Deoxyhypusine synthase in phosphorylated by protein kinase C in vivo as well as in vitro

강기련,김지숙,정수일,박명희,김연웅,임동권,이소영 생화학분자생물학회 2002 Experimental and molecular medicine Vol.34 No.6

Deoxyhypusine synthase catalyzes the first step in the posttranslational synthesis of an unusual ami-no acid, hypusine, in the eukaryotic translation initiation factor 5A (eIF-5A) precursor protein. We earlier observed that yeast recombinant deoxyhy-pusine synthase was phosphorylated by protein kinase C (PKC) in vitro (Kang and Chung, 1999) and the phosphorylation rate was synergistically increased to a 3.5-fold folowing treatment with phosphatidylserine (P.Ser)/diacylglycerol (DAG)/ Ca2+We have extended study on the phosphorylation of deoxyhypusine synthase in vivo in diferent cel lines in order to define its role on the regulation of eIF5A in the cel. Deoxyhypusine synthase was found to be phosphorylated by endogenous ki-nases in CHO, NIH3T3, and chicken embryonic cells. The highest degre of phosphorylation was found in CHO cels. Moreover, phosphorylation of deoxyhypusine synthase in intact CHO cels was revealed and the expression of phosphorylated deoxyhypusine synthase was significantly dimin-ished by diacyl ethylene glycol (DAEG), a PKC in-hibitor, and enhanced by phorbol 12-myristate 13-acetate (PMA) or Ca2+/DAG. Endogenous PKC in CHO cell and cell lysate was able to phosphorylate deoxyhypusine synthase and this modification is enhanced by PMA or Ca2+ plus DAG. Close asso-ciation of PKC with deoxyhypusine synthase in the CHO cells was evident in the immune copre-cipitation and was PMA-, and Ca2+/phospholipid-dependent. These results suggest that phosphory-lation of deoxyhypusine synthase was PKC-de-sible regulation in the interaction with eIF5A pre-cursor for hypusine synthesis.

단백질 티로신 탈인산화 효소들의 특징

김연웅,강기련,김충원,강윤세 경상대학교 유전공학연구소 1997 遺傳工學硏究所報 Vol.16 No.-

지난 30 여년에 걸친 대사과정의 조절에 관한 연구를 통하여 밝혀진 중요한 사실은 이 과정에 참여하고 있는 요소들의 활성은 주로 allosteric 반응 및 단백질 인산화와 같은 공유결합을 이용한 방식으로 조절된다는 것이다. 특히 조절 기작 중 세포의 자극 신호 전달은 주로 인산화를 이용한 공유결합 방식이 이용되고 있다. 호르몬, 성장인자 혹은 신경 전달 물질을 포함하는 일부 리간드들이 자신의 특이 수용체와 결합하면 2차 전령물질의 합성 및 방출을 촉진하여 G 단백질이 관여하는 경로를 활성화하거나 혹은 자신의 수용체가 지니고 있는 단백질 티로신 인산화 효소(protein tyrosine kinases, dlgn PTK로 표시함)활성을 중폭시킨다. 이 후 자극에 의하여 발생한 신호들은 cascade방식을 통하여 초기 자극을 다시 증폭하면서 세포 내로 성장 신호를 전달하게 된다. 일반적으로 신호전달 과정에 참여하고 있는 효소들은 한 효소가 여러 반응 경로에 관여하는 pleiotropic 특성을 보유하고 있으므로 세포기능을 조화 있게 협동적으로 조절할 수 있다. 티로신 인산화 반응은 세포의 성장 및 분화,cell cycle의 조절, 세포 골격구조의 형성 등을 조절하는 중요한 기전으로 사용되고 있다.(1∼3). 세포에서 티로신 인산화의 알짜 수준은 PTK와 단백질 티로신 탈인산화 효소(protein tyrosine phosphatases, 이후 PTP로 표시함)활성 간의 경쟁적인 균형에 의해서 결정된다(4). PTP 의 연구사 PTK에 비하여 활발하게 진행되지 못하였으나 최근 PTP 들이 다수 발견되면서 이 효소들이 종류가 많고 구조가 다양한 것이 알려져 신호전달의 조절 작용에 PTK 와 함께 중요한 역할이 기대되어 연구가 활발히 진행되고 있다. 지금까지 PTP 의 연구가 PTK 보다 효율적이지 못하였던 것은 기술적인 문제로 PTP활성을 용이하게 측정할 기질분자가 적당하지 못하였다. 티로신 인산화 비율은 전체 단백질 가운데 0.1% 정도로 매우 낮아 충분한 양의 기질들이 공급될 수 없었다. 이러한 문제를 극복하기 위하여 고안된 인공기질이 carboxamidomethyled maleylated lysozymen(RCML)이었다. 이 기질을 이용한 assay방법과 thiophophorylate 화한 단백질을 붙인 Sepharose 친화력 크로마토그라피 방법을 이용하여 사람 태반 추출물로부터 순수한 형태의 PTP가 정재되어 PTP1B라 명명하였다(5,6). 분리한 PTP1B 의 아미노산 분석한 결과 PTP1B 의 구조는 이미 알려진 Ser-Thr 탈인산화 효소와 다른 것을 알 수 있었다.(7). 반면에 PTK 는 Ser-Thr인산화 효소들과 구조가 서로 비슷하였다. 이 결과로 PTK는 Ser-Thr인산화 효소들과 같은 조상의 단백질로 부터 진화되어 왔으나 PTP 는 Ser-Thr 탈인산화 효소와 다른 조상으로부터 유래한 것으로 생각된다. 이 글에서는 지금까지 알려진 각 부류에 속하는 PTP들의 구조적인 특성과 기능 및 PTK 와의 상호작용에 관하여 요약하여 PTP 들의 구조 및 PTP들이 관여하는 신호전달 조절 기작을 이해하는 기초로 이용하고자 한다.

Proteomic Analysis of Rat Brains Following Exposure to Electroconvulsive Therapy

이철순,강기련,이지영,박철수,한규희,손진욱,김봉조 대한의학회 2009 Journal of Korean medical science Vol.24 No.1

Electroconvulsive therapy (ECT) is one of the most effective treatments used in psychiatry to date. The mechanisms of ECT action, however, are the least understood and still unclear. As a tool to elucidate the mechanisms of action of ECT, we employed proteomic analysis based on the identification of differentially expressed proteins after exposure to repeated ECT in rat brains. The expression of proteins was visualized by silver stain after two-dimensional gel electrophoresis. Of 24 differentially expressed protein spots (p<0.05 by Student t-test), six different proteins from 7 spots were identified by matrix-assisted laser desorption/ionization time-of flight (MALDI-TOF)/mass spectrometry. Among the identified proteins, there were five dominantly expressed proteins in the ECT-treated rat brain tissues (p<0.05); S100 protein beta chain, 14-3-3 protein zeta/delta, similar to ubiquitin-like 1 (sentrin) activating enzyme subunit 1, suppressor of G2 allele of SKP1 homolog, and phosphatidylinositol transfer protein alpha. The expression of only one protein, ACY1 protein, was repressed (p<0.05). These findings likely serve for a better understanding of mechanisms involved in the therapeutic effects of ECT.

Cloning of chicken elongation factor 2 and its functional domain analysis based on the nucleotide sequence of the molecule

김충원,김연웅,강기련,정은주,안홍준,강윤세 한국생화학분자생물학회 (구 한국생화학회) 1991 추계학술대회집 Vol.- No.-

Molecular Cloning of Chicken Elongation Factor(EF-2) : Sequence Comparison with Mammalian EF-2 and its Expression in Early Developmental Stages of the Embryos 포유류 연장인자 2와의 일차구조 비교 및 초기 배아세포 발육과정에서의 발현

Kim, Choong Won,Kim, Yeon Woong,Kang, Kee Ryeon,Eom, Mi-Ok,Jung, Eun Joo,Kim, Jong Chul,Ahn, Hong Joon,Kang, Yoon-Se 경상대학교 유전공학연구소 1992 遺傳工學硏究所報 Vol.11 No.-

Chicken elongation factor 2(EF-2) cDNA was isolated from a chicken intestine cDNA libary and the sequence was determined. The probe for the screening of library was prepared by polymerase chain reaction(PCR) using two degenerate oligonuckeotide primers encoding NH_2-terminal region (residue 9-14) of chicken EF-2 and a conserved GTP binding domain (residue 80-84) of hamster EF-2. The sequence of amplified 228 bp product was identical to that of hamster EF-2(residue 9-84). One of 5 positive clones, pCEK4, consisted of 3,144 nucleotides with 2,574 bp open reading frame coding for 858 amino acid reaidues. The M_r 95,361 of the protein calculated from the amino acid sequence agreed well with the molecular weight of chicken and other mammalian EF-2(95,000) measured by SDS-PAGE. Sequence identity between chicken and other mammalian EF-2s(human, hamster, and rat) was 97%, while the sequence of GTP-binding/-hydrolysis domain was 99% identical. This result indicates that EF-2 is one of the highly conserved proteins and its structure has not been changed significantly during evolution. When expression of EF-2 in various stage of chicken embryo development was estimated by measuring mRNA and protein of EF-2, the result showed that the levels of its EF-2 mRNA and protein levels were the highest in 3 day embryo and then decreased gradually.

Cloning of Chicken Elongation Factor 2 and its Functional Domain Analysis Based on the Nucleotide Sequence of the Molecule

KIM, CHOONG WON,KIM, YEON WOONG,KANG, KEE RYEON,JUNG, EUN JOO,KIM, JONG CHUL,KANG, YOON SE 경상대학교 유전공학연구소 1991 遺傳工學硏究所報 Vol.10 No.-

Five cDNA clones of elongation factor 2(EF-2) were isolated from chicken intestine cDNA library. All insert in λgt 10 expect one were found to be 3.2 kb. The size should fully encodes for chicken EF-2 with molecular mass of 95kDa. using one of the clones, designated pCEK4, we determined partial amino acid sequence of EF-2 protein. The sequence included NH_2-terminal 169 amino acids correaponding to the common motif of five regions found in GTP-binding proteins. Comparative sequence analysis of each domain among chicken and hamster EF-2 shpwed a striling homology. ADP-ribosylation of chicken EF-2 in the presence of diphtheria toxin and NAD^+ provided an indirect evidence that ADP-ribosylatable diphthamide, 2-[3-carboxyamide-3-(trimethyl ammonio)-propyl]histidine. Finally measurements of EF-2 in chicken embryo of early developmental stage suggest that expression of EF-2 is correlated with the extent of cell growth and development.

Cellular Proliferation and Expression of Proto-Oncogenes in Chicken Embryonic Cells

KIM, YEON WOONG,KIM, CHOOG WON,SUNG, SOON KEE,KANG, KEE RYEON,KANG, YOON-SE 경상대학교 유전공학연구소 1988 遺傳工學硏究所報 Vol.7 No.-

Tyrosine protein kinase has been implicated in the mediation of cellular proliferation and development. Products of several viral oncogenes and growth factors have been shown to be associated with tyrosine kinase activity. Thus we investigated expression of tyrosine kinase-related proto-oncogenes during early chicken embryonic development (from 30 hr to 10 day). Each of four c-onc gene (i.e c-fos, c-src, c-abl, and c-fes/fps) showed characteristic expression depending on the time periods within 10 days, but level of c-erbB appeared very low compared to the other proto-oncogenes. Histone H2B gene was used as control.

Proteomic Analysis of Differentially Expressed Proteins in Bovine Endometrium with Endometritis

조창영,박정원,김은석,이상규,박선영,이정순,조명재,강기련,강다원,한재희 대한약리학회 2010 The Korean Journal of Physiology & Pharmacology Vol.14 No.4

Endometritis is one of the primary reasons for reproductive failure. In order to investigate endometritis-associated marker proteins, proteomic analysis was performed on bovine endometrium with endometritis. In bovine endometritis, desmin, α-actin-2, heat-shock protein (HSP) 27, peroxiredoxin-6, luteinizing hormone receptor isoform 1, collectin-43 precursor, deoxyribonuclease-I (DNase-I), and MHC class I heavy chain (MHC-Ih) were up-regulated. In contrast, transferrin, interleukin-2 precursor, hemoglobin β subunit, and potassium channel tetramerisation domain- containing 11 (KCTD11) were down-regulated in comparison to normal endometrium. The proteomic results were validated by semiquantitative-PCR and immunoblot analysis. The mRNA levels of desmin, transferrin, α-actin-2, HSP27, KCTD11, and MHC-Ih were up-regulated by over 1.5-fold, and showed a pattern similar to their proteomic profiles. Desmin and α-actin-2 protein showed positive correlations between proteomic analysis and immunoblot analysis. These results suggest that desmin and α-actin-2 may play important roles in endometritis-related function, and could be useful markers for the diagnosis of bovine endometritis.

Spinal Cord Injury Markedly Altered Protein Expression Patterns in the Affected Rat Urinary Bladder during Healing Stages

이지영,김봉조,심규진,김규태,강다원,정재훈,화정석,곽연주,최연진,박영숙,한재희,이철순,강기련,박철수 대한의학회 2011 Journal of Korean medical science Vol.26 No.6

The influence of spinal cord injury (SCI) on protein expression in the rat urinary bladder was assessed by proteomic analysis at different time intervals post-injury. After contusion SCI between T9 and T10, bladder tissues were processed by 2-DE and MALDI-TOF/MS at 6 hr to 28 days after SCI to identify proteins involved in the healing process of SCI-induced neurogenic bladder. Approximately 1,000 spots from the bladder of SCI and sham groups were visualized and identified. At one day after SCI, the expression levels of three protein were increased, and seven spots were down-regulated, including heat shock protein 27(Hsp27) and heat shock protein 20 (Hsp20). Fifteen spots such as S100-A11 were differentially expressed seven days post-injury, and seven proteins including transgelin had altered expression patterns 28 days after injury. Of the proteins with altered expression levels, transgelin, S100-A11, Hsp27 and Hsp20 were continuously and variably expressed throughout the entire post-SCI recovery of the bladder. The identified proteins at each time point belong to eight functional categories. The altered expression patterns identified by 2-DE of transgelin and S100-A11 were verified by Western blot. Transgelin and protein S100-A11 may be candidates for protein biomarkers in the bladder healing process after SCI.

Cigarette Smoke Extract-induced Reduction in Migration and Contraction in Normal Human Bronchial Smooth Muscle Cells

윤철호,Hye-Jin Park,Young-Woo Cho,김은진,이종덕,강기련,한재희,강다원 대한약리학회 2011 The Korean Journal of Physiology & Pharmacology Vol.15 No.6

The proliferation, migration, cytokine release, and contraction of airway smooth muscle cells are key events in the airway remodeling process that occur in lung disease such as asthma, chronic obstruction pulmonary disease, and cancer. These events can be modulated by a number of factors, including cigarette smoke extract (CSE). CSE-induced alterations in the viability, migration, and contractile abilities of normal human airway cells remain unclear. This study investigated the effect of CSE on cell viability, migration, tumor necrosis factor (TNF)-α secretion, and contraction in normal human bronchial smooth muscle cells (HBSMCs). Treatment of HBSMCs with 10% CSE induced cell death, and the death was accompanied by the generation of reactive oxygen species (ROS). CSE-induced cell death was reduced by N-acetyl-l-cysteine (NAC), an ROS scavenger. In addition, CSE reduced the migration ability of HBSMCs by 75%. The combination of NAC with CSE blocked the CSE-induced reduction of cell migration. However, CSE had no effect on TNF-α secretion and NF-κB activation. CSE induced an increase in intracellular Ca2+ concentration in 64% of HBSMCs. CSE reduced the contractile ability of HBSMCs, and the ability was enhanced by NAC treatment. These results demonstrate that CSE treatment induces cell death and reduces migration and contraction by increasing ROS generation in normal HBSMCs. These results suggest that CSE may induce airway change through cell death and reduction in migration and contraction of normal HBSMCs.