• 한국동물학회지
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• 제38권2호
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• pp.286-293
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• 1995

The present studies were undertaken to examine the activitites of PKC isoforms in cultures of chick limb mesenchvme. Micromass cultures were prepared using wing buds of stage 23/24 (Hamburger and Hamilton, 19511 chick embryo. The cells were homogenized and DEAE-cellulose column chromatography was performed to get fraction containing protein kinase C (PKC) activity. PKC isoforms were resolved with hvdroxyapatitie column chromatography. Profile of PKC isoforms of cultures were compared with that of rat brain. Activity of $PKC-\beta$ isoform was appeared at the early stage of chondrogenesis. On 3 daw of culture, activities of both PKC a and $\beta$ were observed with remarkable increase but no activity of y isoform was appeared. Treatment of phorbol-12-mvristate-13-acetate (PMA) (10-7 M) to the culture inhibited chondrosenesis and down-regulated a and $\beta$ isoforms. Staurosporine promoted chondro!genesis without any effect on PKC isioforms profile. These data indicate that PKC a and $\beta,$ especiallv $\beta$ isoform is related to chondrosenesis and the promoting effect of staurosporine on chondrogenesis is not related to PKC isoforms activities.

• Bulletin of the Korean Chemical Society
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• 제30권9호
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• pp.1981-1984
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• 2009

Subcellular localization of protein kinase often plays an important role in determining its activity and specificity. Protein kinase C (PKC), a family of multi-gene protein kinases has long been known to be translocated to the particular cellular compartments in response to DAG or its analog phorbol esters. We used C-terminal green fluorescent protein (GFP) fusion proteins of PKC isoforms to visualize the subcellular distribution of individual PKC isoforms. Intracellular localization of PKC-GFP proteins was monitored by fluorescence microscopy after transient transfection of PKC-GFP expression vectors in the HeLa cells. In unstimulated HeLa cells, all PKC isoforms were found to be distributed throughout the cytoplasm with a few exceptions. PKC$\theta$ was mostly localized to the Golgi, and PKC$\gamma$, PKC$\delta$ and PKC$\eta$ showed cytoplasmic distribution with Golgi localization. DAG analog TPA induced translocation of PKC-GFP to the plasma membrane. PKC$\alpha$, PKC$\eta$ and PKC$\theta$ were also localized to the Golgi in response to TPA. Only PKC$\delta$ was found to be associated with the nuclear membrane after transient TPA treatment. These results suggest that specific PKC isoforms are translocated to different intracellular sites and exhibit distinct biological effects.

• 대한수의학회지
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• 제41권3호
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• pp.269-273
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• 2001

The expression of protein kinase C(PKC) isoforms and nitric oxide synthase (NOs) isoforms was studied in the equine vomeronasal organ(VNO), a pheromone receptor organ, using immunohistochemistry. All PKC isoforms including PKC $\alpha$, ${\beta}I$, $\delta$, and $\theta$ were detected in the supporting cells, sensory receptor cells, and basal sensory epithelial cells, while constitutive PKC $\alpha$ and ${\beta}I$ were stained more intensely than novel PKC $\delta$ and ${\theta}$. There was also a varying degree of immunostaining for PKCs in the glandular acini and VNO nerve. Constitutive neuronal and endothelial NOSs, and inducible NOS were detected in the VNO sensory epithelia. There was intense immunoreactivity for endothelial NOS in the VNO sensory epithelia but weak reactivity for neuronal NOS, while inducible NOS showed little immunoreactivity in the adjacent section. These findings suggest that both PKCs and NOSs may be involved in the process of pheromone reception in the horse. Constitutive isoforms of these enzymes may play a more important role in signal trasduction in the VNO of the horse.

• Animal cells and systems
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• 제2권2호
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• pp.229-232
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• 1998

Lysophosphatidylcholine (LPC) has been reported to be responsible for the sustained activation of protein kinase C (PKC). As chondroqenesis is known to be regulated by PKC, this study was performed to investigate the effects of LPC on chondrogenesis of chick limb bud mesenchymes in vitro. LPC treatment of mesenchymes during micromass culture significantly enhanced chondrogenic differentiation. The most effective time of LPC on the stimulation of chondrogenesis was the first day of micromass culture. Analysis of LPC effects on the expression of PKC isoforms revealed that LPC treatment increased expression of PKCa, among the multiple PKC isoforms, in the membrane fraction on day one of culture. The stimulatory effect of LPC on chondrogenesis was abolished if PKCa was down regulated by the prolonged treatment of cells with phorbol ester. The results suqqest that LPC promotes chondrogenesis through the activation of PKCa at the early stage of chondrogenic differentiation.

• Animal cells and systems
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• 제4권4호
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• pp.361-366
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• 2000

In order to investigate the role of protein kinase C (PKC) in chondrogenic differentiation, we examined the localization of PKC isoforms in a limb bud micromass culture system. PKC$\alpha$ is specifically localized in the regions which would become cartilage nodules, while PKC$\lambda/l$ and $\zeta$ display widespread distribution in the whole culture. Distribution of PKC$\alpha$ change along with promotion or inhibition of chondrogenesis by lysophosphatidylcholine or phorbol 12-myristate 13-acetate. On the other hand, localization of PKC$\lambda/l$ or $\zeta$ a was not changed by the modulation of chondrogenesis. Peanut agglutinin binding protein which is associated with cell aggregation during chondrogenesis was present in the cell condensation regions and its expression in those regions was influenced by PKC activity. Expression of fibronectin and N-cadherin in the cell condensing area were also affected by modulation of PKC activity. These results suggest involvement of PKC$\alpha$ in the cell condensation, possibly through regulating expression of fibronectin and N-cadherin.

• 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.

• 대한약학회:학술대회논문집
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• 대한약학회 2003년도 Proceedings of the Convention of the Pharmaceutical Society of Korea Vol.2-2
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• pp.93.3-94
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• 2003

The signaling components of high affinity IgE receptor (Fc RI) were searched by yeast-hybrid screening of the cDNA library constructed from RBL-2H3 cells. The cytoplasmic part of the Fc RI- chain was found to specifically interact with PLC 2, and further comparatives studies were conducted focusing on the differential regulation of two PLC- isoforms through Fc RI. The inhibitors of Src, Syk, and protein kinase C similarly affected the tyrosine phosporylations of PLC 1 and PLC 2 but the inhibitors of PI3-kinase and p42/44 ERK effectively inhibited the activation of PLC 1 but not PLC 2. (omitted)

• 고려인삼학회:학술대회논문집
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• 고려인삼학회 1998년도 Advances in Ginseng Research - Proceedings of the 7th International Symposium on Ginseng -
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• pp.244-252
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• 1998

Ginsenoside Rh, (G-Rh,) from Panax ginseng induced morphological features of apoptosis and DNA fragmentation as a biochemical marker of apoptosis confirmed by TUNEL reaction and agarose gel electrophoresis in human neuroblastoma SK-N-BE(2) and rat glioma C6Bu-1 cells During apoptosis by G-Rh2, protein kinase C (PKC) isoforms were analysed by immunoblotting. In SK-N-BE(2) cells, the levels of a, p and ${\gamma}$ subtypes were increased by undergoing apoptosis, while PKC e isoform increased early in treatment (3 h and 6 h). In addition, PKC s isoform gradually decreased during apoptosis by G-Rh2 and PKC $\theta$ isoform was detected in neither untreated- nor G-Rh1-treated SK-N-BE(2) cells (data not shown). However, no significant changes in the level of S and s isoforms were observed in C6Bu-1 cells undergoing apoptosis by G-Rh2. These results suggest that PKC subtypes may play differential roles in apoptotic signal pathways and their roles can be cell type-specific in apoptosis induced by G-Rh2.

• Journal of Chest Surgery
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• 제36권9호
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• pp.666-673
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• 2003

폐암의 주된 원인으로 알려진 흡연은 그 악성세포 발현기전이 아직 정확히 규명된 바 없다. 이에 저자들은 흡연에 의한 발암성의 지표로 흡연 중에 특이적으로 존재하는 강력한 발암물질인 NNK(4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone)를 이용하여 흡연에 따른 폐암의 발생과 그 Protein kinase C (PKC) isoform과 관련된 기전에 관한 연구를 시도하였다. 대상 및 방법: 인체 상피세포를 NNK에 노출시킨 후 saturation density, soft agar colony formation, cell aggregation 및 foci의 출현 등의 양상을 파악하여 세포 발암성 여부를 관찰하였으며 NNK를 15분간 노출시킨 후 PKC의 변화는 세포 내 PKC isoform의 양을 cytosolic fraction과 membrane fraction으로 분리하여 측정하여 분석하였다. 결과: NNK 투여군에서 saturation density, soft agar colony formation, cell aggregation 및 foci의 출현 시기 등의 세포 발암성을 뚜렷이 나타내었으며 PKC isoform분석의 경우 PKC-$\alpha$의 membrane fraction의 뚜렷한 증가를 보였으며 이러한 활성은 용량-의존적인 형태를 유지하였다. PKC-$\varepsilon$은 NNK 처리 시 용량-의존적으로 cytosol fraction의 감소 및 membrane fraction의 증가를 뚜렷하게 보였고 NNK에 의한 PKC-λ의 변화는 감지되지 않았다. 결론: 본 연구는 화학적 발암물질인 NNK가 인체발암화에 관여함을 재차 확인하면서 초기 과정에 관여하는 PKC isoform의 변화를 분석함으로써 total PKC활성이 아닌 isoform 각각에 대한 변화를 확인하였다는 점에서 앞으로 인체상피세포 기원의 폐암 생성 기전 연구에 기여할 것으로 생각한다.

• Asian Pacific Journal of Cancer Prevention
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• 제13권4호
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• pp.1177-1182
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• 2012

Protein kinase C (PKC) has been implicated in carcinogenesis and displays variable expression profiles during cancer progression. Studies of dietary phytochemicals on cancer signalling pathway regulation have been conducted to search for potent signalling regulatory agents. The present study was designed to evaluate any suppressive effect of maslinic acid on PKC expression in human B-lymphoblastoid cells (Raji cells), and to identify the PKC isoforms expressed. Effects of maslinic acid on PKC activity were determined using a PepTag$^{(R)}$ assay for non-radioactive detection of PKC. The highest expression in Raji cells was obtained at 20 nM PMA induced for 6 hours. Suppressive effects of maslinic acid were compared with those of four PKC inhibitors (H-7, rottlerin, sphingosine, staurosporine) and two triterpenes (oleanolic acid and ursolic acid). The $IC_{50}$ values achieved for maslinic acid, staurosporine, H-7, sphingosine, rottlerin, ursolic acid and oleanolic acid were 11.52, 0.011, 0.767, 2.45, 5.46, 27.93 and $39.29\;{\mu}M$, respectively. Four PKC isoforms, PKC ${\beta}I$, ${\beta}II$, ${\delta}$, and ${\zeta}$, were identified in Raji cells via western blotting. Maslinic acid suppressed the expression of PKC ${\beta}I$, ${\delta}$, and ${\zeta}$ in a concentration-dependent manner. These preliminary results suggest promising suppressive effects of maslinic acid on PKC activity in Raji cells. Maslinic acid could be a potent cancer chemopreventive agent that may be involved in regulating many downstream signalling pathways that are activated through PKC receptors.