• Proceedings of the PSK Conference
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• 2002.10a
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• pp.270.1-270.1
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• 2002

The signaling pathway of D2 dopamine receptor was studied using yeaslt two-hybrid system.. The 3rd cytoplasmic loop of rat D2 dopamine receptor was used to screen the cDNA library of mouse brain. and ALY was found to interact with it. The interaction in the yeast was observed only with the 3rd cytoplasmic loop of D2 dopamine receptor but not with that of D3 or D4 dopamine receptor. The interaction between two proteins was also confirmed by GST pull-down assay. (omitted)

• Journal of Microbiology
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• v.41 no.1
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• pp.46-51
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• 2003

RAD6 in yeast mediates postreplication DNA repair and is responsible for DNA-damage induced mutations. RAD6 encodes ubiquitin-conjugating enzyme that is well conserved among eukaryotic organisms. However, the molecular targets and consequences of their ubiquitination by Rad6 have remained elusive. In Aspergillus nidulans, a RAD6 homolog has been isolated and shown to be an allele of uvs). We screened a CDNA library to isolate UVSJ-interacting proteins by the yeast two-hybrid system. JIPA was identified as an interactor of UVSJ. Their interaction was confirmed in vitro by a GST-pull down assay. JIPA was also able to interact with mutant UVSJ proteins, UVSJl and the active site cysteine mutant UVSJ-C88A. The N- and the C-terminal regions of UVSJ required for the interaction with UVSH, a RAD18 homolog of yeast which physically interacts with Rad6, were not necessary for the JIPA and UVSJ interactions. About 1.4 kb jipA transcript was detected in Northern analysis and its amount was not significantly increased in response to DNA-damaging agents. A genomic DNA clone of the jipA gene was isolated from a chromosome I specific genomic library by PCR-sib selection. Sequence determination of genomic and cDNA of jipA revealed an ORF of 893 bp interrupted by 2 introns, encoding a putative polypeptide of 262 amino acids. JIPA has 33% amino acid sequence identity to TIP41 of Saccharomyces cerevisiae which negatively regulates the TOR signaling pathway.

• Journal of Life Science
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• v.9 no.2
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• pp.34-39
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• 1999

The postsynaptic density (PSD) is a cytoskeletal specialization that is involved in the regulation of synaptic signal transduction. Mainly due to the hydrophobic nature of the PSD proteins, characterization of this intriguing structure at the molecular level has been very intractable until early 1990s. However, recent development in protein microchemistry and molecular cloning techniques allowed identification and characterization of the PSD proteins. As expected, cytoskeletal proteins constitute major components of the PSD. Other major PSD proteins have been identified by protein sequencing, and their genes were used to fish out associating proteins by yeast two-hybrid system expanding our knowledge on the molecular structure of the PSD significantly. In this review, I summarize proteins that are so far identified focusing on the glutamatergic synapses.

• Archives of Pharmacal Research
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• v.26 no.10
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• pp.846-854
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• 2003

The oligomerization of G-proteincoupled receptors (GPCRs) has been shown to occur by various mechanisms, such as via disulfide covalent linkages, non covalent (ionic, hydrophobic) interactions of the N-terminal, and/or transmembrane and/or intracellular domains. Interactions between GPCRs could involve an association between identical proteins (homomers) or non-identical proteins (heteromers), or between two monomers (to form dimers) or multiple monomers (to form oligomers). It is believed that muscarinic receptors may also be arranged into dimeric or oigomeric complexes, but no systematic experimental evidence exists concerning the direct physical interaction between receptor proteins as its mechanism. We undertook this study to determine whether muscarinic receptors form homomers or a heteromers by direct protein-protein interaction within the same or within different subtypes using a yeast two-hybrid system. Intracellular loops (i1, i2 and i3) and the C-terminal cytoplasmic tails (C) of human muscarinic (Hm) receptor subtypes, Hm1, Hm2 and Hm3, were cloned into the vectors (pB42AD and pLexA) of a two-hybrid system and examined for heteromeric or homodimeric interactions between the cytoplasmic domains. No physical interaction was observed between the intracellular domains of any of the Hm/Hm receptor sets tested. The results of our study suggest that the Hm1, Hm2 and Hm3 receptors do not form dimers or oligomers by interacting directly through either the hydrophilic intracellular domains or the C-terminal tail domains. To further investigate extracellular domain interactions, the N-terminus (N) and extracellular loops (o1 and o2) were also cloned into the two-hybrid vectors. Interactions of Hm2N with Hm2N, Hm2o1, Hm2o2, Hm3N, Hm3o1 or Hm3o2 were examined. The N-terminal domain of Hm2 was found to have no direct interaction with any extracellular domain. From our results, we excluded the possibility of a direct interaction between the muscarinic receptor subtypes (Hm1, Hm2 and Hm3) as a mechanism for homo- or hetero-meric dimerization/oligomerization. On the other hand, it remains a possibility that interaction may occur indirectly or require proper conformation or subunit formation or hydrophobic region involvement.

• Journal of Plant Biotechnology
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• v.5 no.3
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• pp.143-148
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• 2003

Bax, a mammalian pro-apoptotic member of the Bcl-2 family induces cell death when expressed in yeast. To investigate whether Bax expression can induce cell death in plant, we produced transgenic Arabidopsis plants that contained murine Bax cDNA under control of a glucocorticoid-inducible promoter. Transgenic plants treated with dexamethasone, a strong synthetic glucocorticoid, induced Bax accumulation and cell death, suggesting that some elements of cell death mechanism by Bax may be conserved among various organisms. Therefore, we developed novel yeast genetic system, and cloned several Plant Bax Inhibitors (PBIs). Here, we report the function of two PBIs in detail. PBI1 is ascorbate peroxidase (sAPX). Fluorescence method of dihydrorhodamine123 oxidation revealed that expression of Bax in yeast cells generated reactive oxygen species (ROS), and which was greatly reduced by co-expression with sAPX. These results suggest that sAPX inhibits the generation of ROS by Bax, which in turn suppresses Baxinduced cell death in yeast. PBI2 encodes nucleoside diphosphate kinase (NDPK). ROS stress strongly induces the expression of the NDPK2 gene in Arabidopsis thaliana (AtNDPK2). Transgenic plants overexpressing AtNDPK2 have lower levels of ROS than wildtype plants. Mutants lacking AtNDPK2 had higher levels of ROS than wildtype. $H_2O_2$ treatment induced the phosphorylation of two endogenous proteins whose molecular weights suggested they are AtMPK3 and AtMPK6. In the absence of $H_2O_2$ treatment, phosphorylation of these proteins was slightly elevated in plants overexpressing AtNDPK2 but markedly decreased in the AtNDPK2 deletion mutant. Yeast two-hybrid and in vitro protein pull-down assays revealed that AtNDPK2 specifically interacts with AtMPK3 and AtMPK6. Furthermore, AtNDPK2 also enhances the MSP phosphorylation activity of AtMPK3 in vitro. Finally, constitutive overexpression of AtNDPK2 in Arabidopsis plants conferred an enhanced tolerance to multiple environmental stresses that elicit ROS accumulation in situ. Thus, AtNDPK2 appears to playa novel regulatory role in $H_2O_2$-mediated MAPK signaling in plants.

• Proceedings of the Korean Society of Plant Biotechnology Conference
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• 2003.04a
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• pp.65-71
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• 2003

Bax, a mammalian pro-apoptotic member of the Bcl-2 family, induces cell death when expressed in yeast. To investigate whether Bax expression can induce cell death in plant, we produced transgenic Arabidopsis plants that contained murine Bax cDNA under control of a glucocorticoid-inducible promoter. Transgenic plants treated with dexamethasone, a strong synthetic glucocorticoid, induced Bax accumulation and cell death, suggesting that some elements of cell death mechanism by Bax may be conserved among various organisms. Therefore, we developed novel yeast genetic system, and cloned several Plant Bax Inhibitors (PBIs). Here, we report the function of two PBIs in detail. PBI1 is ascorbate peroxidase (sAPX). Fluorescence method of dihydrorho-damine 123 oxidation revealed that expression of Bax in yeast cells generated reactive oxygen species (ROS), and which was greatly reduced by co-expression with sAPX. These results suggest that sAPX inhibits the generation of ROS by Bax, which in turn suppresses Baxinduced cell death in yeast. PBI2 encodes nucleoside diphosphate kinase (NDPK). ROS stress strongly induces the expression of the NDPK2 gene in Arabidopsis thaliana (AtNDPK2). Transgenic plants overexpressing AtNDPK2 have lower levels of ROS than wildtype plants. Mutants lacking AtNDPK2 had higher levels of ROS than wildtype. $H_2O_2$ treatment induced the phosphorylation of two endogenous proteins whose molecular weights suggested they are AtMPK3 and AtMPK6. In the absence of $H_2O_2$ treatment, phosphorylation of these proteins was slightly elevated in plants overexpressing AtNDPK2 but markedly decreased in the AtNDPK2 deletion mutant. Yeast two-hybrid and in vitro protein pull-down assays revealed that AtNDPK2 specifically interacts with AtMPK3 and AtMPK6. Furthermore, AtNDPK2 also enhances the MBP phosphorylation activity of AtMPK3 in vitro. Finally, constitutive overexpression of AtNDPK2 in Arabidopsis plants conferred an enhanced tolerance to multiple environmental stresses that elicit ROS accumulation in situ. Thus, AtNDPK2 appears to play a novel regulatory role in $H_2O_2$-mediated MAPK signaling in plants.

• Journal of Life Science
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• v.19 no.8
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• pp.1055-1061
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• 2009

${\alpha}$-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors are widespread throughout the central nervous system and appear to serve as synaptic receptors for fast excitatory synaptic transmission mediated by glutamate. Their modulation is believed to affect learning and memory. To identify the interaction proteins for the AMPA receptor subunit glutamate receptor-interacting protein 1 (GRIPl), GRIP1 interactions with armadillo family protein p0071/plakophilin-4 were investigated. GRIP1 protein bound to the tail region of p0071/plakophilin-4 but not to other armadillo family protein members in a yeast two-hybrid assay. The "S-X-V" motif at the carboxyl (C)-terminal end of p0071/plakophilin-4 is essential for interaction with GRIP1. p0071/plakophilin-4 interacted with the Postsynaptic density-95/Discs large/Zona occludens-1 (PDZ) domains of GRIPI in the yeast two-hybrid assay, as is indicated also by Glutathione S-transferase (GST) pull-down, and co-immunoprecipitated with GRIP1 antibody in brain fraction. The findings of this study provide evidence that p0071/plakophilin-4 is an interactor of GRIP1.

• Proceedings of the Korean Society of Toxicology Conference
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• 2006.11a
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• pp.46-54
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• 2006

The methylmercury (MeHg) is a toxic environmental pollutant, causing serious neurological and developmental effects in humans. Recent epidemiological studies have indicated that ingestion of MeHg in fish during pregnancy can result in neuroethological effects in the offspring. However, the mechanism underlying the MeHg-toxicity is not fully understood. To elucidate the mechanisms of toxicity of MeHg and of defense against MeHg, we searched for factors that determine the sensitivity of yeast cells to MeHg, and found that overexpression of Cdc34, a ubiquitin-conjugating enzyme (E2) that is a component of the ubiquitin-proteasome (UP) system, induces a resistance to MeHg toxicity in both yeast and human cells. The UP system is involved in the intracellular degradation of proteins. When Cdc34 is overexpressed in cells, ubiquitination reactions are activated and the degradation of certain proteins by the UP system is enhanced. Therefore, it seems likely that certain as-yet-unidentified proteins that increase MeHg toxicity might exist in cons and that toxicity might be reduced by the enhanced degradation of such proteins, mediated by the UP system, when Cdc34 is overexpressed. SCF ubiquitin-ligase is a component of UP system and consists of Skpl, the scaffold protein Cdc53, the RING-finger protein Hrt1, and one member of the family of F-box proteins. The F-box proteins directly bind to the substrates and are the determinants of substrate specificity of SCF. Therefore, we searched for the f-box protein that cofers resistance to MeHg, and found that overexpression of Hrt3 or Yi1224w induced resistance to MeHg toxicity in yeast cells. Since the protein(5) that enhance toxicity of MeHg might plausibly be induced in substrates of both f-box proteins, we next searched for substrate proteins that are recognized by Hrt3 or Y1r224w using two-hybrid screen. We found that Did3 or Crsl interacts with Hrt3; and Eno2 interacts with Yir224w. The yeast cells that overexpressed each those proteins showed hypersensitivity to MeHg, respectively, indicating that those proteins enhance the MeHg toxicity. Both Dld3 and Eno2 are proteins involved in the synthesis of pyruvate, and overexpression of both proteins might induce increase in interacellular levels of pyruvate. Deletion of Yi1006w that transports pyruvate into the mitochondria induced aresistance to MeHg. These results suggest that the promotion of the pyruvate irdlowinto the mitochondria might enhance MeHg toxicity. This study providesimportant keyfor the elucidauon of the molecular mechanism of MeHg toxicity.

• IMMUNE NETWORK
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• v.2 no.3
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• pp.125-132
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• 2002

In the past, bioinformatics was often regarded as a difficult and rather remote field, practiced only by computer scientists and not a practical tool available to biologists. However, the various on-going genome projects have had a serious impact on biological sciences in various ways and now there is little doubt that bioinformatics is an essential part of the research environment, with a wealth of biological information to analyze and predict. Fully sequenced genomes made us to have additional insights into the functional properties of the encoded proteins and made it possible to develop new tools and schemes for functional biology on a proteomic scale. Among those are the yeast two-hybrid system, mass spectrometry and microarray: the technology of choice to detect protein-protein interactions. These functional insights emerge as networks of interacting proteins, also known as "pathway informatics" or "interactomics". Without exception it is no longer possible to make advances in the signaling/regulatory pathway studies without integrating information technologies with experimental technologies. In this paper, we will introduce the databases of protein interaction worldwide and discuss several challenging issues regarding the actual implementation of databases.

• Biomedical Science Letters
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• v.10 no.4
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• pp.407-413
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• 2004

Heterotrimeric GTP binding proteins (G proteins) transduce signals of a variety of hormones and neurotransmitters. Go is one of the most abundant G proteins in the brain and classified as the Gi/Go family due to their sequence homology to Gi proteins. While the Gi proteins inhibit adenylyl cyclase and decrease the intracellular cAMP concentration, the functions of Go is not clearly understood despite their sequence homology to Gi. The promeylocytic leukemia zinc finger protein (PLZF) is a DNA binding transcription factor and is expressed highly in central nervous system (CNS). Several studies reported that PLZF may be involved in regulation segmentation/differentiation during CNS development. Here, I report that the alpha subunit of Go (Go ) interacts with PLZF. The interaction between Goa and PLZF was verified by using GST pulldown assay and co-immunoprecipitation. Our findings indicate that Goa could modulate gene expression via interaction with PLZF during neuronal or brain development.