• 대한수의학회지
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• 제38권1호
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• pp.9-15
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• 1998

To investigate the involvement of protein kinase C(PKC) isoenzyme in the testes which control spermatogenesis and hormone secretion, we examined cellular distribution of four types of PKC $\alpha$, ${\beta}I$, ${\delta}$ and ${\theta}$ in the horse testes using PKC antisera by western blot analysis and immunohistochemistry. By the western blot analysis, PKC $\alpha$ and ${\beta}I$ were detected at 82KD, while PKC ${\delta}$ and ${\theta}$ were detected at 80KD in the testes of both juvenile and adult horses. In juvenile horse, PKC $\alpha$, ${\delta}$ and ${\theta}$ except ${\beta}I$ were not detected in the cells of the testes, whereas PKC ${\beta}I$ was immunoreacted with only in spermatocytes. In adult, PKC $\alpha$, ${\beta}I$, ${\delta}$ and ${\theta}$isoenzymes were localized in interstitial cells of the testes. In the seminiferous tubules, PKC ${\beta}I$ is localized in spermatocyte, spermatid and spermatozoa, while PKC ${\delta}$ is localized only in spermatids. We suggest that this is a first report to localize PKC in the testes of horse and PKC isoenzymes are upregulated in the cells of horse testes depending on ages. These findings also suggest that certain PKC isoenzyme plays an important role in the signal transduction of spermatogenic cells and interstitial cells in horse testes.

• The Korean Journal of Physiology and Pharmacology
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• 제4권6호
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• pp.487-496
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• 2000

Insulin stimulates glucose transport in muscle and fat cells by promoting the translocation of glucose transporter (GLUT4) to the cell surface. Phosphatidylinositide 3-kinase (PI3-kinase) has been implicated in this process. However, the involvement of protein kinase B (PKB)/Akt and $PKC-{\zeta}$, those are known as the downstream target of PI3-kinase in regulation of GLUT4 translocation, is not known yet. An interesting possibility is that these protein kinases phosphorylate GLUT4 directly in this process. In the present study, $PKB-{\alpha}$ and $PKC-{\zeta}$ were added exogenously to GLUT4-containing vesicles purified from low density microsome (LDM) of the rat adipocytes by immunoadsorption and immunoprecipitation for direct phosphorylation of GLUT4. Interestingly GLUT4 was phosphorylated by $PKC-{\zeta}$ and its phosphorylation was increased in insulin stimulated state but GLUT4 was not phosphorylated by $PKB-{\alpha}.$ However, the GST-fusion proteins, GLUT4 C-terminal cytoplasmic domain (GLUT4C) and the entire major GLUT4 cytoplasmic domain corresponding to N-terminus, central loop and C-terminus in tandem (GLUT4NLC) were phosphorylated by both $PKB-{\alpha}$ and $PKC-{\zeta}.$ The immunoblots of $PKC-{\zeta}$ and $PKB-{\alpha}$ antibodies with GLUT4-containing vesicles preparation showed that $PKC-{\zeta}$ was co-localized with the vesicles but not $PKB-{\alpha}.$ From the above results, it is clear that $PKC-{\zeta}$ interacts with GLUT4-containing vesicles and it phosphorylates GLUT4 protein directly but $PKB-{\alpha}$ does not interact with GLUT4, suggesting that insulin-elicited signals that pass through PI3-kinase subsequently diverge into two independent pathways, an Akt pathway and a $PKC-{\zeta}$ pathway, and that later pathway contributes, at least in part, insulin stimulation of GLUT4 translocation in adipocytes via a direct GLUT4 phosphorylation.

• 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.

• 생명과학회지
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• 제8권6호
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• pp.638-647
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• 1998

Phorbol 12-myristate 13-acetate (PMA), bryostatin, dioctanoyl glycero1 (DiC8)과 같은 Protein Ki-nase C (PKC)의 활성제는 세포질로부터 막이나 핵으로 PKC 동위효소의 전위를 유도한다. 활성화된 PKC는 일반적으로 암을 유발시키는 역할을 하지만 그와 반대로 사람유방암세포의 성장을 약화시키는 기능을 가지고 있다. PKC의 항증식효과와 전위가 MCF-7 세포에서 조사되었다. PMA, bryostatin, DiC8로 활성화된 PKC 동위효소의 전위는 MCF-7 세포의 여러 장소에서 나타났다. PMA는 PKC $\alpha$ 와 $\beta$는 핵이나 핵막 그리고 PKC $\delta$와 $\varepsilon$은 세포막으로 일부 전위시켰고, 반면 DiC8과 bryostatin은 PKC $\alpha$와 $\beta$를 각각 핵과 핵막으로 전위를 유도하였다. PKC 활성제의 항증식 효과에 있어서 PMA ($IC_{50}$/ values of 1.2$\pm$0.3nM)와 DiC8 ($IC_{50}$/ values of 5.0$\pm$1.1$\mu$M)는 세포의 성장을 억제시켰다. Bryostatin 역시 세포의 성장을 억제시켰지만, PMA로 관찰된 것보다는 낮은 수준이었다. 즉 100nM bryostatin에 의해 16% 정도 성장이 감소되었다. 그러나 PMA는 bryo-stalin과 함께 처리하였을 때 PMA의 항증식 효과는 낮았으나, 10$\mu$M DiC8과 함께 처리하였을 때는 효과가 없었다. 이러한 결과들은 각 PKC 동위효소들이 다른 특이한 위치로 전위되었으며, 특히 PKC $\alpha$ 동위효소가 세포성장의 항증식 기능을 조절하는데 중요한 역할을 함을 시사한다.

• Archives of Plastic Surgery
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• 제34권1호
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• pp.8-12
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• 2007

Purpose: Protein tyrosine kinase(PTK), protein kinase C(PKC), oxidase, as a mediator, have been known to take a role in signal transduction pathway of angiogenesis. The authors confirmed that PKC is the most noticeable mediator for abnormal proliferation of vascular endothelial cells through in vitro study model using the inhibitors, targeting the formation of three co-enzymes. In this study, we would investigate which isoform of PKC play an important role in abnormal angiogenesis of vascular endothelial cell. Methods: In 96 well plates, $10^4$ HUVECs(human umbilical vein endothelial cells) were evenly distributed. Two groups were established; the control group without administration of DMH(1,2-dimethylhydrazine) and the DMH group with administration of $7.5{\times}10^{-9}M$ DMH. RNA was extracted from vascular endothelial cell of each group and expression of the PKC isoform was analyzed by RT-PCR(reverse transcriptase-polymerase chain reaction) method. Results: RT-PCR analysis showed that $PKC{\alpha}$, $-{\beta}I$, $-{\beta}II$, $-{\eta}$, $-{\mu}$ and $-{\iota}$ were expressed in vascular endothelial cells of each group. DMH incresed the expression of $PKC{\alpha}$ and $PKC{\mu}$, and decreased $PKC{\beta}I$, $PKC{\beta}II$ expression dominantly. Conclusion: Based on the result of this study, it was suggested that $PKC{\alpha}$ and $PKC{\mu}$ may have significant role in abnormal proliferation of vascular endothelial cell.

• Genomics & Informatics
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• 제6권1호
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• pp.44-49
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• 2008

Protein kinase C (PKC) is a family of kinases involved in the transduction of cellular signals that promote lipid hydrolysis. PKC plays a pivotal role in mediating cellular responses to extracellular stimuli involved in proliferation, differentiation and apoptosis. Comparative analysis of the PKC-${\alpha},{\beta},{\varepsilon}$ isozymes of 200 recently sequenced microbial genomes was carried out using variety of bioinformatics tools. Diversity and evolution of PKC was determined by sequence alignment. The ser/thr protein kinases of Streptomyces coelicolor A3 (2), is the only bacteria to show sequence alignment score greater than 30% with all the three PKC isotypes in the sequence alignment. S.coelicolor is the subject of our interest because it is notable for the production of pharmaceutically useful compounds including anti-tumor agents, immunosupressants and over two-thirds of all natural antibiotics currently available. The comparative analysis of three human isotypes of PKC and Serine/threonine protein kinase of S.coelicolor was carried out and possible mechanism of action of PKC was derived. Our analysis indicates that Serine/ threonine protein kinase from S. coelicolor can be a good candidate for potent anti-tumor agent. The presence of three representative isotypes of the PKC super family in this organism helps us to understand the mechanism of PKC from evolutionary perspective.

• 대한수의학회지
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• 제38권3호
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• pp.508-517
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• 1998

There is evidence that the sympathetic nervous system exerts a control on thyroid function via an adrenergic innervation of thyroid cells. Although it is clear that the inhibitory effects of catecholamines result from an activation of ${\alpha}_1$-adrenoceptors, the mechanisms involved in ${\alpha}_1$-stimulation are not fully understood. The effects of methoxamine and protein kinase C (PKC) activator on the release of thyroxine ($T_4$) from mouse thyroid were studied to clarify the role of PKC in the regulation of $T_4$ release in vitro. The glands were incubated in the medium, samples of the medium were assayed for $T_4$ by EIA kits. Methoxamine inhibited the TSH-stimulated $T_4$ release. This inhibition was reversed by prazosin, an ${\alpha}_1$-adrenergic antagonist. Futhermore, the inhibitory effect of methoxamine on the $T_4$ release stimulated by TSH was prevented by chloroethylclonidine, an ${\alpha}_{1b}$-adrenoceptor antagonist, but not by WB4101, an ${\alpha}_{1a}$-adrenoceptor antagonist. Also methoxamine inhibited the forskolin-, cAMP- or IBMX-stimulated $T_4$ release. These inhibition were reversed by PKC inhibitors, such as staurosporine and $H_7$. PMA, a PKC activator, completely inhibited the TSH-stimulated $T_4$ release, and its inhibition was reversed by staurosporine and $H_7$, but not by chelerythrine. R59022 (a diacylglycerol kinase inhibitor), like methoxamine, also inhibited the TSH-stimulated $T_4$ release, and its inhibition was also reversed by staurosporine. The present study suggests that methoxamine inhibition of $T_4$ release from mouse thyroid can be induced by activation of the ${\alpha}_{1b}$-adrenoceptors and that it is mediated through the ${\alpha}_1$-adrenoceptor-stimulated PKC formation.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제34권1호
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• pp.28-35
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• 2008

Purpose: The nitric oxide (NO) release by inducible nitric oxide synthase (iNOS) is the key events in macrophage response to lipopolysaccharide (LPS) which is suggested to be a crucial mediator for inflammatory and innate immune responses. NO is an important mediator involved in many host defense action and may also lead to a harmful host response to bacterial infection. However, given the importance of iNOS in a variety of pathophysiological conditions, control of its expression and signaling events in response to LPS has been the subject of considerable investigation. Materials and Methods: The Raw264.7 macrophage cell line was used to observe LPS-stimulated iNOS expression. The expression of iNOS is observed by Western blot analysis and real-time RT-PCR. Protein kinase C $(PKC)-{\alpha}$ overexpressing Raw264.7 cells are established to determine the involvement of $PKC-{\alpha}$ in LPS-mediated iNOS expression. $NF-{\kappa}B$ activity is measured by $I{\kappa}B{\alpha}$ degradation and $NF-{\kappa}B$ luciferase activity assay. Results: We found that various PKC isozymes regulate LPS-induced iNOS expression at the transcriptional and translational levels. The involvement of $PKC-{\alpha}$ in LPS-mediated iNOS induction was further confirmed by increased iNOS expression in $PKC-{\alpha}$ overexpressing cells. $NF-{\kappa}B$ dependent transactivation by LPS was observed and $PKC-{\alpha}$ specific inhibitory peptide abolished this activation, indicating that $NF-{\kappa}B$ activation is dependent on $PKC-{\alpha}$. Conclusion: Our data suggests that $PKC-{\alpha}$ is involved in LPS-mediated iNOS expression and that its downstream target is $NF-{\kappa}B$. Although $PKC-{\alpha}$ is a crucial mediator in the iNOS regulation, other PKC isozymes may contribute LPS-stimulated iNOS expression. This finding is needed to be elucidated in further study.

• 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.

• 대한의생명과학회지
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• 제6권2호
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• pp.83-91
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• 2000

Protein kinase C는 세포의 신호전달계에 관여하는 중요한 조절효소로서 여러 가지 세포의 분화와 증식과도 밀접한 관련이 있다. 신생아의 포피 keratinocyte를 농도 200 ng/ml의 human recombinant epidermal growth factor (hrEGF)와 human recombinant insulin-like growth factor-1 (hrIGF-1) 그리고 hrEGF와 hrIGF-1의 혼합액을 각각 첨가하여 24시간 배양한후 세포질과 세포막의 PKC단백질을 추출하여 그 농도를 측정하고, Western blot analysis를 이 용하여 각 growth factor들의 PKC isoenzyme에 대한 영향을 분석하였다. 세포질의 총 PKC 단백질의 농도는 hrIGF-1을 처리한 keratinocyte에서 가장 높았으며, 세포막에서는 대조군의 단백질 농도가 가장 높게 나타났다. EGF를 처리한 keratinocyte의 세포질에서 는 PKC-$\beta$II, -$\delta$, -$\theta$가 막성분에서는 PKC-$\alpha$, -$\beta$I, -$\delta$, -$\Im$, -$\theta$가 증가하였다. IGF-1을 처리한 군의 세포질성분에는 PKC-$\beta$I, -$\Im$, -$\theta$, 막성분에서는 PKC-$\alpha$, -$\beta$I, -$\delta$, -$\Im$, -$\varepsilon$, -$\theta$가 증가하였다 EGF와 IGF-1의 혼합처리 군에서는, PKC-$\alpha$, -$\beta$I, -$\Im$, -$\theta$이 세포질에서, PKC-$\alpha$, -$\delta$, -$\Im$, -$\varepsilon$, -$\theta$은 세포막에서 증가하였다.