핵 내에서 분리한 Mitogen-Activated Protein (MAP) Kinase의 Transcription Factor에 대한 인산화

김태우,김소영,김윤석 THE KOREAN SOCIETY FOR BIOMEDICAL LABORATORY SCIEN 1996 Journal of biomedical laboratory sciences Vol.2 No.2

모든 진핵세포에 존재하며 세포의 성장 및 분화에 주로 관계되는 신호전달물질의 하나인 Mitogen-activated protein(MAP) kinase의 mitogen에 의한 핵내 활성화와 기질 인산화에 대해 알아보기 위해 본 실험을 수행하였다. P388 세포를 10% fetal bovine serum이 첨가된 DMEM 배지에 배양한 후, 혈청이 들어있지 않은 배지에서 24시간 더 배양하고 serum 및 PMA를 농도별로 처리하여 세포성장을 위한 최적농도를 확인한 결과 serum은 5-20% 농도에서 세포성장을 촉진시켰고 PMA는 실험한 모든 농도에서 세포성장을 거의 촉진시키지 못하는 경향을 확인하였다. 이어 P388 세포를 serum 및 PMA로 10 분간 활성화하여 파쇄한 후 세포질분획과 핵분획으로 분리하여 각 분획을 10% gel상에서 전기영동 하여 nitrocellulose paper에 옮긴 후 anti-ERK1 antibody를 이용해 확인해본 결과 serum, PMA로 처리된 세포 모두에서 MAP kinase의 핵내 이동이 관찰되었으며 특히 세포질 내에 주로 존재하는 42, 44 Kd의 MAP kinase isoform중 42 Kd의 isoform이 주로 핵내로 이동되는 것이 관찰되었다. MAP kinase의 기질인산화실험을 위해 serum으로 활성화시킨 세포를 파쇄하여 SP-sephadex C-50, Phenyl superose, Mono Q column의 순서로 chromatography를 시행하여 MAP kinase를 부분분리 하였다. 이와 같이 얻은 MAP kinase를 가지고 면역 T 세포에 존재하는 tyrosine kinase인 p56lck의 N-terminal peptide로 구성된 GST-fusion protein에 대한 인산화를 확인하였다. 또한 세포에서 분리한 MAP kinase를 가지고 transcription factor의 하나인 c-Jun protein에 대한 인산화실험을 실시한 결과 MAP kinase에 의해 인산화 됨이 확인되었다. 이상의 결과를 통해 P388 세포는 (1)세포 성장시 외부 신호를 G-protein-coupled receptor/protein kinase C/MAP kinase의 경로보다는 주로 tyrosine kinase receptor protein/Ras/MAP kinase의 경로를 이용하여 핵으로 전달하는 것으로 추측되며 (2) mitogen의 처리로 활성화된 MAP kinase중 주로 42 Kd isoform이 핵내로 이동하고, 분리한 MAP kinase가 GST-fusion protein과 transcription factor인 c-Jun을 모두 인산화 시키는 결과로 보아 MAP kinase의 isoform에 따라 표적 compartment가 다르고 결과적으로 표적 기질에 차이가 있을지 모른다고 간접적으로 추론할 수 있다. The mitogen-activated protein(MAP) kinase signal transduction pathway represents an important mechanism by which mitogen, such as serum and PMA, regulate cell proliferation and differentiation. Target substrates of the MAP kinase are located within several compartments containing plasma membranes and nucleus. We now report that serum addition induces proliferation of the P388 murine leukemia cell, but PMA does not, while both serum and PMA treatment cause translocation of the MAP kinase, mainly p42mapk isoform, from cytosol into the nucleus, which was monitored by immunoblot analysis using polyclonal anti-ERK1 antibodies. We investigated whether the MAP kinase was capable of phosphorylating c-Jun protein and GST-fusion proteins, the P56lckN-terminal peptides (1-77 or 1-123 domain) of the T cell tyrosine kinase, using the partially purified MAP kinase by SP-sephadex C-50, phenyl superose and Mono Q column chromatography. We found that the partially purified MAP kinase was able to phosphorylate c-Jun protein and the GST-fusion protein expressed using E.coli DH5α which is transformed with pGEX-3Xb plasmid vector carrying of p56lck N-terminal peptide-encoding DNA. These results imply that tyrosine kinase receptor/Ras/Raf/MAP kinase pathway is a major mechanism for mitogen-induced cell proliferation in P388 murine leukemia cell and that the various MAP kinase isoforms may have their own target substrates located in distinct subcellular compartments.

Salmonella sp.의 신속한 동정을 위한 증진배양의 개선에 관한 연구

김기태,김태우,옥순학,이영호,백운화 한국미생물생명공학회 ( 구 한국산업미생물학회 ) 1996 한국미생물·생명공학회지 Vol.24 No.6

식품 및 생활폐수내의 존재하는 Salmonella spp.에 대한 효율적이고 신속한 동정을 위한 증진배야업을 개발하고자 하였다. 본 연구에 사용된 균주인 S. enteritidis 생육을 촉진시키기 위한 방법으로 cAMP 및 yeast extract를 사용하였는데 전자인 경우는 배지내의 농도가 10 mM 이상일 때 10^-3 cfu/ml의 균수로 7시간 배양후 균수가 control보다 약5배의 증가를 보였으며 후자인 경우는 0.6% 첨가시 10배의 증가를 보였다. 다른 균주들에 대한 선택적인 성장효과를 보기위하여 selenite broth와 bile salts를 사용하였고 이때 사용된 균주는 Staphylococcus aureus, Pesudomonas aeruginosa, Lactobacillus plantarum 및 Escherichia coli이었고 bile salts의 농도가 0.1%일때 네 가지 균주의 증식에 대한 억제 효과가 있었다. 두 단계의 증진배양법으로서 1차 증진배양에서는 selenite broth에 0.6% yeast extract를 첨가한 것으로 2차 증진배양에서는 0.1% bile salts를 첨가한 것으로 하였는데 타균과의 혼합배양에서 Salmonella의 초기균수가 10^0.3일 때 14시간 증진배양으로 10^8.5 cfu/ml까지 증식을 보였으며 초기균수가 1 cfu/100 ml인 경우는 10시간의 1차 증진배양과 6시간의 증진배양으로 약 10^7의 증식속도를 나타내었다. The development of an enrichment method for the rapid and effective identification of Salmonella spp. in sewage or food was studied. As a growth factor for Salmonella, 10 mM cyclic adenosine monophosphate (cAMP) in trypticase soy broth with 0.6% yeast extract (TSBYE) increased cell number five-folds and 0.6% yeast extract in selenite broth increased cell number ten-folds of control. Bile salts in slenite broth was tested for the selection of S. enteritidis in a mixture with Staphylococcus aureus, Pesudomonas aeruginosa, Lactobacillus plantarum and Escherichia coli. The latter four strains were effectively inhibited at 0.1% bile salt. A two-step culture method was used to enrich Salmonella spp.; a primary-enrichment and secondary-enrichment culture. At a primary-enrichment step, selenite broth with 0.6% yeast extract and 10 mM cAMP was used, and at a secondary-enrichment step, -0.1% bile salt was additionally used. Culture times of a primary-enrichment and a secondary-enrichment step were 8 hr and 6 hr, respectively. In this procedure, cell number increased from 10^0.3 to 10^8.5 with inhibition of other strains within 14 hr. In the case of an initial cell concentrarion as low as 10^-2 cfu/ml, a cell number increased to 10^7 cfu/ml by using a 10 hr primary-enrichment and 6 hr secondary-enrichment procedure.

동물 조직세포로부터 Mitogen-activated Protein (MAP) Kinase의 분리 및 성격규명

김태우,김윤석,정동주 THE KOREAN SOCIETY FOR BIOMEDICAL LABORATORY SCIEN 1996 Journal of biomedical laboratory sciences Vol.2 No.1

Mitogen-activated protein (MAP) kinase는 여러 세포증식 촉진인자들에 의하여 자신이 인산화됨으로써 할성화되어 다른 protein kinase를 인산화시키는 역할을 하는 세포내 신호전달의 중요한 효소이다. 본 연구에서는 P388 murine leukemia 세포 파쇄액에서 SP sephadex C-50, phenyl superose, Mono Q column을 통하여 MAP kinase를 분리한 결과, 44kD 와 66kD의 isoform을 확인할 수 있었다. 면역 T 세포의 P56kk의 N-terminal로 부터 유전자 재조합 방법을 통하여 glutathion-s-transferase(GST) fusion protein을 얻은 후 분리한 MAP kinase의 기질로 사용하여 본 결과, wild type과 mutant 간에 인산화 정도의 차이를 확인할 수 있어 MAP kinase의 또다른 기질로 이용할 수 있는 가능성을 제시하였다. MAP kinases are a family of serine/threonine specific protein kinases becoming activated in response to different proliferative stimuli by phosphory lation at both threonine and tyrosine residue. Present study shows that MAP kinase was purified from P388 murine leukema cells by SP sephadex C-50, phenyl superose and Mono Q column chromatography and identified with anti-ERK1 antibody by western blotting. Immublotting analysis to the crude extract of P388 cell lysate shows 44 kD and other minor bands but partial purified fraction eluted from phenyl supherose column have 44 kD and 66 kD isoform. Subcloned GST-fusion protein from N-terminal of p56kk was tested as a substrate for MAP kinase phosphorylation. It was showed that the wild type and mutant forms(S42A) were fully phosporylated by purified MAP kinase fraction as compare with the other mutant from(S59A). This finding suggest that those GST-fusion proteins may be used as substrate for the in vitro test of MAP kinase.

Cytotoxic and Apoptotic Activites of Echinomycin Derivative (Echinomycin-7) on P388 Murine Leukemia Cells

Kim, Tae-Ue,Jeon, Hyang,Kim, Sung-Su,Kim, Yoon-Suk,Park, Yil-Sung,Kim, Yong-Hae,Choi, Sun-Ju,Kim, Soo-Kie The Korea Science and Technology Center 1998 BMB Reports Vol.31 No.6

Echinomycin-7 is an echinomycin derivative, S-methylated sulfonium perchlorate of echinomycin. We studied the in vitro cytotoxicity and in vivo antitumor activity of echinomycin-7 against P388 leukemia cells and compared the results with echinomycin. With respect to the cytotoxic effects, echinomycin-7 had cell line-dependent IC50 values while echinomycin had similar values to several tumor cell lines. Also, in vivo antitumor activities were observed in tumor-bearing mice treated with both agents, which showed that echinomycin-7 had a broad therapeutic dose range. We also observed the apoptosis on leukemia cells treated with echinomycin-7 which exihibited the ladder pattern of DNA on electrophoresis. In addition to apoptosis, echinomycin-7 arrested G₁/S phases of the cell cycle at the same time. We then examined the signaling pathway of echinomycin-7-induced apoptosis and showed that ERK of the MAP kinase family was activated and translocatd into the nucleus by echinomycin-7 stimulation. This study suggests that echinomycin-7 acts as an antitumor agent through in vitro cytotoxicity and has in vivo antitumor activity against leukemia cells, and that the echinomycin-7-induced apoptosis might involve signal transduction via MAP kinases.

Taxol-Induced Apoptosis and Nuclear Translocation of Mitogen-Activated Protein (MAP) Kinase in HeLa Cells

Kim, Tae-Ue,Kim, Sung-Su,Kim, Yoon-Suk,Jung, Yon-Woo,Choi, Hyun-Il,Shim, Moon-Jeong The Korea Science and Technology Center 1999 BMB Reports Vol.32 No.4

Taxol, a natural product with significant anti-tumor activity, stabilizes microtubules and arrests cells in the G2/M phase of the cell cycle. It has been reported that taxol has additional effects on the cell such as an increase in tyrosine phosphorylation of proteins and activation of mitogen-activated protein (MAP) kinase. This phosphorylated kinase translocates into the uncleus and phosphorylates its substrate c-jun, c-fos, ATF2, and ATF3. The MAP kinase family is comprised of key regulatory proteins that control the cellular response to both proliferation and stress signals. First examination was cytotoxicity and apoptosis-induced concentration with paclitaxel in HeLa cell. A half-maximal inhibition of cell proliferation (IC?) occurred at 13 nM paclitaxel. When DNA fragmentation was analyzed by agarose gel electrophoresis, a nucleosomal ladder became evident 24 h after a taxol (50 nM) addition to the cells. In addition, an apoptotic body was detected by electron microscopy. Taxol-treated cells were arrested at the S phase at 10 nM. Treatment of 50 nM taxol activated the extracellular signal-regulated protein kinase (ERK1), and a fraction of the activated MAP kinases entered the nucleus. It was also discovered that nucleus substrates c-jun was phosphorylated and activated in the cell. The activated ERK1 could subsequently translocate into the nucleus and phosphorylate its substrate c-jun as well. This study suggests that taxol-induced apoptosis might be related with signal transduction via MAP kinases.

Cytotoxic and Apoptotic Effects of Echinomycin on Murine Leukemia Cells

Kim, Tae-Ue,Yang, Se-Hwan,Kim, Soo-Kie Korean Society for Biochemistry and Molecular Biol 1996 Journal of biochemistry and molecular biology Vol.29 No.6

A number of anticancer-chemotherapeutic agents induce cell death through the process of apoptosis. Effects of echinomycin, an anticancer agent on cancer progression, were investigated in P388 murine leukemia cells. First, according to the results of cytotoxicity measurement. $IC_{50}$ of echinomycin was 1.12 nM, a relatively lower value than the other examined anticancer agents, mitomycin-C and etoposide Second, the DNA fragmentation assay for echinomycin-treated cells exhibited that echinomycin was able to induce apoptosis in a shorter period of time and with a lower dose than mitomycin-C or etoposide. The data of DNA fragmentation were quite comparable to those of cytotoxicity measurement. Finally we showed that mitogen-activated protein (MAP) kinase, a key protein in cell mitosis, was translocated into the nucleus from the cytosol after treatment with echinomycin. These findings suggest that a MAP kinase-related process may be involved in apoptosis induced by echinomycin.

Neuroprotective effects of 2-cyclopropylimino-3-methyl-1,3-thiazoline hydrochloride against oxidative stress.

Kim, Hanwook,Son, Hyo Jeong,Ha, Seung Cheol,Kim, Eun-A,Kim, Tae Ue,Choi, Soo Young,Ahn, Jee-Yin,Cho, Sung-Woo Kluwer Academic/Plenum Publishers 2011 Cellular and molecular neurobiology Vol.31 No.7

<P>Oxidative stress, glutamate excitotoxicity, and inflammation are the important pathological mechanisms in neurodegenerative diseases. Recently, we reported that 2-cyclopropylimino-3-methyl-1,3-thiazoline hydrochloride protects rat glial cells against glutamate-induced excitotoxicity. In this study, we report the effects of 2-cyclopropylimino-3-methyl-1,3-thiazoline hydrochloride on primary cultured cortical astrocytes after exposure to hydrogen peroxide (H2O2). Pretreatment of cells with 2-cyclopropylimino-3-methyl-1,3-thiazoline hydrochloride prior to H2O2 exposure attenuated the H2O2-induced reductions in cell survival and superoxide dismutase, catalase, glutathione, and glutathione peroxidase activities. It also reduced H2O2-induced increases in reactive oxygen species levels, malondialdehyde content, and production of nitric oxide. These effects were all concentration-dependent. Our results suggest that 2-cyclopropylimino-3-methyl-1,3-thiazoline hydrochloride protects against oxidative stress.</P>

Reaction Mechanism of Thymidylate Syntase from Lactobacillus casei

Kim, Tae Ue,Lee, Jongweon,Cho, Sung Woo 연세대학교 보건과학연구소 1994 보건과학논집 Vol.4 No.-

Cytotoxic and Apoptotic Effects of Echinomycin on Murine Leukemia Cells

Kim, Tae Ue,Yang, Se Hwan,Kim, Soo Kie 생화학분자생물학회 1982 BMB Reports Vol.29 No.6

A number of anticancer-chemotherapeutic agents induce cell death through the process of apoptosis. Effects of echinomycin, an anticancer agent on cancer progression, were investigated in P388 murine leukemia cells. First, according to the results of cytotoxicity measurement, IC_(50) of echinomycin was 1.12 nM, a relatively lower value than the other examined anticancer agents, mitomycin-C and etoposide. Second, the DNA fragmentation assay for echinomycin-treated cells exhibited that echinomycin was able to induce apoptosis in a shorter period of time and with a lower dose than mitomycin-C or etoposide. The data of DNA fragmentation were quite comparable to those of cytotoxicity measurement. Finally we showed that mitogen-activated protein (MAP) kinase, a key protein in cell mitosis, was translocated into the nucleus from the cytosol after treatment with echinomycin. These findings suggest that a MAP kinase-related process may be involved in apoptosis induced by echinomycin.

Taxol-Induced Apoptosis and Nuclear Translocation of Mitogen-Activated Protein (MAP) Kinase in HeLa Cells

Kim, Yoon Suk,Kim, Tae Ue,Kim, Sung Su,Jung, Yon Woo,Choi, Hyun il,Shim, Moon Jeong 생화학분자생물학회 2000 BMB Reports Vol.32 No.4

Taxol, a natural product with significant anti-tumor activity, stabilizes microtubules and arrests cells in the G2/M phase of the cell cycle. It has been reported that taxol has additional effects on the cell such as an increase in tyrosine phosphorylation of proteins and activation of mitogen-activated protein (MAP) kinase. This phosphorylated kinase translocates into the nucleus and phosphorylates its substrate c-jun, c-fos, ATF2, and ATF3. The MAP kinase family is comprised of key regulatory proteins that control the cellular response to both proliferation and stress signals. First examination was cytotoxicity and apoptosis-induced concentration with paclitaxel in HeLa cell. A half-maximal inhibition of cell proliferation (IC_(50)) occurred at 13 nM paclitaxel. When DNA fragmentation was analyzed by agarose gel electrophoresis, a nucleosomal ladder became evident 24 h after a taxol (50 nM) addition to the cells. In addition, an apoptotic body was detected by electron microscopy. Taxol-treated cells were arrested at the S phase at 10 nM. Treatment of 50 nM taxol activated the extracellular signal-regulated protein kinase (ERKl), and a fraction of the activated MAP kinases entered the nucleus. It was also discovered that nucleus substrates c-jun was phosphorylated and activated in the cell. The activated ERK1 could subsequently translocate into the nucleus and phosphorylate its substrate c-jun as well. This study suggests that taxol-induced apoptosis might be related with signal transduction via MAP kinases.