under stress, little is known about its role in death-inducing signal pathways. In this study, we addressed the effects of RCAN1.4 knockdown on cellular susceptibility to apoptosis and the activation of death pathway proteins. Transfection of siRNAs against RCAN1.4 resulted in enhanced Fas- and etoposide-induced apoptosis, which was associated with increased expression and translocation of Bax to mitochondria. Our results suggest that enhanced expression and activation of p53 was responsible for the upregulation of Bax and the increased sensitivity to apoptosis, which could be reversed by p53 knockdown. To explain the observed upregulation of p53, we propose a downregulation of the ubiquitin ligase HDM2, probably translationally. These findings show the importance of appropriate RCAN1.4 expression in the modulation of cell survival and reveal a link between RCAN1.4 and p53.

• BMB Reports
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• v.51 no.10
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• pp.484-485
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• 2018

Receptor-interacting protein kinase-3 (RIP3 or RIPK3) is a serine-threonine kinase largely essential for necroptotic cell death; it also plays a role in some inflammatory diseases. High levels of RIP3 are likely sufficient to activate necroptotic and inflammatory pathways downstream of RIP3 in the absence of an upstream stimulus. For example, we have previously detected high levels or RIP3 in the skin of Toxic Epidermal Necrolysis patients; this correlates with increased phosphorylation of MLKL found in these patients. We have long surmised that there are molecular mechanisms to prevent anomalous activity of the RIP3 protein, and so prevent undesirable cell death and inflammatory effects when inappropriately activated. Recent discovery that Carboxyl terminus of Hsp 70-Interacting Protein (CHIP) could mediate ubiquitylation- and lysosome-dependent RIP3 degradation provides a potential protein that has this capacity. However, while screening for RIP3-binding proteins, we discovered that pellino E3 ubiquitin protein ligase 1 (PELI1) also interacts directly with RIP3 protein; further investigation in this study revealed that PELI1 also targets RIP3 for proteasome-dependent degradation. Interestingly, unlike CHIP, which targets RIP3 more generally, PELI1 preferentially targets kinase active RIP3 that has been phosphorylated on T182, subsequently leading to RIP3 degradation.

• Interdisciplinary Bio Central
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• v.4 no.2
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• pp.3.1-3.7
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• 2012

Epidermal growth factor receptor (EGFR) is a member of the ErbB family (ErbB1-4) of receptor tyrosine kinases (RTKs). EGFR controls numerous physiological functions, including cell proliferation, migration, differentiation and survival. Importantly, aberrant signaling by EGFR has been linked to human cancers in which EGFR and its various ligands are frequently overexpressed or mutated. EGFR coordinates activation of multiple downstream factors and is subject of various regulatory processes as it mediates biology of the cell it resides in. Therefore, many studies have been devoted to understanding EGFR biology and targeting the protein for the goal of controlling tumor in clinical settings. Endocytic regulation of EGFR offers a promising area for targeting EGFR activity. Upon ligand binding, the activated receptor undergoes endocytosis and becomes degraded in lysosome, thereby terminating the signal. En route to lysosome, the receptor becomes engaged in activating various signaling pathways including PI-3K, MAPK and Src, and endocytosis may offer both spatial and temporal regulation of downstream target activation. Therefore, endocytosis is an important regulator of EGFR signaling, and increasing emphasis is being placed on endocytosis in terms of cancer treatment and understanding of the disease. In this review, EGFR signaling pathway and its intricate regulation by endocytosis will be discussed.

• Biomolecules & Therapeutics
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• v.26 no.1
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• pp.57-68
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• 2018

Nuclear factor E2-related factor 2 (NRF2) plays an important role in redox metabolism and antioxidant defense. Under normal conditions, NRF2 proteins are maintained at very low levels because of their ubiquitination and proteasomal degradation via binding to the kelch-like ECH associated protein 1 (KEAP1)-E3 ubiquitin ligase complex. However, oxidative and/or electrophilic stresses disrupt the KEAP1-NRF2 interaction, which leads to the accumulation and transactivation of NRF2. During recent decades, a growing body of evidence suggests that NRF2 is frequently activated in many types of cancer by multiple mechanisms, including the genetic mutations in the KEAP1-NRF2 pathway. This suggested that NRF2 inhibition is a promising strategy for cancer therapy. Recently, several NRF2 inhibitors have been reported with anti-tumor efficacy. Here, we review the mechanisms whereby NRF2 is dysregulated in cancer and its contribution to the tumor development and radiochemoresistance. In addition, among the NRF2 inhibitors reported so far, we summarize and discuss repurposed NRF2 inhibitors with their potential mechanisms and provide new insights to develop selective NRF2 inhibitors.

• Proceedings of the Korea Crystallographic Association Conference
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• 2003.05a
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• pp.15-15
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• 2003

To discover new drugs more quickly and more efficiently, pharmaceutical companies and biotechnology firms are increasingly turning to the genomics and the structural proteomics technologies. Structural-proteomics can provide a foundation for this through the determination and analysis for protein structure on a genomics scale. Among many structures determined by CGI, we will present with the representative examples drawn from our work on novel structures or complex structures of the disease-related proteins. The alpha subunit of Hypoxia-inducible factor (HIF) is targeted for degradation under normoxic conditions by an ubiquitin-ligase complex that recognizes a hydroxylated proline residue in HIF. Hydroxylation is catalysed by HIF prolyl 4-hydroxylases (HIFPH) which are fe(II) and 2-oxoglutarate (2-OG) dependent oxygenases. Here, we discuss the first crystal structure of the catalytic domain of HIFPH in complexes, with the Fe(II)/2-OG at 1.8Å. These structures suggest that the Ll region (residues 236-253), which is also conserved in mammals, form a 'lid' that closes over the active site. The structural and mutagenesis analyses allow us to provide a focus for understanding cellular responses to hypoxia and a target for the therapeutic manipulation.