• Journal of Life Science
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• v.15 no.6 s.73
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• pp.904-908
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• 2005

Recently the pathological actions of CagA of Helicobacter pylori (H. pylori) on gastric epithelial cells have been reported. CagA is directly injected into the host cytoplasm and undergoes tyrosine phosphorylation in the cells. In addition, translocated CagA forms a physical complex with SHP-2. There are two major CagA subtypes according to the amino acid sequence in the 3'region of CagA; i) the East Asian type (A-B-D of EPIYA motifs) and ii) the Western type (A-B-C of EPIYA motifs). Repeated EPIYA motifs in the 3'region of CagA are involved in the interaction with SHP-2. The East Asian type conferred stronger SHP-2 binding activity than the Westrrn type of CagA. Here we analyzed the amino acid sequences of the SHP-2 binding site of cagA gene in H. pyzori, and investigated whether there is my relationship between the diversities of cagA and the disease out-come in Korea. Most of Korean H. pylori strains showed A-B-D motifs(the East Asian type), and only one strain showed A-B-B-D motifs. In Korea, the incidence of atrophic gastritis and gastric cancer is significantly high compared with Western countries. The high frequency of the East Asian type CagA among Korean H. pylori strains would be involved in increasing the risk of gastric cancer in Korean populations.

• The Korean Journal of Pharmacology
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• v.30 no.1
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• pp.117-124
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• 1994

Phospholamban is the regulator of $Ca^{2+}-ATPase$ in cardiac sarcoplasmic reticulum(SR). The mechanism of regulation appears to involve inhibition by dephosphorylated phospholamban. Phosphorylation of phospholamban relieves this inhibition. Recently, there has been a report that the cytoplasmic domain (amino acids 1-25) of phospholamban is insufficient to inhibit the $Ca^{2+}$ pump. To explore the domains of phospholamban responsible for $Ca^{2+}-ATPase$ inhibitory activity, we examined the effect of a synthetic phospholamban peptide consisting of amino acid residues 1-25 on $Ca^{2+}$ uptake by reconstituted skeletal SR $Ca^{2+}-ATPase$. The $Ca^{2+}-ATPase$ of skeletal SR was purified and reconstituted in proteoliposomes containing phosphatidylcholine (PC) or phosphatidylcholine: phosphatidylserine (PC:PS). Inclusion of a phospholamban peptide in PC proteoliposomes was associated with significant inhibition of the initial rates of $Ca^{2+}$ uptake at pCa 6.0, and phosphorylation of this peptide by the catalytic subunit of cAMP-dependent protein kinase reversed the inhibitory effect on the $Ca^{2+}$ pump. Similar effects of phospholamban peptide were also observed using PC:PS proteoliposomes. Based on these results, we could conclude that the cytoplasmic domain of phospholamban, containing the phosphorylation sites, by itself is sufficient to inhibit the $Ca^{2+}$ pump of SR.

• BMB Reports
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• v.44 no.7
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• pp.490-495
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• 2011

Transcription factor AP-$2{\alpha}$ involves in the process of mammalian embryonic development and tumorigenesis. Many studies have shown that AP-$2{\alpha}$ functions in association with other interacting proteins. In a two-hybrid screening, the regulatory subunit ${\beta}$ of protein casein kinase 2 ($CK2{\beta}$) was identified as an interacting protein of AP-$2{\alpha}$; we confirmed this interaction using in-vitro GST pull-down and in-vivo co-immunoprecipitation assays; in an endogenous co-immunoprecipitation experiment, we further found the catalytic subunit ${\alpha}$ of protein casein kinase 2 ($CK2{\alpha}$) also exists in the complex. Phosphorylation analysis revealed that AP-$2{\alpha}$ was phosphorylated by CK2 kinase majorly at the site of Ser429, and such phosphorylation could be blocked by CK2 specific inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB) in a dose-dependent manner. Luciferase assays demonstrated that both $CK2{\alpha}$ and $CK2{\beta}$ enhanced the transcription activity of AP-$2{\alpha}$; moreover, $CK2{\beta}$ increased the stability of AP-$2{\alpha}$. Our data suggest a novel cellular function of CK-2 as a transcriptional co-activator of AP-$2{\alpha}$.

• KSBB Journal
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• v.10 no.5
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• pp.499-509
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• 1995

PU.1, a tissue-specific transcription activator, binds to a purine-rich sequence(5'-GAGGAA-3') called PU box. The PU.1 cDNA consists of an open reading frame of 816 nucleotides coding for 272 amino acids. The amino terminal end is highly acidic, while the carboxyl terminal end is highly basic. Transcriptional activation domain is located at the amino terminal end, while DNA binding domain is located at the carboxyl terminal end. Activation of PU.1 transcription factor is supposed to be accomplished by the phosphorylation of serine residue(s). There exist 22 serines in the PU.1. Five(the 41, 45, 132$.$133, and 148th) of the serines(plausible phosphorylation site by casein kinase II), are the primary targets of interest in elucidating the molecular mechanism(s) of the action of the PU.1 gene. In this study, PU.1 cDNA coding for the five serine residues(41th AGC, 45th AGC, 132$.$133th AGC$.$TCA, and 148th TCT), was mutated to alanine codon(41th GCC, 45th GCC, 132$.$133th GCC$.$GCA, and 1481h GCT), respectively, by Splicing-Overlapping-Extension(SOE) using Polymerase Chain Reaction(PCR). And each mutated cDNA fragments was ligated into pBluescript KS＋ digested with HindIII and Xba I, to generate mutant clones named pKKS41A, pRKS45A, pMKS132$.$133A, and pMKS148A. The clones will be informative to study the "Structure and Function" of the immu-nologically important gene, PU.1.

• Journal of Life Science
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• v.31 no.7
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• pp.671-676
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• 2021

A cDNA encoding the protein lethal (2) essential for life [l(2)efl] was cloned from Gryllus bimaculatus and named GBl(2)efl. This protein is composed of 189 amino acids, including an N-glycosylation site and 15 phosphorylation sites. Its predicted molecular mass is 21.19 kDa, with a theoretical isoelectric point of 6.2. The secondary structure of GBl(2)efl was predicted from the identification of random coils (56.08%), alpha helices (22.22%), extended strands (17.99%), and beta turns (3.7%) through sequence analyses. A homology analysis revealed that GBl(2)efl exhibited a high similarity with other species at the amino acid level, ranging from 52% to 69%. While GBl(2)efl mRNA expression was higher in the dorsal longitudinal flight muscle following a three-day starvation and in the Malpighian tubules following a one-day starvation, no changes in expression were detected in other tissues. Furthermore, tunicamycin-induced endoplasmic reticulum (ER) stress resulted in an approximately 1.8-fold higher expression in the fat body compared with the wild type.

• IMMUNE NETWORK
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• v.2 no.2
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• pp.72-78
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• 2002

Background: The precise mechanism by which CTLA-4 regulates T cell immune responses is still not fully understood. Previously we proposed that CTLA-4 could downregulate T cell function by modulating a signaling cascade initiated from the T cell receptor complex. The evidence for this notion comes from our findings that CTLA-4 associated with the T cell receptor zeta (TCR zeta) chain, and hence regulated TCR zeta phosphorylation by co-associated SHP-2 tyrosine phosphatase (1). In this report, we investigated whether any other signaling molecules could be involved in the CTLA-4 signaling pathway. Methods: We have taken biochemical approaches, such as immunoprecipitation followed by autoradiography or immunoblotting, to identify the molecules associated with CTLA-4. To perform these assays, we used activated primary T cells and ectopically transfected 293 cells. Various truncation mutants of CTLA-4 were used to map the interaction site on CTLA-4. Results: We found that in addition to TCR zeta and SHP-2, a recently cloned small adaptor molecule, SAP (SLAM-associated protein), was also able to associate with CTLA-4. We identified the domain of SAP association in CTLA-4 being a motif involving GVYVKM. This motif has been previously found to bind SHP-2 through its phosphorylated tyrosine interaction with SH-2 domain of SHP-2. Indeed, co-expression of SAP and SHP-2 reduced their binding to CTLA-4 significantly, suggesting that SAP and SHP-2 compete for the common binding site, GVYVKM. Thus, by blocking SHP-2 recruitment SAP could function as a negative regulator of CTLA-4. Conclusion: Taken together, our data suggest the existence of complicate signaling cascade in regulating CTLA-4 function, and further provide evidence that SAP can act either as a positive or negative regulator depending on the nature of the associating receptors.

• Proceedings of the Botanical Society of Korea Conference
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• 1999.07a
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• pp.57-62
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• 1999

Light-regulated translation of chloroplast mRNAs requires nuclear-encoded trans-acting factors that interact with the 5' untranslated region (UTR) of these mRNAs. A set of four proteins (60, 55, 47, and 38 kDa) that bind to the 5'-UTR of the psbA mRNA had been identified in C. reinhardtii. 47 kDa protein (RB47) was found to encode a chloroplast poly (A)-binding protein (cPABP) that specifically binds to the 5'-UTR of the psbA mRNA, and essential for translation of this mRNA, cDNA encoding 60 kDa protein (RB60) was isolated, and the amino acid sequence of the encoded protein was highly homologous to plants and mammalian protein disulfide isomerases (PDI), normally found in the endoplasmic reticulum (ER). Immunoblot analysis of C. reinhardtii proteins showed that anti-PDI recognized a distinct protein of 56 kDa in whole cell extract, whereas anti-rRB60 detected a 60 kDa protein. The ER-PDI was not retained on heparin-agarose resin whereas RB60 was retained. In vitro translation products of the RB60 cDNA can be transported into C. reinhardtii chloroplast in vitro. Immunoblot analysis of isolated pea chloroplasts indicated that higher plant also possess a RB60 homolog. In vitro RNA-binding studies showed that RB60 modulates the binding of cPABP to the 5'-UTR of the psbA mRNA by reversibly changing the redox status of cPABP using redox potential or ADP-dependent phosphorylation. Site-directed mutagenesis of -CGHC- catalytic site in thioredoxin-like domain of RB60 is an unique PDI located in the chloroplast of C. reinhardtii, and suggest that the chloroplast PDI may have evolved to utilize the redox-regulated thioredoxin like domain as a mechanism for regulating the light-activated translation of the psbA mRNA.

• Proceedings of the Botanical Society of Korea Conference
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• 1985.08b
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• pp.7-18
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• 1985

Light-regulated translation of chloroplast mRNAs requires nuclear-encoded trans-acting factors that interact with the 5' untranslated region (UTR) of these mRNAs. A set of four proteins (60, 55, 47, and 38 kDa) that bind to the 5'-UTR of the psbA mRNA had been identified in C. reinhardtii. 47 kDa protein (RB47) was found to encode a chloroplast poly (A)-binding protein (cPABP) that specifically binds to the 5'-UTR of the psbA mRNA, and essential for translation of this mRNA, cDNA encoding 60 kDa protein (RB60) was isolated, and the amino acid sequence of the encoded protein was highly homologous to plants and mammalian protein disulfide isomerases (PDI), normally found in the endoplasmic reticulum (ER). Immunoblot analysis of C. reinhardtii proteins showed that anti-PDI recognized a distinct protein of 56 kDa in whole cell extract, whereas anti-rRB60 detected a 60 kDa protein. The ER-PDI was not retained on heparin-agarose resin whereas RB60 was retained. In vitro translation products of the RB60 cDNA can be transported into C. reinhardtii chloroplast in vitro. Immunoblot analysis of isolated pea chloroplasts indicated that higher plant also possess a RB60 homolog. In vitro RNA-binding studies showed that RB60 modulates the binding of cPABP to the 5'-UTR of the psbA mRNA by reversibly changing the redox status of cPABP using redox potential or ADP-dependent phosphorylation. Site-directed mutagenesis of -CGHC- catalytic site in thioredoxin-like domain of RB60 is an unique PDI located in the chloroplast of C. reinhardtii, and suggest that the chloroplast PDI may have evolved to utilize the redox-regulated thioredoxin like domain as a mechanism for regulating the light-activated translation of the psbA mRNA.

• Journal of the Korean Chemical Society
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• v.41 no.4
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• pp.205-209
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• 1997

The site specificity of dephosphorylation of myelin basic protein(MBP) was studied in vitro. To assign amino acid site of dephosphorylation, MBP was phosphorylated by protein kinase C(PKC) and dephosphorylated by protein phosphatase PP2A. The phosphorylated MBP was digested by trypsine and the digested peptides were separated by a reverse phase HPLC chromatography. The radioactivity of each fraction was counted and partially sequenced. Seven radioactive peptides were observed and $Ser^{55}$ in the second peak($P_2$) shows the best susceptibility for the phosphorylation. However in the dephosphorylation, the fifth peak($P_5$) appeared to release it's phosphate group most rapidly. This result demonstrates that MBP is a suitable substrate for protein phosphatase.

• BMB Reports
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• v.50 no.11
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• pp.539-545
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• 2017

The Integrated Stress Response (ISR) refers to a signaling pathway initiated by stress-activated $eIF2{\alpha}$ kinases. Once activated, the pathway causes attenuation of global mRNA translation while also paradoxically inducing stress response gene expression. A detailed analysis of this pathway has helped us better understand how stressed cells coordinate gene expression at translational and transcriptional levels. The translational attenuation associated with this pathway has been largely attributed to the phosphorylation of the translational initiation factor $eIF2{\alpha}$. However, independent studies are now pointing to a second translational regulation step involving a downstream ISR target, 4E-BP, in the inhibition of eIF4E and specifically cap-dependent translation. The activation of 4E-BP is consistent with previous reports implicating the roles of 4E-BP resistant, Internal Ribosome Entry Site (IRES) dependent translation in ISR active cells. In this review, we provide an overview of the translation inhibition mechanisms engaged by the ISR and how they impact the translation of stress response genes.