• Proceedings of the Korean Society of Applied Pharmacology
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• 1994.04a
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• pp.259-259
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• 1994

Protein kinase C (PKC) participates in many cellular signal transduction. Previously we found that PKC activity of whole rat brain was altered after an exposure to cold temperature of 4 $^{\circ}C$ (Lee and Choi, Exp. Neurobiol., 2, 6, 1993). In this time PKC activity in each region of rat brain was investigated in order to know each regions is affected mostly by the stress.

• BMB Reports
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• v.32 no.4
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• pp.379-384
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• 1999

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 (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.

• Journal of Life Science
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• v.17 no.6 s.86
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• pp.783-790
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• 2007

G protein coupled receptors (GPCRs) transmit various extracellular signals into the cells. Upon binding of the ligands, conformational changes in the extracellular and/or transmembrane (TM) domains of CPCRs were propagated into the cytoplasmic (CP) domain of the molecule leading to the activation of their cognate heterotrimeric C proteins and kinases. Constitutively active GPCR mutants causing the activation of C Protein signaling even in the absence of ligand binding are of interest for the study of activation mechanism of GPCRs. Two classes of constitutively active mutations, categorized by their effects on the salt bridge between Ell3 and K296, were found in the TM domain of rhodopsin. Opsin mutants containing combinations of the mutations were constructed to study the conformational changes required for the activation of rhodopsin. Rhodopsin chromophore regenerated with 11-cis-retinal showed a thermal stability inversely correlated with its constitutive activity. In contrast, rhodopsin mutants exhibited a binding affinity to an agonist, all-trans-retinal, in a constitutive activity-dependent manner. In order to test whether the conformational changes responsible for the activation of trans-ducin (Gt) are the same as the conformation required for the recognition of rhodopsin kinase, analysis of the mutants were carried out with phosphorylation by rhodopsin kinase. Rhodopsin mutants containing combinations of different classes of the mutations showed a strong synergistic effect on the phosphorylation of the mutants in the dark as similar to that of Gt activation. The results suggest that at least two or three kinds of segmental and independent conformational changes are required for the activation of rhodopsin and the conformational changes responsible for activating rhodopsin kinase and Gt are similar to each other.

• The Korean Journal of Physiology
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• v.29 no.1
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• pp.39-50
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• 1995

This study was designed to clarify the action of cyclic nucleotides, cyclic AMP and cyclic GMP, on phorbol 12,13-dibutyrate (PDBu)-induced contraction in rings isolated from rabbit carotid artery. Arterial rings, 2 mm in width, were myographied isometrically in an isolated organ bath. PDBu produced slowly developing, sustained contraction in rabbit carotid artery, in a dose dependent manner, which was independent of extracellular $Ca^{2+}$ PDBu-induced contraction was relaxed by staurosporine, which suggests that PDBu-induced contraction is mediated by protein kinase C (PKC). $^{45}Ca^{2+}$ uptake by rabbit carotid artery was increased by PDBu during depolarization, but not in control. Isoproterenol and sodium nitroprusside (SNP) relaxed phenylephrine-induced contraction. However, SNP but not isoproterenol relaxed the contraction induced by PDBu. Acetylcholine relaxed PDBu-induced contraction in the presence of the endothelium. 8-bromo-cyclic AMP, a permeable analogue of cyclic AMP, suppressed phenylephrine-induced contraction but not PDBu-induced contraction. 8-bromo cyclic GMP relaxed both of them with dose dependency. A large dose of forskolin relaxed PDBu-induced contraction. PDBu increased cyclic AMP without considerable change in the level of cyclic GMP. Based on these findings, PDBu-induced contraction of rabbit carotid artery was relaxed by cyclic GMP more effectively than cyclic AMP, and the action of cyclic AMP could be mediated by cyclic GMP dependent protein kinase. Therefore it is suggested that the antagonistic action between protein kinase C and cyclic GMP-dependent protein kinase plays a major role in the regulation of vascular tone.

• International Journal of Oral Biology
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• v.45 no.4
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• pp.169-178
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• 2020

L-ascorbic acid (L-AA; vitamin C) induces apoptosis in cancer cells. This study aimed to elucidate the molecular mechanisms of L-AA-induced apoptosis in human laryngeal epidermoid carcinoma Hep-2 cells. L-AA suppressed the viability of Hep-2 cells and induced apoptosis, as shown by the cleavage and condensation of nuclear chromatin and increased number of Annexin V-positive cells. L-AA decreased Bcl-2 protein expression but upregulated Bax protein levels. In addition, cytochrome c release from the mitochondria into the cytosol and activation of caspase-9, -8, and -3 were enhanced by L-AA treatment. Furthermore, apoptosis-inducing factor (AIF) and endonuclease G (EndoG) were translocated into the nucleus during apoptosis of L-AA-treated Hep-2 cells. L-AA effectively inhibited the constitutive nuclear factor-κB (NF-κB) activation and attenuated the nuclear expression of the p65 subunit of NF-κB. Interestingly, L-AA treatment of Hep-2 cells markedly activated Akt and mitogen-activated protein kinase (MAPK; extracellular signal-regulated kinase 1/2, p38, and c-Jun N-terminal kinase [JNK]) and and LY294002 (Akt inhibitor), SB203580 (p38 inhibitor) or SP600125 (a JNK inhibitor) decreased the levels of Annexin V-positive cells. These results suggested that L-AA induces the apoptosis of Hep-2 cells via the nuclear translocation of AIF and EndoG by modulating the Bcl-2 family and MAPK/Akt signaling pathways.

• Bulletin of the Korean Chemical Society
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• v.30 no.9
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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.

• BMB Reports
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• v.29 no.1
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• pp.11-16
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• 1996

The present study showed that receptor-mediated activation of rabbit kidney proximal tubule cells by angiotensin II, the $Ca^{2+}$ ionophore A23187, or the protein kinase C activator phorbol myristate acetate (PMA) all stimulated phospholipase D (PLD). This was demonstrated by the increased formation of phosphatidic acid, and in the presence of 0.5% ethanol, phosphatidylethanol (PEt) accumulation. Angiotensin II leads to a rapid increase in phosphatidic acid and diacylglycerol, and phosphatidic acid formation preceeded the formation of diacylglycerol. This result suggests that some phosphatidic acid seems to be formed directly from phosphatidylcholine hydrolyzed by Pill. On the other hand, EGTA substantially attenuated angiotensin II and A23187-induced PEt formation, and when the cells were pretreated with verapamil angiotensin II-induced Pill activation was completely abolished. These results provide the evidence that calcium ion influx is essential for the agonist-induced Pill activation. In addition, staurosporine, an inhibitor of protein kinase C, strongly inhibited PMA-induced PEt formation, but was ineffective on angiotensin II-induced PEt accumulation. $GTP{\gamma}S$ also stimulates PEt formation in digitonin-permeabilized cells, but pretreatment of the cells with pertussis toxin failed to suppress angiotensin II-induced PEt formation. From these results, we conclude that in the rabbit kidney proximal tubule cells the mechanisms of angiotensin II- and PMA-induced Pill activation are different from each other and mediated via a pertussis toxin-insensitive trimeric G protein.

• Childhood Kidney Diseases
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• v.21 no.1
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• pp.1-7
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• 2017

Mitogen-activated protein kinases (MAPKs) play important roles in various cellular functions including proliferation, differentiation, and apoptosis. We showed that MAPKs are developmentally regulated in the rat kidney. p38 MAPK (p38) and extracellular signal-regulated kinase (ERK) were strongly expressed in the fetal kidney, whereas c-Jun N-terminal kinase (JNK) was detected predominantly in the adult kidney. The inhibition of p38 or ERK in organ culture resulted in reduced nephron formation with or without reduced kidney size. On the other hand, persistent fetal expression pattern of MAPKs, i.e., upregulation of p38 and ERK and downregulation of JNK, was observed in the cyst epithelium of human renal dysplasia, ovine fetal obstructive uropathy, and pcy mice, a model of polycystic kidney disease. Furthermore, activated p38 and ERK induced by cyclic stretch mediated proliferation and $TGF-{\beta}1$ expression in ureteric bud cells, probably leading to cyst formation and dysplastic changes. Inhibition of ERK slowed the disease progression in pcy mice. Finally, ERK and p38 were inactivated in the early embryonic kidney subjected to maternal nutrient restriction, characterized by reduced ureteric branching and nephron number. Thus, MAPKs mediate the development of normal and diseased kidney. Their modulation may result in novel therapeutic strategies against developmental abnormalities of the kidney.

• Natural Product Sciences
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• v.15 no.1
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• pp.32-36
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• 2009

Glutamate causes neurotoxicity through formation of reactive oxygen species and activation of mitogen-activated protein kinase (MAPK) pathways. MAPK phosphatase-1 (MKP-1) is one of the phosphatases responsible for dephosphorylation/deactivation of three MAPK families: the extracellular signal-regulated kinase-1/2 (ERK-1/2), the c-Jun N-terminal kinase-1/2 (JNK-1/2), and the p38 MAPK. In this report, the potential involvement of MKP-1 in neuroprotective effects of curcumin, the active ingredient of turmeric (Curcuma longa), was examined using HT22 cells. Glutamate caused cell death and activation of ERK-1/2 but not p38 MAPK or JNK-1/2. Blockage of ERK-1/2 by its inhibitor protected HT22 cells against glutamate-induced toxicity. Curcumin attenuated glutamate-induced cell death and ERK-1/2 activation. Interestingly, curcumin induced MKP-1 activation. In HT22 cells transiently transfected with small interfering RNA against MKP-1, curcumin failed to inhibit glutamate-induced ERK-1/2 activation and to protect HT22 cells from glutamate-induced toxicity. These results suggest that curcumin can attenuate glutamate-induced neurotoxicity by activating MKP-1 which acts as the negative regulator of ERK-1/2. This novel pathway may contribute to and explain at least one of the neuroprotective actions of curcumin.

• Archives of Pharmacal Research
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• v.27 no.3
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• pp.324-333
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• 2004

To determine whether Insulin-like growth factor (IGF-I) treatment represents a potential means of enhancing the survival of cardiac muscle cells from adriamycin (ADR)-induced cell death, the present study examined the ability of IGF-I to prevent cell death. The study was performed utilising the embryonic, rat, cardiac muscle cell line, H9C2. Incubating cardiac muscle cells in the presence of adriamycin increased cell death, as determined by MTT assay and annexin V-positive cell number. The addition of 100 ng/mL IGF-I, in the presence of adriamycin, decreased apoptosis. The effect of IGF-I on phosphorylation of PI, a substrate of phosphatidylinositol 3-kinase (PI 3-kinase) or protein kinase B (AKT), was also examined in H9C2 cardiac muscle cells. IGF-I increased the phosphorylation of ERK 1 and 2 and $PKC{\;}{\zeta}{\;}kinase$. The use of inhibitors of PI 3-kinase (LY 294002), in the cell death assay, demonstrated partial abrogation of the protective effect of IGF-I. The MEK1 inhibitor-PD098059 and the PKC inhibitor-chelerythrine exhibited no effect on IGF-1-induced cell protection. In the regulatory subunit of PI3K-p85- dominant, negative plasmid-transfected cells, the IGF-1-induced protective effect was reversed. This data demonstrates that IGF-I protects cardiac muscle cells from ADR-induced cell death. Although IGF-I activates several signaling pathways that contribute to its protective effect in other cell types, only activation of PI 3-kinase contributes to this effect in H9C2 cardiac muscle cells.