• Tuberculosis and Respiratory Diseases
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• 제43권2호
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• pp.123-127
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• 1996

Secretion of surfactant phospholipid can be stimulated by a variety of agonists acting via at least three different signal transduction mechanisms. These include the adenylate cyclase system with activation of cAMP-dependent protein kinase; activation of protein kinase C either directly or subsequent to activation of phosphoinositide-specific phospholipase C and generation of diacylglycerols and inositol trisphosphate; and a third mechanism that involves incresed $Ca^{2+}$ levels and a calmodulin-dependent step. ATP stimulates secretion via all three mechanisms. The protein kinase C pathway is also coupled to phopholipase D which, acting on relatively abundant cellular phospholipids, generates diacylglycerols that further activate protein kinase C. Sustained protein kinase C activation can maintain phosphatidylcholine secretion for a prolonged period of time. It is likely that interactions between the different signaling pathways have an important role in the overall physiological regulation of surfactant secretion.

• 한국미생물·생명공학회지
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• 제37권2호
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• pp.105-109
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• 2009

단백질 인산화를 통한 세포내 신호전달기구 중 serine/threonine kinase에 속하는 Akt/PKB는 세포 생존과 사멸, 당대사 등을 조절하는 것으로 알려져 있다. 이러한 이유로, Akt/PKB 단백질은 천연물질들로부터 항암제를 탐색하기 위한 한 가지 target으로 사용되어 왔다. 본 연구에서는 Akt/PKB 단백질을 대량으로 생산하기 위하여 대장균의 단백질 발현 시스템을 이용하여 human Akt/PKB 단백질을 발현시켰다. 대장균에서 대량 발현된 Akt는 일반적인 조건에서는 inclusion body를 형성하였다. 배양온도 $27^{\circ}C$에서 0.01-0.09 mM IPTG로 발현 유도 시 발현된 human Akt/PKB 단백질 상당 부분이 가용화 되었다. 발현된 Akt kinase를 $Ni^{2+}$-NTA agarose column으로 정제하고, anti-Akt antibody를 이용하여 정제된 단백질이 Akt kinase 임을 확인하였다. 정제된 human Akt/PKB 단백질은 세포추출물에 존재하는 인산화 단백질을 이용하여 in vitro에서 인산화 되었으며, 인산화된 활성형 human Akt/PKB protein kinase는 human Akt/PKB protein kinase 특이 형광 peptide를 특이적으로 인산화하였다.

• 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.

• Journal of Ginseng Research
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• 제20권3호
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• pp.219-225
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• 1996

This study was performed to investigate the inhibition mechanism of cancer cell proof iferation caused by the petroleum-ether extract of ginseng against human rectum (HRT-18), colon (HT-29), llepatoma (Hep G2) and prostate (LNCaP) cancer cells and monkey kidney cells (Vero 76). Cells were treated with the petroleum-ether extract of ginseng (50 to 200 $\mu\textrm{g}$/ml) in G1 or S phase of the cell cycle, and proliferation and protein kinase C activity were measured. The petroleum-eth or extract of ginseng inhibited proliferation of HRT-18, HT-29, Hep G2 and LNCaP when treated in Gl phase, but not in S phase. This result shows that the ginseng extract arrests the cell cycle in G1 phase, resulting in the inhibition of cell proliferation. At the same concentrations, treatment of the ginseng extract in G1 phase decreased protein kinase C activity, while the treatment in S phase had no effect. This reault suggests that protein kinase C might be involved in the inhibition of the cell cycle and proliferation of cancer cells caused by the petroleum-ether extract of ginseng.

• 한국의학물리학회지:의학물리
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• 제11권1호
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• pp.29-38
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• 2000

지금까지 각종 신경전달물질의 칼슘통로 억제 효과는 일반적으로 protein kinase 의 관여없이 G-protein mediated, membrane-delimited mechanism$^{1)}$ 으로 설명되어왔다. 그러나 최근들어 protein kinase C (PKC)의 활성화가 몇몇 신경전달물질에 의한 칼슘통로 억제효과를 야기하는 중요한 세포내 기전으로 보고되고 있다 그러므로 본 연구에서는 흰쥐 교감신경뉴론을 대상으로 하여 whole cell patch clamp technique을 사용하여 칼슘전류를 기록하고, 세포밖에 norepinephrine (NE)과 함께 PKC agonist 인 phorbol-12, 13-dibutyrate (PDBu)을 투여하면서 PDBu 전 처치로 인하여 NE 에 의한 칼슘전류 억제에 어떤 변화가 초래되는 지를 분석함으로써, 신경전달물질의 칼슘전류 억제효과시 PKC의 역할을 밝히고자 하였다. PDBu (500 nM) 처치는 칼슘전류의 크기를 증가시켰으며 이는 막전압 의존성을 보여 -10 mV ~ +10 mV 의 저분극 자극시 가장 크게 전류크기가 증가하였다. 또한 PDBu 처치는 tail current 의 deactivation을 느리게 하였다. PDBu 는 NE 에 의하여 활성화되는 pertussis toxin 예민성 G protein pathway를 통한 칼슘전류 억제를 감소시켰다. 비특이적인 protein kinase 길항제인 staurosporine (1 $\mu$M) 을 전처치 하고 PDBu를 투여하면 PDBu의 칼슘전류 크기 증가 효과가 소실되었으며 또한 NE에 의한 칼슘전류 억제를 해제하는 PDBu 의 조절효과도 소실되었다. 이상의 결과로부터 Protein Kinase C 가 활성되면 G protein을 경유하여 나타나는 칼슘전류 억제 효과가 소실된다고 결론지을 수 있다. Protein Kinase C 에 의하여 인산화되는 부위가 G-protein 인지 혹은 칼슘통로인 지에 관한 해답을 얻기 위하여는 추후 연구가 진행되어야 한다.

• Journal of Plant Biology
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• 제36권2호
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• pp.171-176
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• 1993

Protein kinase C, a protein related in PI cascade, was partially purified from the cytosol protein of etiolated plants of Glycine max by DEAE-52 cellulose chromatography and phenylsepharose chromatography. When the DEAE column was eluted with 0-0.8 M linear gradient KCl, tow fractions were found that increased the phosphorylation of histon H1 about five and nine-fold in the presence of 5 $\mu\textrm{g}$/mL phosphatidylserine and 0.5 $\mu\textrm{g}$/mL diolein, respectively. These fractions were used as DEAE pool. The reaction eluted with relatively high concentration of KCl was loaded on phyenylsepharose column with 5 mM CaCl2 and eluted with 1 mM EGTA. A fraction contained the protein kinase C, which increased the phosphorylation of the histon H1 was fractionated. To determine the molecular weight of PKC, the fraction eluted from phenylsepharose column was analyzed by 5~15% polyacrylamide gel electrophoresis after concentrated with the Amicon membrane (YM10). That revealed two bands corresponding to 60 and 65 kGy by silver staining of the gel, respectively.

• 한국동물학회지
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• 제32권1호
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• pp.40-47
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• 1989

계배 근원세포의 배양 배지에 calcium ionophore A23187이나 EGTA를 배양 24시간에 첨가함으로서 초래된 세포내 칼슘 농도의 변화는 근원세포의 분화과정에 상당한 영향을 미쳤다. 배양 24시간에 A23187이나 EGTA를 첨가한 후 배양 48시간, 72시간, 및 96시간에 각각 세포를 [35S]methionine으로 1시간 표지시킨 후 수확하여 2차원 전기영동법으로 단백질을 분리시켰을 때, 일부 단백질은 배양 조건에 따라 합성 양상을 달리함을 보였다. 배양 24시간에 처리한 A23187과 calcium-activated neutral protease는 대조군에 비해 세포융합을 촉진시켰으나 동일 시기에 처리된 phosphoprotein을 정량함으로써 조사하였을 때, A23187이 배양 초기에는 대조군에 비해 약간 이 효소의 활성도를 높이는 효과를 보였으나 세포융합이 완성된 시기인 96시간에는 대조군에 비해 활성도를 높이는 효과를 보였으나 세포융합이 완성된 시기인 96시간에는 대조군에 비해 활성도의 차이를 나타내지 않았다. A23187 및 calcium-activated neutral protease에 의한 세포융합의 촉진, 그리고 A23187에 의한 protein kinase C 활성도의 증가가 모두 근원세포의 융합이 활발히 진행되는 시기인 배양 48-72 시간에 관찰됨을 볼 때, 세포내 칼슘의 농도는 protein kinase C 및 calcium-activated neutral protease와 상호연관을 가지면서 세포분화에 관여하는 것으로 사료된다.

• BMB Reports
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• 제34권6호
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• pp.517-525
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• 2001

Six renaturable protein kinases that utilize the myelin basic protein (MBP) as a substrate were activated during prolonged exposure of cardiac myocytes to okadaic acid (OA). We characterized the substrate preference and activation of these kinases, with particular emphasis on 3 novel kinases-MBPK-55, MBPK-62 and MBPK-87. The transcription factors c-Jun, Elk, ATF2, and c-Fos that are used to assess mitogen-activated protein kinase activation were all poor substrates for these three kinases. MAPKAPK2 was also not phosphorylated. In contrast, Histone IIIS was phosphorylated by MBPK-55 and MBPK-62. These protein kinases were activated in cultured cardiac fibroblasts, H9c2 cardiac myoblasts, and Cos cells. High concentrations (0.5 to $1\;{\mu}M$) of OA were essential for the activation of the protein kinases in all of the cell types examined, whereas calyculin A [an inhibitor of protein phosphatase 1 (PP1) and PP2A], cyclosporin A (a PP2B inhibitor), and an inactive OA analog all failed to activate these kinases. The high dose of okadaic acid that is required for kinase activation was also required for phosphatase inhibition, as assessed by immunoblotting whole cell lysates with anti-phosphothreonine antibodies. A variety of chemical inhibitors, including PD98059 (MEK-specific), genistein (tyrosine kinase-specific) and Bisindolylmaleimide I (protein kinase C-specific), failed to inhibit the OA activation of these kinases. Thus, MBPK-55 and MBPK-62 are also Histone IIIS kinases that are widely expressed and specifically activated upon exposure to high OA concentrations.

• 대한의생명과학회지
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• 제2권1호
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• pp.21-30
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• 1996

Mitogen-activated protein (MAP) kinase는 여러 세포증식 촉진인자들에 의하여 자신이 인산화됨으로써 활성화되어 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, 44 kD와 66kD의 isoform을 확인할 수 있었다. 면역 T 세포의 $p56^{kk}$의 N-terminal로부터 유전자 재조합 방법을 통하여 glutathion-s-transferase(GST) fusion protein을 얻은 후 분리한 MAP kinase의 기질로 사용하여 본 결과, wild type과 mutant간에 인산화 정도의 차이를 확인할 수 있어 MAP kinase의 또 다른 기질로 이용할 수 있는 가능성을 제시하였다.

• Journal of Nutrition and Health
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• 제27권5호
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• pp.419-428
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• 1994

To investigate the effect of dietary menhaden oil on protein kinase C (PKC) activity and membrane phospholipid composition in epidermal cells, female BALB/C mice were fed either menhaden oil or corn oil with two different levels(5% or 20%) for 6 weeks. Membrane phosphatidycholine(PC) was decreased in menhaden oil-fed group. Eicosapentaenoic acid(EPA) and Docosahexaenoic acid(DHA) were only presented in the acyl chain of membrane phospholipid of menhaden oil-fed mice, so that membrane fluidity of the group could be different from the other group. Both cytosolic and membrane-associated PKC activity in epidermal cells were decreased in menhaden oil-fed mice when compare with corn oil-fed mice. Furthermore, rate of PKC transfer from cytosol to membrane in menhaden oil-fed group was not as fast as in corn oil-fed group. Based on these observations, dietary menhaden oil might act differently from other dietary fat in carcinogenesis.