• Journal of Yeungnam Medical Science
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• v.7 no.1
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• pp.39-50
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• 1990

The one event on signalling mechanism is the cleavage by adenyl cyclase of ATP into second messenger, cyclic AMP. The other transfer system of inositol metabolism. it is widely recognized that hydrolysis of the minor membrane lipid phosphoinositide bisphosphate($PIP_2$) initiated by occupation of certain receptors and catalyzed by phospholipase C, lead to toe generation of the two intracellular messengers, inositol triphosphate($IP_3$) and diacylglycerol(DG). $IP_3$ is converted to inositol tetrakisphosphate($IP_4$) by $IP_3$ kinase. In the present study, it is that purification of calmodulin is used by phenyl-Sepharose CL-4B chromatography. it's molecular weigh, 17.000 in SDS-polyacrylamide gel electrophoresis. In order to observe the affinity between calmodulin (CaM)-Affigel 15 and $IP_3$ kinase, and isolated $IP_3$ kinase, was applied in CaM-Affigel with $Ca^{2+}$ equilibirum buffer and EGTA equilibirum buffer. We compared with binding and elution effect of $IP_3$ kinase in several condition of buffer. In affinity of binding. $Ca^{2+}$ equilibrium buffer was in the most proper condition. and elution, CaM/$Ca^{2+}$ buffer(CE1 10.36, CE2 12. 76pM/min/mg of protein) was effected much more than EGTA buffer(E2 1.48, E3 2.43pM/min/mg of protein), but CaM/$Ca^{2+}$ stimulate the activity of $IP_3$ kinase. And then, several detergents such as sodium deoxycholate, tween 20. cholic acid, polyethylene glycol, chaps were applied. The 0.2% chaps buffer(E2 23.19, E3 8.05pM/min/mg of protein) was the most effective in elution of $IP_3$ kinase.

• Journal of Microbiology and Biotechnology
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• v.5 no.3
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• pp.125-131
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• 1995

A 3, 346 bp of the cdd upstream region in Bacillus subtilis was sequenced from the pSO1 (Song BH and J Neuhard. 1989. Mol. Gen. Genet 216: 462-468) and sequence homology was searched to the known genes in Genbank and European Molecular Biology Laboratory databanks. Five complete and one truncated putative coding sequences deduced from the nucleotide sequence were found through the ORF searching by Genetyx and Macvector software, and one of them was identified as the dgk (diacylglycerol kinase) gene and another, a truncated one, as the phoH (phosphate starvation-inducible gene) gene. The B. subtilis dgk gene, having a role for response to several environmental stress signals, revealed an open reading frame of 134 amino acids with 43.1% of sequence identity to the Streptococcus mutans dgk gene. The carboxy terminal 59 residues of the truncated phoH gene showed 52.7% and 34.5% of sequence identity in amino acids with the corresponding genes of Mycobacterium leprae and Escherichia coli. The four remaining coding sequences consisting of 115, 421, 91, and 91 residues were thought to be unknown ORFs because they have no significant similarity to known genes.

• Journal of Life Science
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• v.20 no.10
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• pp.1451-1457
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• 2010

Serotonin (5-hydroxytryptamine, 5-HT) is an important mediator of cell-cell signaling in neuronal systems. The serotonin transporter (SERT) on the plasma membrane controls the extracellular 5-HT level by reuptake of released 5-HT from the synaptic cleft, but the underlying regulation mechanism is unclear. Here, we used the yeast two-hybrid system to identify the specific binding protein(s) that interacts with the carboxyl (C)-terminal region of SERT and found a specific interaction with protein kinase C-$\varepsilon$ (PKC-$\varepsilon$), a PKC isotype that is characterized as a calcium-independent and phorbol ester/diacylglycerol-sensitive serine/threonine kinase. PKC-$\varepsilon$ bound to the tail region of SERT but not to other members of the $Na^+/Cl^-$ dependent SLC6 gene family in the yeast two-hybrid assay. The C-terminal region of PKC-$\varepsilon$ is essential for interaction with SERT. In addition, these proteins showed specific interactions in the glutathione S-transferase (GST) pull-down assay. PKC-$\varepsilon$ phosphorylated the peptide of the SERT amino (N)-terminus in vitro. These results suggest that the phosphorylation of SERT by PKC-$\varepsilon$ may regulate SERT activity in plasma membrane.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1993.04a
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• pp.63-63
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• 1993

Phospoinositide-specific phospholipase C (PLC)는 세포막의 phosphoinositide를 분해하여 inositol phosphates와 diacylglycerol을 전달하는데 핵심적인 효소이다. PLC는 분자량과 1차구조의 비교에 의하여 type (PLC-$\beta$, ${\gamma}$, $\delta$)로 구분되며, 각 type마다 2-4종의 subtype이 존재하고 PLC isozyme들에 대한 현재가지의 각종 신호 전달 및 조절에 대한 연구를 종합하면: (1) PLC-$\beta$ type은 G-protein과 연결되어 신호를 전달받고, (2) PLC-${\gamma}$ type은growth factor receptor tyrosine kinase에 의하여 인산화 되어 활성화됨으로, 세포의 성장 신호를 전달하며. (3) PLC-$\delta$ type에 대한 신호 전달이나 조절은 밝혀지지 않고 있다.

• 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.

• The Korean Journal of Physiology and Pharmacology
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• v.5 no.4
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• pp.287-297
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• 2001

Contraction of smooth muscle is initiated by an increase in cytosolic $Ca^{2+}$ leading to activation of $Ca^{2+}$/ calmodulin-dependnet myosin light chain (MLC) kinase and phosphorylation of MLC. The types of contraction and signaling mechanisms mediating contraction differ depending on the region. The involvement of these different mechanisms varies depending on the source of $Ca^{2+}$ and the kinetic of $Ca^{2+}$ mobilization. $Ca^{2+}$ mobilizing agonists stimulate different phospholipases $(PLC-{\beta},\;PLD\;and\;PLA_2)$ to generate one or more $Ca^{2+}$ mobilizing messengers $(IP_3\;and\;AA),$ and diacylglycerol (DAG), an activator of protein kinase C (PKC). The relative contributions of $PLC-{\beta},\;PLA_2$ and PLD to generate second messengers vary greatly between cells and types of contraction. In smooth muscle cell derived form the circular muscle layer of the intestine, preferential hydrolysis of $PIP_2$ and generation of $IP_3$ and $IP_3-dependent\;Ca^{2+}$ release initiate the contraction. In smooth muscle cells derived from longitudinal muscle layer of the intestine, preferential hydrolysis of PC by PLA2, generation of AA and AA-mediated $Ca^{2+}$ influx, cADP ribose formation and $Ca^{2+}-induced\;Ca^{2+}$ release initiate the contraction. Sustained contraction, however, in both cell types is mediated by $Ca^{2+}-independent$ mechanism involving activation of $PKC-{\varepsilon}$ by DAG derived form PLD. A functional linkage between $G_{13},$ RhoA, ROCK, $PKC-{\varepsilon},$ CPI-17 and MLC phosphorylation in sustained contraction has been implicated. Contraction of normal esophageal circular muscle (ESO) in response to acetylcholine (ACh) is linked to $M_2$ muscarinic receptors activating at least three intracellular phospholipases, i.e. phosphatidylcholine-specific phospholipase C (PC-PLC), phospholipase D (PLD) and the high molecular weight (85 kDa) cytosolic phospholipase $A_2\;(cPLA_2)$ to induce phosphatidylcholine (PC) metabolism, production of diacylglycerol (DAG) and arachidonic acid (AA), resulting in activation of a protein kinase C (PKC)-dependent pathway. In contrast, lower esophageal sphincter (LES) contraction induced by maximally effective doses of ACh is mediated by muscarinic $M_3$ receptors, linked to pertussis toxin-insensitive GTP-binding proteins of the $G_{q/11}$ type. They activate phospholipase C, which hydrolyzes phosphatidylinositol bisphosphate $(PIP_2),$ producing inositol 1, 4, 5-trisphosphate $(IP_3)$ and DAG. $IP_3$ causes release of intracellular $Ca^{2+}$ and formation of a $Ca^{2+}$-calmodulin complex, resulting in activation of myosin light chain kinase and contraction through a calmodulin-dependent pathway.

• The Journal of Korean Physical Therapy
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• v.20 no.3
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• pp.61-68
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• 2008

Purpose: Protein kinase C (PKC) is a member of a family of serine/threonine kinases that are activated by diacylglycerol (DG) and PKC stimulants. PKC play a key role in signal transduction, including muscle contraction, cell migration, apoptosis, cell proliferation and differentiation. However, the mechanism relating mitogen-activated protein kinases (MAPKs) and PKC, especially in the volume-dependent hypertensive state, remains unclear. Methods: In the present study, I investigated the relationship between PKC and MAPKs for isometric contraction, PKC translocation, and enzymatic activity from normotensive sham-operated rats (NSR) and aldosterone-analogue deoxycorticosterone acetate (DOCA) hypertensive rats (ADHR). Results: Systolic blood pressure was significantly increased in ADHR than in NSR. Physiological salt solution (PSS)-induced resting tension and the intracellular $Ca^{2+}$ concentration ([$Ca^{2+}{_i}$]) were different in the ADHR and NSR. The expression of PKC$\alpha$, PKC$\beta$II, PKC$\delta$, PKC$\varepsilon$ and PKC$\xi$ were different between the cytoplasmic and membranous fractions. However, expression of the PKC isoforms did not differ for the ADHR and NSR. The use of 12-deoxyphorbol 13-isobutyrate (DPB, a PKC stimulant) induced isometric contraction in $Ca^{2+}$-free medium, which was diminished in muscle strips from ADHR as compared to NSR. Increased vasoconstriction and phosphorylation induced by the use of 1 ${\mu}$M DPB were inhibited by treatment with 10 ${\mu}$M PD098059 and 10 ${\mu}$M SB203580, inhibitors of extracellular-regulated protein kinase 1/2 (ERK1/2) and p38 MAPK from ADHR, respectively. Conclusion: These results suggest that the development of aldosterone analogue-induced hypertension is associated with an altered blood pressure, resting tension, [$Ca^{2+}{_i}$], and that the $Ca^{2+}$-independent contraction evoked by PKC stimulants is due to the activation of ERK1/2 and p38 MAPK in volume-dependent hypertension. Therefore, it is suggested that PKC activity affects volume-dependent hypertension and the need to develop cardiovascular disease-specialized physical therapy.

• Korean Journal of Veterinary Research
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• v.38 no.3
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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.

• Korean Journal of Veterinary Research
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• v.39 no.6
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• pp.1073-1080
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• 1999

Previous studies suggested that the inhibition of thyroxine ($T_4$) release by ${\alpha}_1$-adrenoceptor and muscarinic receptor stimulation results in activated protein kinase C (PKC) from mouse and guinea pig thyroids. In the present study, the effect of carbachol, methoxamine, phorbol myristate acetate (PMA), and R59022 on the release of $T_4$ from the mouse, rat, and guinea pig thyroids was compared to clarify the role of PKC in the regulation of the release of $T_4$. The thyroids were incubated in the medium containing the test agents, samples of the medium were assayed for $T_4$ by EIA kits. Forskolin, an adenylate cyclase activator, chlorophenylthio-cAMP sodium, a membrane permeable analog of cAMP, and isobutyl-methylxanthine, a phosphodiesterase inhibitor, like TSH (thyroid stimulating hormone), enhaced the release of $T_4$ from the mouse, rat, and guinea pig thyroids. Methoxamine, an ${\alpha}_1$-adrenoceptor agonist, inhibited the TSH-stimulated release of $T_4$ in mouse, but not rat and guinea pig thyroids. In contrast, carbachol, a muscarinic receptor agonist, inhibited the release of $T_4$ in guinea pig, but not mouse and rat thyroids. These inhibition were reversed by prazosin, an ${\alpha}_1$-adrenoceptor antagonist or atropine, a muscarinic antagonist or $M_1$- and $M_3$-muscarinic antagonists, in mouse or guinea pig thyroids. In addition, staurosporine, a PKC inhibitor, reversed methoxamine or carbachol inhibition of TSH stimulation. Furthermore, PMA, a PKC activator, and R59022, a diacylglycerol (DAG) kinase inhibitor, inhibited the TSH-stimulated release of $T_4$ in mouse, rat, and guinea pig thyroids. These inhibition were blocked by staurosporine. These findings suggest that the activation of receptor or DAG inhibits TSH-stimulated $T_4$ release through a PKC-dependent mechanism in thyroid gland.

• Journal of Plant Biology
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• v.35 no.1
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• pp.77-84
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• 1992

In view of the well-established role of protein kinase C effector element in signal transduction of animal systems, the possibility of diacylglycerol (DAG) and its analog 12-O-tetradecanoylphorbol13-acetate (TPA) having an effect on auxin-induced growih of com coleoptiles was explored. Both DAG and TP A were found to promote cell elongation in the coleoptile tissue_ Treatment of tissue with these protein kinase C-activating agents resulted in increase in the growth rate over the control by about 300%. When 1M was applied to TPA-pretreated coleoptiles. auxin effect appeared synergistic. Morever. coleoptile growth was found to be inhibited by staurosporine and methylated TPA, both of which are known to specifically inhibit protein kinase C. Electrophoretic and autoradiographic patterns of soluble proteins from the coeoptiles indicated that either 1M or TPA tereatment resulted in increased phosphorylation of certain proteins of 205 Kd. 66 Kd and 32 Kd in size. The results obtained from the present work suggest that protein kinase C may be associated with auxin action on cell elongation in the corn coleoptile segments.gments.