• The Korean Journal of Zoology
• /
• v.36 no.4
• /
• pp.439-451
• /
• 1993

Protein kinase C isozymes in a human prostate adenocarcinoma PC-3 cell line were characterized. Immunoreactive bands and immunocytochemical stains were obsenred in PC-3 cells with antibodies raised against protein kinase C ${\alpha}$, ${\beta}$, ${\gamma}$, $\delta$, $\varepsilon$, and ζ types, respectively. Protein kinase C ${\alpha}$ corresponded to a immunoreactive band at a molecular weight of 80,000-dalton, whereas molecular weights of other immunoreactive isozvmes of protein kinase C were detected at 68,000-dalton. Protein kinHse C $\delta$ and ζ antibodies detected additional bands at 55,000-dalton and 80,000-dalton, respectively Immunocvtochemical study confirmed the results of the immunoblotting experiments qualitatively: all six protein kinase C isozymes were detected in the cytoplasm of PC-3 cells. Translocation of protein kinase C in PC-3 cells were also examined with phorbol 12-myristate 13-acetate (PMA), bryostatin 2, diolein, and 1-oleoyl-2-acetyl glycerol (OAG). Differential reactions of protein kinase C isozvmes to these activators were obsenred. When PC-3 cells were treated with 10mM bryostatin 2, protein kinase C isozyme u was translocated into the nucleus, whereas s type was translocated into the plasma membrane and the nucleus. Protein kinase C ${\alpha}$ and ζ types were translocated into the nucleus following the treatment with 101M diolein, whereas protein kinase C ${\alpha}$, ${\beta}$, ${\gamma}$, and $\varepsilon$ types were translocated into the nucleus by the treatment with 10mM OAG. Protein kinase C ${\alpha}$ and $\varepsilon$ types were translocated into the nucleus in the presence of 100nM PMA. Protein kinase C $\delta$ type was translocated to the nuclear membrane by these activators, however, only PMA-induced translocation was inhibited by protein kinase C inhibitor, 1-(5-isoquinolinesulfonyll-2-methvlpiperazine dihvdrochloride (H7) . H7 inhibited translocation of protein kinase C ${\alpha}$ type induced by PMA, ${\beta}$ type by OAG and s type by PMA and OAG, whereas it did not affect translocations induced by bryostatin and diolein, respectively. These results suggest that there exist six isoformes of protein kinase C (${\alpha}$, ${\beta}$, ${\gamma}$, $\delta$, $\varepsilon$ and ζ types) in PC-3 cells and that each of these isozvmes distinctivelv reacts to bryostatin, diolein, OAG and PMA, in part due to an altered molecular size and conceivably discrete binding site(s).

• Tuberculosis and Respiratory Diseases
• /
• v.44 no.2
• /
• pp.338-348
• /
• 1997

Background : Endotoxin, the component of outermembrane of gram negative organism, plays an important role in the initiation and amplification of inflammatory reaction by its effects on inflammatory cells. Until recently, there have been continuing efforts to delinate the mechanisms of the signal trasduction pathway of endotoxin stimuli on inflammatory cells. By uncovering the mechanisms of signal transduction pathway of endotoxin stimuli, we can expect to have tools to control the excessive inflammatory responses which sometimes may be fatal to the involved host. It was generally accepted that endotoxin exerts its inflammatory effects through inflammatory cytokines that are produced by endotoxin-stimulated inflammatory cells and there were some reports on the importance of protein kinase C and protein tyrosine kinase activation in the production of inflammatory cytokines by endotoxin So we evaluated the effect of pretreatment of protein kinase C inhibitors (H7, Staurosporin) and protein tyrosine kinase inhibitors(Herbimycin, Genistein) on the endotoxin-stimulated cytokines(IL-8 & TNF-$\alpha$) mRNA expression. Method : Peripheral blood monocytes were isolated from healthy volunteers by Ficoll-Hypaque density gradient method and purified by adhesion to 60mm Petri dishes. Endotoxin(LPS 100ng/ml) was added to each dishes except one control dish, and each endotoxin-stimulated dishes was preincubated with H7, Staurosporin(protein kinase C inhibitor), Herbimycin or Genistein(protein tyrosine kinase inhibitor) respectively except one dish. Four hours later the endotoxin stimulation, total RNA was extracted and Northern blot analysis for IL-8 mRNA and TNF-$\alpha$ mRNA was done. Result : Endotoxin stimulation increased the expression of IL-8 mRNA and TNF-$\alpha$ mRNA expression in human peripheral blood monocyte as expected and the stimulatory effect of endotoxin on TNF-$\alpha$ mRNA expression was inhibited by protein kinase C inhibitors(H7, Staurosporin) and protein tyrosine kinase inhibitors (Herbimycin, Genistein). The inhibitory effect of each drugs was increased with increasing concentration. The stimulatory effect of endotoxin on IL-8 mRNA was also inhibited by H7 and protein tyrosine kinase inhibitors (Herbimycin, Genistein) dose-dependently but not by Staurosporin. Conclusion : Protein kinase C and protein tyrosine kinase are involved in the endotoxin induced signal transduction pathway in human peripheral blood monocyte.

• Korean Journal of Veterinary Research
• /
• v.38 no.3
• /
• pp.508-517
• /
• 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.

• Biomedical Science Letters
• /
• v.14 no.2
• /
• pp.77-81
• /
• 2008

Testis brain RNA-binding protein (TB-RBP) is a DNA/RNA binding protein. TB-RBP is mainly expressed in testis and brain and highly conserved protein with several functions, including chromosomal translocations, DNA repair, mitotic cell division, and mRNA transport, stabilization, and storage. In our previous study, we identified TB-RBP as an interacting partner for the catalytic subunit $(C{\alpha})$ of protein kinase A(PKA) and verified their interaction with several biochemical analyses. Here, we confirmed interaction between $C{\alpha}$. and TB-RBP in mammalian cells and determined the effect of $C{\alpha}$. on the function of TB-RBP. The activation of $C{\alpha}$. increased the TB-RBP function as a DNA-binding protein. These results suggest that the function of TB-RBP can be modulated by PKA and provide insights into the diverse role of PKA.

• The Korean Journal of Physiology and Pharmacology
• /
• v.3 no.1
• /
• pp.11-18
• /
• 1999

Nuclear factor $_{\kappa}B\;(NF-_{\kappa}B)$ activation is modulated by various protein kinases. Activation of $NF-_{\kappa}B$ is known to be important in the regulation of cell viability. The present study investigated the effect of inhibitors of protein tyrosine kinase (PTK), protein kinase C (PKC) and protein kinase A (PKA) on $NF-_{\kappa}B$ activity and the viability of bovine cerebral endothelial cells (BCECs). In serum-deprivation-induced BCEC death, low doses of $TNF{\alpha}$ showed a protective effect. $TNF{\alpha}$ induced $NF-_{\kappa}B$ activation within 4 h in serum-deprivation. PTK inhibitors (herbimycin A and genistein) and PKC inhibitor (calphostin C) prevented $NF-_{\kappa}B$ activation stimulated by $TNF{\alpha}.$ Likewise, these inhibitors prevented the protective effect of $TNF{\alpha}.$ In contrast to $TNF{\alpha}-stimulated\;NF-_{\kappa}B$ activity, basal $NF-_{\kappa}B$ activity of BCECs in media containing serum was suppressed only by calphostin C, but not by herbimycin A. As well BCEC death was also induced only by calphostin C in serum-condition. H 89, a PKA inhibitor, did not affect the basal and $TNF{\alpha}-stimulated\;NF-_{\kappa}B$ activities and the protective effect of $TNF{\alpha}$ on cell death. These data suggest that modulation of $NF-_{\kappa}B$ activation could be a possible mechanism for regulating cell viability by protein kinases in BCECs.

• Korean Journal of Veterinary Research
• /
• v.38 no.1
• /
• pp.9-15
• /
• 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.

• Biomedical Science Letters
• /
• v.13 no.4
• /
• pp.305-311
• /
• 2007

cAMP-dependent protein kinase A (PKA) is the best-characterized protein kinases and has served as a model of the structure and regulation of cAMP-binding protein as well as of protein kinases. To determine the function of PKA in development, we employed the yeast two-hybrid system to screen for catalytic subunit of PKA $(C\alpha)$ interacting partners in a cDNA library from mouse embryo. A Testis-brain RNA-binding protein (TB-RBP), specifically bound to $C\alpha$. This interaction was verified by several biochemical analysis. Our findings indicate that $C\alpha$ can modulate nucleic acid binding proteins of TB-RBP and provide insights into the diverse role of PKA.

• YAKHAK HOEJI
• /
• v.47 no.1
• /
• pp.31-36
• /
• 2003

To investigate the effect of protein kinase on melanin production via cAMP-dependent pathway, we measured the melanin amount and tyrosinase activity in B16 melanoma cells stimulated by alpha-melanocyte stimulating hormone (MSH), forskolin and 8-Br-cAMP. MSH, forskolin and 8-Br-cAMP significantly increased both melanin production and tyrosinase activity in B16 cells. Melanin production and tyrosinase activity by MSH are significantly inhibited by cyclic AMP-dependent protein kinase inhibitor (KT5720) and protein kinase C down-regulation treated with PMA. Bisindolmaleimide (1$\mu$M), protein kinase C inhibitor, significantly inhibited melanin production and tyrosinase activity stimulated by MSH, forskolin and 8-Br-cAMP with the following order of potency: MSH＞forskolin＞8-Br-cAMP. Tyrosine kinase inhibitor, genistein and DHC, significantly inhibited both, but the inhibitory effect was more potent in 8-Br-cAMP-stimulated B16 cells than MSH-stimulated cells. NFkB inhibitor (parthenolide) significantly inhibited melanin production and tyrosinase activity. Neither melanin production nor tyrosinase activity induced by MSH, forskolin and 8-Br-cAMP were affected by KN-62 (calmodulin-dependent protein kinase II inhibitor), PD098059 (mitogen-activated protein kinase inhibitor, MAPKK) and worthmannin (phosphatidylinositol 3-kinase inhibitor). These results suggest that both protein kinase C and tyrosine kinase are involved in melanin production by cyclic AMP-dependent pathway and NFkB pathway may play an important role in cyclic AMP-dependent melanin production in B16 melanoma cells.

• Korean Journal of Acupuncture
• /
• v.32 no.2
• /
• pp.51-58
• /
• 2015

Objectives : Pinus densiflora gnarl, called Song-Jeol in Korean medicine, has been used to cure inflammatory diseases such as arthritis. In the present study, we evaluated inhibitory property of Song-Jeol pharmacopuncture(SJ) on C6 glioma cell migration. Methods : To evaluate cell viability on C6 glioma cells of SJ, the viability was assessed by using Ez-cytox assay kit. The cell migration was assessed by wound-healing assay and Boyden chamber assay, respectively. LPS-induced NO productions were determined by using the Griess reagent. The expression of iNOS and protein kinase $C(PKC)-{\alpha}$ were estimated by western blotting assay. Results : In the wound-healing assay and Boyden chamber assay, SJ showed a significant inhibition on serum-induced C6 glioma cell migration. In addition, NO production was decreased by SJ through suppression of iNOS expression in LPS-stimulated C6 glioma cell. Futhermore, LPS-induced protein kinase $C(PKC)-{\alpha}$ expression was effectively inhibited by SJ. Conclusions : These results demonstrated that SJ was useful for the suppression of the C6 glioma cell migration.

• The Korean Journal of Physiology and Pharmacology
• /
• v.4 no.6
• /
• pp.487-496
• /
• 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.