• Title/Summary/Keyword: protein kinase C

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Immunocytochemical Localization of c-raf Protein Kinase in EC-4 Cell (EC-4 세포에 있어서 c-raf Protein Kinase의 면역세포화학적 위치)

  • 최원철
    • The Korean Journal of Zoology
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    • v.33 no.3
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    • pp.266-275
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    • 1990
  • c-raf protein kinase, a kind of oncogene, is a cytopiasmic serine / threonine-specific protein and is activated by mitogenic or oncogenic signals. The strncture and functions of c-raf protein kinase are considered very similar to those of protein kinase C. Using immunocytochemical approach, the time course of singal transduction of c-raf protein kinase in EC-4 cell was examined with 12-0-tetradecanoylphorbol-13-acetate (TPA) as tumor promotor and plateletderived growth factor (PDGF) as mitogenic factor. Immunoreactive c-raf was initially bound to the perinuclear membrane and then moved into the nucleus. The effect of the long-term treatment with TPA or PDGF was taken place down regulation at different time point. These results indicate that TPA and PDGF give rise to the translocation of c-raf protein kinase through the two different pathways.

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Immunocytochemical Localization Qf raf Protein Kinase in Cerebrum of Geoclemys reevesii (Gray) (남생이(Geoclemys reevesii) 대뇌에 있어서 raf Protein Kinase의 면역세포화학적 분포)

  • 최원철;문현근
    • The Korean Journal of Zoology
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    • v.33 no.2
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    • pp.141-151
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    • 1990
  • Raf protein kinases and protein kinase C belong to serine/threonine-specific proteins in the cytoplasin, and are similar to each other in functional structure and the aspect of the distribution of celI. The distribution of raf protein kinase in the cerebrum of Geoclemys reevesfi as studied by using the antibodies against a-raf and c-raf protein kinase which induce the expression of raf fainily oncogenes. In general, raf protein kinases were distributed in such restricted regions as the general pallium, hippocampal formation, pdmordiuin hippocampi,nucleus of lateral olfactory tract, basal amygdaloid nucleus, and bed of stria terminalis. Immunological labeling of c-raf protein kinase was more widespread than that of a-raf. However, the intensity of the labeling of c-raf was lower than that of a-raf. The spherical cells of basal amygdaloid nucleus is a ring-like form, because only the cytoplasm was imunolabeled. Especially, c-raf protein kinase occurred in the cells which contained protein kinase C abundandy such as pyramidal cells and Purkinje cells. This suggests that a- and e-raf protein kinases may synegistically induce carclnoma with myc gene which is activated by protein kinase C.

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Protein Kinase C (PKC) in Cellular Signalling System: Translocation of Six Protein Kinase C Isozymes in Human Prostate Adenocarcinoma PC-3 Cell Line (세포신호계에 있어서 Protein Kinase C: 사람의 전입선 adenocarcinoma PC-3 세포내의 여섯개의 Protein kinase C 동립효소의 translocation)

  • Park, Won-Chul;Ahn, Chang-Ho
    • The Korean Journal of Zoology
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    • v.36 no.4
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    • pp.439-451
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    • 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).

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Action of Protein Kinase A and C Activators on Germinal Vesicle Breakdown and One-Cell Embryos in the Mouse (생쥐 GV난자와 1-세포기 배아의 핵막붕괴에 미치는 Protein Kinase A와 C의 작용)

  • 이대기;김경진;조완규
    • The Korean Journal of Zoology
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    • v.32 no.2
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    • pp.153-162
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    • 1989
  • Expedments were perfonned to examine the role of cAMP-dependent protein kinase (PK-A) and diacylglycerol-dependent protein kinase (PK-C) during the meiodc resumption and the first mitotic cell cycle of mouse embryogenesis. Mejoric GV oocytes and one-cell embryos derived from in vitro fertilization were cultured in vitro, and morphological changes in response to activators of PK-A and PK-C were examined. Treatments with a membrane-permeable cAMP analog, dbcAMP (0.1 mg/mi), phosphodiesterase inhibitor, IBMX (0.1 mM), biologically active phorbol ester, WA (10 nglmi), or a synthetic diacylglycerol, sn-diC8 inhibited resumption of melosis. Combination of PK-A and PK-C activator brought about furiher inhibition. On the contrary, dbcAMP (0.1 mg/mi), IBMX (0.2 mM), WA (10 nglml), and sn-diC8 (0.5 mM) did not inhibit pronucleus membrane breakdown (PNBD) when added S or G2 phase of cell cycle. However, activators of PK-C inhibited cleavage of one-cefl embryos. This result indicates that the action mechanism of PK-A and PK-C on dissolution of nuclear membrane in primary meiotic arrest oocytes may be different from that of mitotic one-cell embryos.

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The Effects of Chronic Carbamazepine Administration on Protein Kinase A and Protein Kinase C Activities in Rat Brain (카바마제핀 장기 투여가 백서(白鼠) 뇌의 Protein Kinase A와 Protein Kinase C 활성도에 미치는 영향)

  • Rheem, Doo-Won;Kim, Leen;Suh, Kwang-Yoon
    • Korean Journal of Biological Psychiatry
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    • v.5 no.2
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    • pp.227-234
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    • 1998
  • Objective : Many evidences suggest that patients with bipolar disorder have functional abnormalities in their postreceptor signal transduction pathways, and mood stabilizing effect of lithium is exerted by modulating this dysfunctioning system. Carbamazepine, an antiepileptic agent, is also known to be effective in the treatment and prevention of bipolar disorder. But the precise mechanism of action of the drug is still poorly understood. This study was performed to elucidate the possible therapeutic mechanism of carbamazepine. Method : The effects of chronic carbamazepine administration on protein kinase A and protein kinase C activities in frontal cortex of rat brain after 2 weeks of drug administration were measured and compared with those of control subjects. Results : Mean(${\pm}SE$) value of activity(phosphate transfer ${\mu}mol/mg$ of $protein{\cdot}min$) of protein kinase A in control and test group was $0.249563{\pm}0.036$ and $0.539853{\pm}0.078$, and that of protein kinase C was $0.654817{\pm}0.053$ and $1.146205{\pm}0.052$ respectively, being increased in test group. And differences between the two groups were statistically significant for both enzymes(protein kinase A ; p<0.01, protein kinase C ; p<0.001). Conclusion : These results show that chronic carbamazepine administration increases protein kinase A and C activities, and concerning the possible mode of therapeutic action in bipolar disorder it is suggested that enhanced enzymes phosphorylate receptor-G-protein-effector complexes to dampen hyperfunctioning neuronal activity and thus stabilize the system.

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Phosphorylation of Transcriptional Factor by Mitogen-activated Protein (MAP) Kinase Purified from Nucleus (핵 내에서 분리한 Mitogen-Activated Protein (MAP) Kinase의 Transcription Factor에 대한 인산화)

  • 김윤석;김소영;김태우
    • Biomedical Science Letters
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    • v.2 no.2
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    • pp.175-185
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    • 1996
  • The mitogen-activated protein(MAP) kinase signal transduction pathway represents an important mechanism by which mitogen, such as serum and PMA, regulate cell proliferation and differentiation. Target substrates of the MAP kinase are located within several compartments containing plasma membranes and nucleus. We now report that serum addition induces proliferation of the P388 murine leukemia cell, but PMA does not, while both serum and PMA treatment cause translocation of the MAP kinase, mainly p42$^{mapk}$ isoform, from cytosol into the nucleus, which was monitored by immunoblot analysis using polyclonal anti-ERK1 antibodies. We investigated whether the MAP kinase was capable of phosphorylating c-Jun protein and GST-fusion proteins, the P562$^{kk}$N-terminal peptides (1-77 or 1-123 domain) of the T cell tyrosine kinase, using the partially purified MAP kinase by SP-sephadex C-50, phenyl superose and Mono Q column chromatography. We found that the partially purified MAP kinase was able to phosphorylate c-Jun protein and the GST-fusion protein expressed using E.coli DH5$\alpha$ which is transformed with pGEX-3Xb plasmid vector carrying of p562$^{kk}$N-terminal peptide-encoding DNA. These results imply that tyrosine kinase receptor/Ras/Raf/MAP kinase pathway is a major mechanism for mitogen-induced cell proliferation in P388 murine leukemia cell and that the various MAP kinase isoforms may have their own target substrates located in distinct subcellular compartments.

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Molecular cloning and nucleotide sequence of schizosaccharomyces pombe Homologue of the receptor for activated protein kinase C gene

  • Park, Seung-Keil;Yoo, Hyang-Sook
    • Journal of Microbiology
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    • v.33 no.2
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    • pp.128-131
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    • 1995
  • Using differential hybridization, we selected the prk gene fortuitously from Schizosaccharomyces pombe homologous to RACK1 of rat which encodes the receptor for activated protein kinase C. The cDNA sequence of prk was determined and its deduced amino acid sequence was 76% homologous to RACK1 and had the feature of trimeric G protein bata subunit. The specific amino acid sequences required for the protein kinase C binding were also present in Prk as in the case of RACK1 protein. From these similarities, we suggest that the Prk is protein kinase C binding protein of S. prombe. The involvement of Prk in signal transduction mediated by protein kinase C remained to be studied.

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Effects of Protein Kinase Inhibitors on Melanin Production in B16 Melanoma Cells Stimulated via Cyclic AMP-dependent Pathway (B16 Melanoma 세포에서 Protein Kinase 억제제들이 Cyclic AMP 경로를 통한 멜라닌 생성에 미치는 영향)

  • 차상복;조남영;윤미연;임혜원;김경원;박영미;이지윤;이진희;김창종
    • YAKHAK HOEJI
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    • v.47 no.1
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    • pp.31-36
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    • 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.

Alteration of the Activated Responses in Platelet-Activating Factor-Stimulated Neutrophils by Protein Kinase Inhibitors (Protein Kinase 억제제 첨가 후 Platelet-Activating Factor에 의하여 자극된 호중구반응의 변경)

  • Lee, Kang-Kun;Ko, Ji-Young;Ham, Dong-Suk;Shin, Yong-Kyoo;Lee, Chung-Soo
    • The Korean Journal of Pharmacology
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    • v.32 no.1
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    • pp.103-112
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    • 1996
  • Roles of protein kinase C and protein tyrosine kinase in the activation of neutrophil respiratory burst, degranulation and elevation of cytosolic $Ca^{2+}$ in platelet-activating factor (PAF)-stimulated neutrophils were investigated. Superoxide and $H_2O_2$ production and myeloperoxidase and acid phosphatase release in PAF-stimulated neutrophils were inhibited by protein kinase C inhibitors, staurosporine and H-7 and protein tyrosine kinase inhibitors, genistein and tyrphostin. The PAF-induced elevation of $[Ca^{2+}]_i$ in neutrophils was inhibited by staurosporine, genistein and methyl-2,5-dihydroxycinnamate. Staurosporine inhibited both intracellular $Ca^{2+}$ release and $Mn^{2+}$ influx in PAF-stimulated neutrophils. Genistein and methyl-2,5-dihydroxycinnamate inhibited $Mn^{2+}$ influx induced by PAF, whereas their effects on intracellular $Ca^{2+}$ release were not detected. In neutrophils preactivated by PMA, the stimulatory effect of PAF on the elevation of $[Ca^{2+}]_i$ was reduced. Protein kinase C and protein tyrosine kinase may be involved in respiratory burst, lysosomal enzyme release and $Ca^{2+}$ mobilization in PAF-stimulated neutrophils. The elevation of $[Ca^{2+}]_i$ appears to be accomplished by intracullular $Ca^{2+}$ release and $Ca^{2+}$ influx which are differently regulated by protein kinases. Preactivation of protein kinase C appears to attenuate the stimulatory action of PAF on intracellular $Ca^{2+}$ mobilization.

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Isolation and Biological Activity of Verbascoside, A Potent Inhibitor of Protein Kinase C from the Calyx of Campsis grandiflora (능소화의 꽃받침으로부터 Protein Kinase C 저해물질인 Verbascoside의 분리 및 그 생물활성)

  • 이현선;박문수;오원근;안순철;김보연;김환묵;오구택;민태익;안종석
    • YAKHAK HOEJI
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    • v.37 no.6
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    • pp.598-604
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    • 1993
  • The calyx extract of Campsis grandiflora displayed inhibitory activity against protein kinase C from the bovine brain. Separation guided by protein kinase C enzyme assay and bleb forming assay led to isolation of a potent protein kinase C inhibitor that was identified as a known phenylpropanoid glycoside, verbascoside. It suppressed completely bleb-formation of K562 cell surface induced by phorbol 12,13-dibutylate at the concentration of 60 $\mu\textrm{g}$/ml and IC$_{50}$ of the protein kinase C occured at 20 $\mu{M}$. This compound was tested for cytotoxic activity against ten human tumor cell lines in vitro. it exhibited moderate cytotoxic activity against skin tumor cell line M14 (IC$_{50}$ 2.2 $\mu\textrm{g}$/ml) and very weak cytotoxicity against other cell lines (IC$_{50}$>10 $\mu\textrm{g}$/ml)

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