• Molecules and Cells
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• v.40 no.4
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• pp.271-279
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• 2017

Ran-binding protein family member, RanBP9 has been reported in various basic cellular mechanisms and neuropathological conditions including schizophrenia. Previous studies have reported that RanBP9 is highly expressed in the mammalian brain and retina; however, the role of RanBP9 in retinal development is largely unknown. Here, we present the novel and regulatory roles of RanBP9 in retinal development of a vertebrate animal model, zebrafish. Zebrafish embryos exhibited abundant expression of ranbp9 in developing brain tissues as well as in the developing retina. Yeast two-hybrid screening demonstrated the interaction of RanBP9 with Mind bomb, a component of Notch signaling involved in both neurogenesis and neural disease autism. The interaction is further substantiated by co-localization studies in cultured cells. Knockdown of ranbp9 resulted in retinal dysplasia with defective proliferation of retinal cells, downregulation of neuronal differentiation marker huC, elevation of neural proliferation marker her4, and alteration of cell cycle marker p57kip2. Expression of the $M{\ddot{u}}ller$ glial cell marker glutamine synthase was also affected in knockdown morphants. Our results suggest that Mind bomb-binding partner RanBP9 plays a role during retinal cell development of zebrafish embryogenesis.

• BMB Reports
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• v.43 no.7
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• pp.485-490
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• 2010

Neurotrophins regulate many aspects of neuronal function through activation of the high affinity Trk receptors. Shc family proteins are implicated in the coupling of RTK to the Ras/mitogen-activated protein kinase signaling cascade. Here we report that the fourth Shc family member, ShcD, associates with TrkB receptor and regulates BDNF-induced MAPK activation. Yeast two-hybrid assay and Co-IP experiments demonstrate ShcD interacts with TrkB in a kinase-activity-dependent manner. Confocal analysis shows ShcD cololizes well with TrkB in transfected 293T cells. Subsequent mapping experiments and mutational analysis indicate that both PTB and SH2 domains are capable of binding to TrkB and PTB domain binds to TrkB NPQY motif. Furthermore, ShcD is involved in BDNF-induced MAPK activation. In summary, we demonstrate that ShcD is a substrate of TrkB and mediates TrkB downstream signaling pathway.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.83.2-83
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• 2003

A full length cDNA, OsLEUZIP, encoding leucine zipper containing protein from rice EST of rice (0ryza sativa L. cv. Dongjin) treated Xanthomonas oryzae pv. oryzae 10331. OsLEUZIP contains 1,227 bp nucleotides and encodes a protein of 408 amino acid residues with predicted molecular weight of 47,229 Da. The deduced amino acid sequence of OsLEUZIP has consensus sequence of leucine zipper from PROSITE (PDOC00029), L-X(6)-L-X(6)-L-X(6) -L. OsLEUZIP gene were preferentially induced in rice during incompatible interaction with Xanthomonas oryzae pv. oryzae 10331 and Pyracuraria grisea KJ-301. Expression of OsLEUZIP gene was also induced by treatment of abiotics such as ethephon and ABA. Our data represented in this study suggesting that OsLEUZIP gene may play an important role in the rice defense-related. Further studies of this gene, overexpression in rice, yeast-two hybrid assay, electrophoretic mobility shift assay and northern blot analyses of transgenic plant, would be useful to elucidate the role of the OsLEUZIP gene in defense responses of rice.

• Journal of Plant Biotechnology
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• v.2 no.2
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• pp.79-82
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• 2000

We have isolated a cDNA encoding a calcium-dependent protein kinase (CDPK) in Nicotiana tabacum, which was designated NtCDPK1. Accumulation of the NtCDPK1 mRNA was stimulated by various stimuli, including phytohormones, CaCl$_2$ wounding, fungal elicitors, chitin and methyl jasmonate. The NtCDPK1 gene encodes a functional Ser/Thr protein kinase of which phosphorylation activity is strongly induced by calcium. By analyzing expression of the NtCDPK1-GFP fusion protein and by immunoblotting with antibody which reacts with NtCDPK1, we found that NtCDPK1 is localized in membrane and nucleus in plant cells. Silencing expression of the NtCDPK1 transgene resulted in marked decrease of lateral root development in the transgenic tobacco plants. Yeast two hybrid screening using NtCDPK1 as a bait identified a tobacco homologue of proteasome regulatory subunit 21D7, designated Nt21D7. The 21D7 mRNA has been shown to be predominantly expressed in proliferating tissues in the cell cycledependent manner in carrot. The recombinant NtCDPK1 protein associated with Nt21D7 in vitro, and could phosphorylate the Nt21D7 protein in vitro in the presence of calcium, suggesting that Nt21D7 protein is a natural substrate of NtCDPK1 in tobacco. These results suggest that NtCDPK1 may regulate tell proliferation processes, such as lateral root formation, by regulating specificity and/or activity of proteasome-mediated protein degradation pathway.

• Microbiology and Biotechnology Letters
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• v.33 no.4
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• pp.260-266
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• 2005

IgE-dependent histamine releasing factor (HRF), previously known as P23/P21 or translationally controlled tumor protein (TCTP), induces the degranulation of histamine in mast cell and basophil. Yeast two hybrid results showed that HRF interacts with the alpha subunit of Na, K-ATPase, suggesting that HRF is a regulator for governing the activity of Na, K-ATPase. In this study, we examined the interaction of HRF and Wa,K-ATPase after treatments of various PKC isotype inhibitors. Membrane fractionation, pull-down assay and immunoprecipitation results showed that PKC $\alpha,\;PKC\;\beta,\;\delta$ subunits are involved in the phosphorylation of HRF. However, these results did not correlate with the results of histamine release assay since histamine release assay results suggested that some PKC isotype inhibitors induced the histamine release in RBL-2H3 cell.

• BMB Reports
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• v.35 no.6
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• pp.629-636
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• 2002

Protein kinase CKII (CKII) is required for progression through the cell division cycle. We recently reported that the $\beta$ subunit of protein kinase CKII ($CKII{\beta}$) associates with CKBBP1 that contains the Ring-H2 finger motif in the yeast two-hybrid system. We demonstrate here that the Ring-H2 finger-disrupted mutant of CKBBP1 does not interact with purified $CKII{\beta}$ in vitro, which shows that the Ring-H2 finger motif is critical for direct interaction with $CKII{\beta}$. The CKII holoenzyme is efficiently co-precipitated with the wild-type CKBBP1, but not with the Ring-H2 finger-disrupted CKBBP1, from whole cell extracts when epitope-tagged CKBBP1 is transiently expressed in HeLa cells. Disruption of the Ring-H2 finger motif does not affect the cellular localization of CKBBP1 in HeLa cells. The increased expression of either the wild-type CKBBP1 or Ring-H2 finger-disrupted CKBBP1 does not modulate the protein or the activity levels of CKII in HeLa cells. However, the stable expression of Ring-H2 finger-disrupted CKBBP1 in HeLa cells suppresses cell proliferation and causes the accumulation of the G1/G0 peak of the cell cycle. The Ring-H2 finger motif is required for maximal CKBBP1 phosphorylation by CKII, suggesting that the stable binding of CKBBP1 to CKII is necessary for its efficient phosphorylation. Taken together, these results suggest that the complex formation of $CKII{\beta}$ with CKBBP1 and/or CKII-mediated CKBBP1 phosphorylation is important for the G1/S phase transition of the cell cycle.

• The Journal of Korean Society of Virology
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• v.28 no.1
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• pp.39-52
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• 1998

HIV-1 tat, a strong transactivator, is essential for the HIV-1 replication and AIDS progression. The Tat function is markedly inhibited by human anti-oncogene p53. This work was initiated to identify the p53-associated inhibitory mechanism on tat-mediated transactivation. Inhibitory function of p53 was confirmed by co-transfection of tat-expressing Jurkat cells with LTR-CAT plasmid, or H3T1 cells (LTR-CAT integrated HeLa cells) with different ratio of pSV-tat/pCDNA-p53 plasmids. Results from the direct protein-protein interaction between soluble p53 and tat, and yeast two-hybrid experiments showed that the co-suppression mechanism is unlikely to be due to the direct interaction. CAT activity was not affected by tat in Jurkat cells which were transfected with p53-promoter-CAT or p53-enhancer-CAT, suggesting that the tat-mediated p53 suppression is not directly associated with p53-promoter. Finally, we have tested protein kinase activity in p53-tranfected Jurkat cells, which might phosphorylate HIV-1 tat, resulting in inhibition of tat function. Some of our data lead us to assume that the p53-mediated tat inhibition is likely to be associated with p53-associated, signaling-mediated phosphorylation of tat, resulting in the dysfunction of tat. This study is now under investigation.

• The Plant Pathology Journal
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• v.30 no.1
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• pp.58-67
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• 2014

The multifunctional triple gene block protein 1 (TGB1) of the Potexvirus Alternanthera mosaic virus (AltMV) has been reported to have silencing suppressor, cell-to-cell movement, and helicase functions. Yeast two hybrid screening using an Arabidopsis thaliana cDNA library with TGB1 as bait, and co-purification with TGB1 inclusion bodies identified several host proteins which interact with AltMV TGB1. Host protein interactions with TGB1 were confirmed by biomolecular fluorescence complementation, which showed positive TGB1 interaction with mitochondrial ATP synthase delta' chain subunit (ATP synthase delta'), light harvesting chlorophyll-protein complex I subunit A4 (LHCA4), chlorophyll a/b binding protein 1 (LHB1B2), chloroplast-localized IscA-like protein (ATCPISCA), and chloroplast ${\beta}$-ATPase. However, chloroplast ${\beta}$-ATPase interacts only with $TGB1_{L88}$, and not with weak silencing suppressor $TGB1_{L88}$. This selective interaction indicates that chloroplast ${\beta}$-ATPase is not required for AltMV movement and replication; however, TRV silencing of chloroplast ${\beta}$-ATPase in Nicotiana benthamiana induced severe tissue necrosis when plants were infected by AltMV $TGB1_{L88}$ but not AltMV $TGB1_{L88}$, suggesting that ${\beta}$-ATPase selectively responded to $TGB1_{L88}$ to induce defense responses.

• Proceedings of the Korean Society for Bioinformatics Conference
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• 2000.11a
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• pp.56-58
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• 2000

Post-genomics may be defined in different ways depending on how one views the challenges after the genome. A popular view is to follow the concept of the central dogma in molecular biology, namely from genome to transcriptome to proteome. Projects are going on to analyze gene expression profiles both at the mRNA and protein levels and to catalog protein 3D structure families, which will no doubt help the understanding of information in the genome. However complete, such catalogs of genes, RNAs, and proteins only tell us about the building blocks of life. They do not tell us much about the wiring (interaction) of building blocks, which is essential for uncovering systemic functional behaviors of the cell or the organism. Thus, an alternative view of post-genomics is to go up from the molecular level to the cellular level, and to understand, what I call, the "interactome"or a complete picture of molecular interactions in the cell. KEGG (http://www.genome.ad.jp/kegg/) is our attempt to computerize current knowledge on various cellular processes as a collection of "generalized"protein-protein interaction networks, to develop new graph-based algorithms for predicting such networks from the genome information, and to actually reconstruct the interactomes for all the completely sequenced genomes and some partial genomes. During the reconstruction process, it becomes readily apparent that certain pathways and molecular complexes are present or absent in each organism, indicating modular structures of the interactome. In addition, the reconstruction uncovers missing components in an otherwise complete pathway or complex, which may result from misannotation of the genome or misrepresentation of the KEGG pathway. When combined with additional experimental data on protein-protein interactions, such as by yeast two-hybrid systems, the reconstruction possibly uncovers unknown partners for a particular pathway or complex. Thus, the reconstruction is tightly coupled with the annotation of individual genes, which is maintained in the GENES database in KEGG. We are also trying to expand our literature surrey to include in the GENES database most up-to-date information about gene functions.

• BMB Reports
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• v.41 no.2
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• pp.158-163
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• 2008

Hepatitis B virus X protein (HBx) is essential for hepatitis B virus infection and exerts a pleiotropic effect on various cellular machineries. HBx has been also demonstrated as an indirect transcriptional transactivator of various different viral and cellular promoters. In addition, HBx is involved in the development of various liver diseases including hepatocellular carcinoma. However the mechanism of HBx in hepatocellular carcinogenesis remains largely unknown. In this study, to identify possible new cellular proteins interacting with HBx, we carried out yeast two-hybrid assay. We obtained several possible cellular partners including VBP1, a binding factor for VHL tumor suppressor protein. The direct physical interaction between HBx and VBP1 in vitro and in vivo was confirmed by immunoprecipitation assay. In addition, we found that VBP1 facilitates HBx-induced $NF{\kappa}B$ activation and cell proliferation. These results implicate the important role of HBx in the development of hepatocellular carcinoma through its interaction with VBP1.