• 약학회지
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• 제45권3호
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• pp.287-292
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• 2001

To investigate whether phospholipase $A_2$pathway is involved in histamine release of rat peritoneal mast cells, we measured histamine release in the presence of various enzyme inhibitors involved in eicosanoid pathway, such as phospholipase $A_2$, cyclooxygenase and lipoxygenase. Phospholipase $A_2$inhibitors, manoalide and OPC, significantly inhibited histamine release induced by 100 $\mu$M ATP and 1$\mu$g/ml compound 48/80. Cyclooxygenase inhibitors, ibuprofen and indomethacin, significantly inhibited ATP-induced histamine release and lipoxygenase inhibitors, baicalein and caffeic acid, also significantly inhibited. To investigate the involvement of protein kinase in ATP- and compound 48/80-induced histamine release, we observed effects of protein kinase inhibitors on histamine release. Bisindolmaleimide (protein kinase C antagonist) dose-dependently inhibited both ATP and compound 48/80-induced histamine release. Tyrosine kinase inhibitors (methyl 2,5-dihydroxy cinnamate and genistein) dose-dependently inhibited ATP and compound 48/80-induced histamine release. Protein kinase C and tyrosine kinase seem to be involved in histamine release induced by ATP and compound 48/80. These results suggest that phospholipase $A_2$pathway as well as protein kinase C and tyrosine kinase are involved in histamine release of rat peritoneal mast cells by ATP and compound 48/80.

• BMB Reports
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• 제39권5호
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• pp.469-478
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• 2006

Vascular endothelial growth factor (VEGF)-A, a major regulator for angiogenesis, binds and activates two tyrosine kinase receptors, VEGFR1 (Flt-1) and VEGFR2 (KDR/Flk-1). These receptors regulate physiological as well as pathological angiogenesis. VEGFR2 has strong tyrosine kinase activity, and transduces the major signals for angiogenesis. However, unlike other representative tyrosine kinase receptors which use the Ras pathway, VEGFR2 mostly uses the Phospholipase-$C{\gamma}$-Protein kinase-C pathway to activate MAP-kinase and DNA synthesis. VEGFR2 is a direct signal transducer for pathological angiogenesis including cancer and diabetic retinopathy, thus, VEGFR2 itself and the signaling appear to be critical targets for the suppression of these diseases. VEGFR1 plays dual role, a negative role in angiogenesis in the embryo most likely by trapping VEGF-A, and a positive role in adulthood in a tyrosine kinase-dependent manner. VEGFR1 is expressed not only in endothelial cells but also in macrophage-lineage cells, and promotes tumor growth, metastasis, and inflammation. Furthermore, a soluble form of VEGFR1 was found to be present at abnormally high levels in the serum of preeclampsia patients, and induces proteinurea and renal dysfunction. Therefore, VEGFR1 is also an important target in the treatment of human diseases. Recently, the VEGFR2-specific ligand VEGF-E (Orf-VEGF) was extensively characterized. Interestingly, the activation of VEGFR2 via VEGF-E in vivo results in a strong angiogenic response in mice with minor side effects such as inflammation compared with VEGF-A, suggesting VEGF-E to be a novel material for pro-angiogenic therapy.

• IMMUNE NETWORK
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• 제6권3호
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• pp.117-122
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• 2006

Background: Caveolin, a family of integral membrane proteins are a principal component of caveolae membranes. In this study, we investigated the effect of p38 kinase on differentiation and on inflammatory responses in sodium nitroprusside (SNP)-treated chondrocytes. Methods: Rabbit articular chondrocytes were prepared from cartilage slices of 2-week-old New Zealand white rabbits by enzymatic digestion. SNP was used as a nitric oxide (NO) donor. In this experiments measuring SNP dose response, primary chondrocytes were treated with various concentrations of SNP for 24h. The time course of the SNP response was determined by incubating cells with 1mM SNP for the indicated time period $(0{\sim}24h)$. The cyclooxygenase-2 (COX-2) and type II collagen expression levels were determined by immunoblot analysis, and prostaglandin $E_2\;(PGE_2)$ assay was used to measure the COX-2 activity. The tyrosine phosphorylation of caveolin-1 was determined by immunoblot analysis and immunostaining. Results: SNP treatment stimulated tyrosine phosphorylation of caveolin-1 and activation of p38 kinase. SNP additionally caused dedifferentiation and inflammatory response. We showed previously that SNP treatment stimulated activation of p38 kinase and ERK-1/-2. Inhibition of p38 kinase with SB203580 reduced caveolin-1 tyrosine phosphorylation and COX-2 expression but enhanced dedifferentiation, whereas inhibition of ERK with PD98059 did not affect caveolin-1 tyrosine phosphorylation levels, suggesting that ERK at least is not related to dedifferentiation and COX-2 expression through caveolin-1 tyrosine phosphorylation. Conclusion: Our results indicate that SNP in articular chondrocytes stimulates dedifferentiation and inflammatory response via p38 kinase signaling in association with caveolin-1 phosphorylation.

• BMB Reports
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• 제34권1호
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• pp.33-38
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• 2001

The human PTK6 (also known as Brk) polypeptide, which is deduced from its full-length cDNA, represents a non-receptor protein tyrosine kinase (PTK). It contains SH3, SH2, and tyrosine kinase catalytic domains that are closely related to Src family members. We generated an antihuman PTK6 antibody by immunizing rabbits with a PTK6-specific oligopeptide conjugated to BSA, which corresponds to 11 amino acid residues near the C-terminus. An immunoblot analysis with the antibody detected an expected 52-kDa band in various mammalian transformed cell lines. Immunoprecipitation and immunoblot analyses demonstrated that PTK6 is phosphorylated on the tyrosine residues) and interacts with approximately a 23-kDa tyrosine-phosphorylated polypeptide (most likely a substrate of PTK6) in breast carcinoma T-47D cells. An immunofluorescence analysis demonstrated that PTK6 is localized throughout the cytoplasm of T-47D cells. These results support a possible role for PTK6 in the intracellular signal transduction through tyrosine phosphorylation.

• The Korean Journal of Physiology and Pharmacology
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• 제4권3호
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• pp.211-217
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• 2000

Paxillin is a regulatory component of the complex of cytoskeletal proteins that link the actin cytoskeleton to the plasma membrane. However, the role of paxillin during smooth muscle contraction is unclear. We investigated a possible role for the membrane-associated dense plaque protein paxillin in the regulation of contraction in rat aortic vascular smooth muscle. The tyrosine phosphorylation of paxillin, which was increased by norepinephrine, reached a peak level after 1 min stimulation and then decreased with time. However, norepinephrine induced a sustained contraction that reached a steady state 30 min after application. Pretreatment with tyrphostin, an inhibitor of tyrosine kinase, inhibited the tyrosine phosphorylation of paxillin and also the contraction stimulated by norepinephrine. Both inhibitions were concentration-dependent, and the degree of correlation between them was high. These results show that, in rat aortic smooth muscle, tyrosine kinase(s) activated by norepinephrine may phosphorylate the tyrosine residues of paxillin, thereby providing a source of regulation during vascular smooth muscle contraction.

• 한국미생물·생명공학회지
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• 제46권2호
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• pp.91-101
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• 2018

The aim of this study was to transfer the 18.5 kb gene clusters coding for 17 genes from Lactobacillus rhamnosus to Lactococcus lactis subsp. cremoris MG1363 in order to determine the effect of host on exopolysaccharide (EPS) production and to provide a model for studying the phosphorylation of proteins which are proposed to be involved in EPS polymerization. Lactobacillus rhamnosus RW-9595M and ATCC 9595 have 99% identical operons coding for EPS biosynthesis, produced different amounts of EPS (543 vs 108 mg/l). L. lactis subsp. cremoris MG1363 transformed with the operons from RW-9595M and ATCC 9595 respectively, produced 326 and 302 mg/l EPS in M17 containing 0.5% glucose. The tyrosine protein kinase transmembrane modulator (Wzd) was proposed to participate in regulating chain elongation of EPS polymers by interacting with the tyrosine protein kinase Wze. While Wzd was found in phosphorylated form in the presence of the phosphorylated kinase (Wze), no phosphorylated proteins were detected when all nine tyrosines of Wzd were mutated to phenylalanine. Lactococcus lactis subsp. cremoris could produce higher amounts of EPS than other EPS-producing lactococci when expressing genes from L. rhamnosus. Phosphorylated Wzd was essential for the phosphorylation of Wze when expressed in vivo.

• Archives of Pharmacal Research
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• 제29권11호
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• pp.1006-1017
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• 2006

A series of 3-(substituted-benylidene)-1, 3-dihydro- indolin-2-one, 3-(substituted-benylidene)-1, 3-dihydro- indolin-2-thione and 2, 2'-dithiobis 3-(substituted-benylidene)-1, 3-dihydro-indole derivatives was investigated as inhibitor of $p60^{c-Src}$tyrosine kinase by performing receptor docking studies and inhibitory activity toward tyrosine phosphorylation. Some compounds were shown to be docked at the site, where the selective inhibitor PP1 [1-tert-Butyl-3-p-tolyl-1H-pyrazolo[3,4-d]pyrimidine-4-yl-amine] was embedded at the enzyme active site. Evaluation of all compounds for the interactions with the parameters of lowest binding energy levels, capability of hydrogen bond formations and superimposibility on enzyme active site by docking studies, it can be assumed that 3-(substituted-benzylidene)-1, 3-dihydro-indolin-2-one and thione derivatives have better interaction with enzyme active site then 2, 2'-dithiobis 3-(substituted-benzylidene)-1, 3-dihydro indole derivatives. The test results for the inhibitory activity against tyrosine kinase by Elisa method revealed that 3-(substituted-benylidene)-1, 3-dihydro- indolin-2-thione derivatives have more activity then 3-(substituted-benylidene)-1, 3-dihydro- indolin-2-one derivatives.

• Tuberculosis and Respiratory Diseases
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• 제75권6호
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• pp.236-237
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• 2013

Many attempts have been made to find genetic abnormalities inducing carcinogenesis after the development of epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor targeting EGFR in lung cancer. New target therapies have been already commercialized and studied along with the recent discovery of gene rearrangement involved in the carcinogenic process of non-small cell lung cancer. This study aims to investigate anplastic lymphoma kinase, c-ros oncogene 1, and receptor tyrosine kinase, in particular.

• 대한의생명과학회지
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• 제2권2호
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• pp.175-185
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• 1996

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. P388세포를 10% fetal bovine serum이 첨가된 DMEM배지에 배양한 후, 혈청이 들어있지 않은 배지에서 24시간 더 배양하고 serum 및 PMA를 농도별로 처리하여 세포성 장을 위한 최적 농도를 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 파쇄한후 세포질분획과 핵분획으로 분리하여 각 분획을 10% gel 상에서 전기영동 하여 nitrocellulose paper에 옳긴 후 anti-ERKI 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 기질인산화 실험을 위해 serum으로 활성화시킨 세포를 파쇄하여 SP-sephadex C-50, Phenyl superose, Mono Q column의 순서로 chromatography를 시 행하여 MAP kinase를 부분분리 하였다. 이와 같이 얻은 MAP kinase를 가지고 면역 T세포에 존재하는 tyrosine kinase인 $p56^{lck}$ 의 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 P388세포는 (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 추측되 며 (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 Korean Journal of Physiology and Pharmacology
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• 제1권5호
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• pp.547-553
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• 1997

It has been known that activation of tyrosine kinases is involved in signal transduction. Role of the tyrosine kinase in vascular smooth muscle contraction was examined in deoxycorticosterone acetate (DOCA)-salt hypertensive rats. Male Sprague-Dawley rats underwent uninephrectomy, one week after which they were subcutaneously implanted with DOCA (200 mg/kg) and supplied with 1% NaCl and 0.2% KCl drinking water for $4{\sim}6$ weeks. Control rats were treated the same except for that no DOCA was implanted. Helical strips of carotid arteries were mounted in organ baths for measurement of isometric force development. Genistein was used as a tyrosine kinase inhibitor. Concentration-response curves to 5-hydroxytryptamine (5-HT) shifted to the right by genistein in both DOCA-salt hypertensive and control rats. Although the sensitivity to genistein was similar between the two groups, the maximum force generation by 5-HT was less inhibited by genistein in arteries from DOCA-salt hypertensive rats than in those from controls. Genistein-induced relaxations were attenuated in arteries from DOCA-salt rats. Genistein affected the contraction to phorbol 12, 13-dibutyrate (PDBu) neither in DOCA-salt nor in control arteries. These observations suggest that tyrosine kinase is involved in 5-HT-induced vascular contraction, of which role is reduced in DOCA-salt hypertension.