• 한국동물학회지
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• 제33권2호
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• pp.141-151
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• 1990

Raf protein kinases and protein kinase C는 세포질내 serine/threonine-specific protein에 속한다. 그리고 기능적인 구조와 세포내의 분포 양상은 서로 비슷하다. Raf family oncogene를 발현시키는 a-raf와 c-raf protein kinase에 대한 antibodies로써 남생이 대뇌의 raf protein kinase의 분포를 조사하였다. 일반적으로 raf protein kinase는 제한된 지역에서 즉,general pallium,hippocampal formation, pdmordiuin hippocampi,nucleus of lateral olfactory tract, basal amygdaloid nucleus와bed of stria terminalis에 나타났으며, c-raf protein kinase의 면역학적 labeling은 a-raf보다 그 범위가 넓었다. 그렇지만 labeling되는 intensity는 오히려 a-raf보다 낮았다. 그런데 a-raf에서 가장 명확한 좋은 예는 basal amygdaloid nucleus내의 구형모양의 세포인데, 이 세포는 세포질이 매우 강하게 labeling되어 지므로 ring모양과 같이 나타났다. 특히 c-raf는 protein kinase C 가 많이 나타나는 pyramidal 세포나 Purkinje세포에 많이 존재하는 것을 볼 때 protein kinase에 의하여 활성화되는 myc와 서로 상협작용을 유도한다고 제안하는 바이다.

• 한국동물학회지
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• 제33권3호
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• pp.266-275
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• 1990

Onocogene의 일종인 c-raf protein kinase는 세포질 속에 존재하는 serine / threonine-speccific protein이며, 이것은 mitogene signal에 의해 활성화된다. c-raf protein kinase의 구조와 기능은 protein kinase C와 매우 유사한 것으로 생각된다. 면역세포화학적으로 c-raf protein kinase의 signal transduction을 조사하기 위하여 EC-4 세포에 tumor promotor인 12-0-tet-radecanoylphorbol-13-acetae와 mitogenic gactor인 platelet-derived growth factor로 time-course에 따라서 처리하였다. Translocotion되는 c-raf는 먼저 perinuclear membrane에 모이고 그 후에 핵내로 이동되었다. 그런데 TPA와 PDGF로 처리한 c-raf의 translocotion은 각각의 다른 경로를 가짐을 알 수 있었다. TPA와 PAGF을 장기간 처리하였을 때, c-raf protein kinase의 down regulation이 유도됨을 알 수 있었다.

• 생물정신의학
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• 제5권2호
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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.

• 한국동물학회지
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• 제36권4호
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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).

• 한국동물학회지
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• 제32권2호
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• pp.153-162
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• 1989

난자성숙 재개와 1-세포기 배아의 세포주기에서, cAMP-의존성 protein kinase A와 diacylglycerol-의존성 protein kinase C가 핵막붕괴에 미치는 영향을 조사하였다. 난자성숙 재기는 dbcAMP, IBMX, TPA, 또는 diacyllycerol에 의해 억제되었다. 또한 protein kinase A와 protein kinase C 활성제를 같이 처리하면 난자성숙이 더욱 억제되었다. 그러나 1-세포기 배아의 전핵막붕괴에는 아무런 영향도 미치지 못하였으며, 단지 protein kinase C 활성제만이 세포질 분열을 억제하였다. 이상의 결과로부터, protein kinase A와 protein kinase C에 의한 단백질 인산화 양상이 GV난자의 핵막붕괴와 1-세포기 배아의 전핵막붕괴에 미치는 세포내 작용기작은 상이함을 알 수 있었으며, 전기영동 결과, 81 KD 단백질이 난자성숙 재개에 중요한 역할을 하리라 사료되었다.

• 대한의생명과학회지
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• 제2권2호
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• pp.175-185
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• 1996

모든 진핵세포에 존재하며 세포의 성장 및 분화에 주로 관계되는 신호전달물질의 하나인 Mitogen-activated protein(MAP)kinase의 mitogen에 의한 핵내 활성화와 기질 인산화에 대해 알아보기 위해 본 실험을 수행하였다. P388세포를 10% fetal bovine serum이 첨가된 DMEM배지에 배양한 후, 혈청이 들어있지 않은 배지에서 24시간 더 배양하고 serum 및 PMA를 농도별로 처리하여 세포성 장을 위한 최적 농도를 확인한 결과 serum은 5-20% 농도에서 세포성장을 촉진시켰고 PMA는 실험한 모든 농도에서 세포성장을 거의 촉진시키지 못하는 경향을 확인하였다. 이어 P388 세포를 serum 및 PMA로 10 분간 활성화하여 파쇄한후 세포질분획과 핵분획으로 분리하여 각 분획을 10% gel 상에서 전기영동 하여 nitrocellulose paper에 옳긴 후 anti-ERKI antibody를 이용해 확인해본 결과 serum, PMA로 처리된 세포 모두에서 MAP kinase의 핵내 이동이 관찰되었으며 특히 세포질 내에 주로 존재하는 42, 44 Kd의 MAP kinase isoform중 42 Kd의 isoform이 주로 핵내로 이동되는 것이 관찰되었다. MAP kinase의 기질인산화 실험을 위해 serum으로 활성화시킨 세포를 파쇄하여 SP-sephadex C-50, Phenyl superose, Mono Q column의 순서로 chromatography를 시 행하여 MAP kinase를 부분분리 하였다. 이와 같이 얻은 MAP kinase를 가지고 면역 T세포에 존재하는 tyrosine kinase인 $p56^{lck}$ 의 N-terminal peptide로 구성된 GST-fusion protein에 대한 인산화를 확인하였다. 또한 세포에서 분리한 MAP kinase를 가지고 transcription factor의 하나인 c-Jun protein에 대한 인산화실험을 실시한 결과 MAP kinase에 의해 인산화 됨이 확인되었다. 이상의 결과를 통해 P388세포는 (1)세포 성장시 외부 신호를 G-protein-coupled receptor/protein kinase C/MAP kinase의 경로보다는 주로 tyrosine kinase receptor protein/Ras/MAP kinase의 경로를 이용하여 핵으로 전달하는 것으로 추측되 며 (2) mitogen의 처리로 활성화된 MAP kinase중 주로 42 Kd isoform이 핵내로 이동하고, 분리한 MAP kinase가 GST-fusion protein과 transcription factor인 c-Jun을 모두 인산화 시키는 결과로 보아 MAP kinase의 isoform에 따라 표적 compartment가 다르고 결과적으로 표적 기질에 차이가 있을지 모른다고 간접적으로 추론할 수 있다.

• 한국동물학회지
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• 제36권3호
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• pp.367-372
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• 1993

재생쥐간에서 분리한 topoisomerase II에서 protein kinase 활성이 발견되었다. ,topo II 활성 및 kinase 활성은 hydroxyapatite, phosphocellulose, double strand DNA cellulose chromatography 등의 순수 분리 과정 중에도 서로 분리되지 않았으며 glycerol gradient sedimentation 분석에서도 같은 분획에서 활성이 존재하였다. Kinase는 topo II 저해제인 N-ethylmaleimide와 novobiocin 등에 의해 그 활성이 저해되었다. 그러나 이러한 증거들 만으로 kinase 활성이 topo II가 아닌 다른 polypeptide에 의한 것일 가능성을 완전히 배제 할 수는 없다. Topo II와 결합된 kinase 활성에는 Mg++가 절대적으로 필요하였으며 다른 일가 또는 이가 이온으로는 그 효과가 대체되지 않았다. Histone H1은 kinase 활성을 증가 시키며 또 kinase에 의해 강하게 인산화된다. 이러한 효과는 다른 histone 류 및 casein 등에 의해 대체되지 않았다.

• Animal cells and systems
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• 제1권1호
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• pp.135-142
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• 1997

Random sequencing of expressed sequence tags in roots of Chinese cabbage led to isolation of a partial cDNA clone, BR77, which encoded a putative protein kinase. Using the BR77 cDNA as a probe, we isolated a full-length cDNA encoding the Brassica campestris protein kinase 1 (Bcpk1). The Bcpt1 cDNA contained one open reading frame encoding a polypeptide of 439 amino acids. The putative polypeptide consisted of a short N-terminal region and a protein kinase catalytic domain. The catalytic domain of Bcpkl showed a high homology to cAMP- and calcium- phospholipid-dependent subfamilies of serine/threonine protein kineses. Eleven major catalytic domains in protein kineses were well conserved in Bcpk1. However, Bcpk1 contained a unique nonhomologous intervening sequence between subdomains VII and VIII, which was not found in protein kineses of animals and lower eukaryotes. Genomic DNA gel blot analysis showed that Bcpt1 genes might be present as three copies in the Chinese cabbage genome. These imply that Bcpk1 belongs to a plant-specific serine/threonine protein kinase subfamily.

• 약학회지
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• 제37권6호
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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)

• BMB Reports
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• 제31권3호
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• pp.221-226
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• 1998

Phosphorylation of phytochrome may play important functional roles to control plant photomorphogenesis. Many attempts have failed to identify the protein kinase that phosphorylates phytochrome in vivo. It has been reported that a polycation-stimulated protein kinase activity was associated with the purified phytochrome. However, it is not known if the kinase activity is an intrinsic property of phytochrome or whether it comes from a contaminant of the purified phytochrome. In the present study, three protein kinases that phosphorylate phytochrome have been identified from etiolated oat seedlings. A polycationstimulated protein kinase that had very similar enzymatic properties with that associated with the purified phytochrome was identified in the cytosolic extract. It phosphorylated several contaminant proteins in the kinase preparation as well as phytochrome and had a broad substrate specificity. A CK II-type protein kinase phosphorylated phytochrome and the exogenously added casein. It is likely that this kinase may not be a feasible candidate for the kinase phosphorylating phytochrome in vivo since the content of the kinase seemed to well exceed the content of phytochrome in the etiolated oat seedlings. Another protein kinase that had unique enzymatic properties phosphorylated phytochrome very specifically and seemed to be present in a small quantity in the etiohlted seedlings. It is expected that one of three kinases may be responsible for the phytochrome phosphorylation in vivo.