• 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.

• 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.

• Journal of Life Science
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• v.8 no.1
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• pp.14-26
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• 1998

a- and c-raf protein kinase in the brain of rat, the protein pattern of cerebellum during postnatal development of rat by polyacryamide gel electrophoresis, and the existence of c-raf protein kinase by using Western blotting method. The results were as follows: The cytoplasm of Purkinje cells was, in general, strongly labeled with the antibodies of a- and c-raf protein kinases in the cortex regions such as Pyramis cerebelli, Unula, Nodulus, Paraflocculus, and Flocculus. C-raf protein kinase appeared stronger immunoreactivity than a-raf protein kinase. In peripheral of cytoplasm of Nucleus emboliformis, A-raf Protein kinase was labeled markedly. During postnatal development, the protein of 38,000 dalton increased gradually in the cytosolic fraction of cerebellum, and the protein of 260,600 dalton appeared in the membrane fraction of cerebellum. By immunoblotting method, the protein band of 74,000 dalton was detected in crude and cytosolic fractions, but it was not exhibited in membrane fraction, In this fact, it was identified that a - and c-raf proteins were distributed throughout neuronal cells, especially in the Purkinje cells, in normal cerebellum cortex of rat. Also, this phenomenon was assumed that raf protein kinase in cytoplasm of neuronal cell had to do with a certain functional mechanism and signal transduction of neurotransmitter as Protein kinase C.

• Animal cells and systems
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• v.1 no.4
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• pp.637-640
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• 1997

Raf-1, a cytoplasmic serine/threonine protein kinase, serves as a central intermediate in many signaling pathways in cell proliferation, differentiation, and development. In this study, we investigated that B-raf, Drosophila homolog of the human c-raf-1, is involved in ultraviolet (UV) responsive events by using hypomorphic mutant $D-raf^{c110}$ and Draf-lacZ transgenic fly. At first, effect of UV damage on the survival of wild-type and $D-raf^{C110}$ strains was examined. In terms of $1/LD_{50}$ value, the relative ratio of UV sensitivities of wild-type versus $D-raf^{C110}$ strain was 1 : 2.2. By using quantitative $\beta$-galactosidase activity analysis, transcriptional activity of the D-raf gene promoter was also examined in UV-irradiated Draf-lacZ transgenic larvae. UV irradiation increased the expression of lacZ reporter gene in Draf-lacZ transgenic fly. However, in $D-raf^{C110}$ strain the transcriptional activity of D-raf gene promoter by UV irradiation was extensively reduced. Results obtained in this study suggest that D-raf plays a role in UV response, leading to better survival of Drosophila to UV damage.

• Journal of Life Science
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• v.9 no.2
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• pp.52-56
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• 1999

The Raf, a cytoplasmic serine/thereonine protein kinase, acts as an important mediator of signals involving cell proliferation, differentiation and development. Multiple regulatory elements should participate in the expression of D-raf, Drosophila homolog of human c-raf-1. In order to search regulatory factors involved in the D-raf promoter activation, we accomplished the yeast one-hybrid screening using D-raf promoter region from bp-330 to -309 with respect to the transcription initiation site as bait. After screening, sixteen independent positive clones of ${\beta}$-galactosidase activties were identified and sequenced. Two clones having 94-98% identity with daughterless and one clone having 93% identity with escargot by Blast search among these clones were screened.

• 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.

• IMMUNE NETWORK
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• v.5 no.4
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• pp.237-246
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• 2005

Background: Little information is available on the identification and characterization of the upstream regulators of the signal transduction cascades for Mycobacterium tuberculosis (M. tbc)-induced ERK 1/2 activation and chemokine expression. We investigated the signaling mechanisms involved in expression of CCL3 /MIP-1 and CCL4/MIP-1 in human primary monocytes infected with M. tbc. Methods: MAP kinase phosphorylation was determined using western blot analysis with specific primary antibodies (ERK 1/2, and phospho-ERK1/2), and the upstream signaling pathways were further investigated using specific inhibitors. Results: An avirulent strain, M. tbc H37Ra, induced greater and more sustained ERK 1/2 phosphorylation, and higher CCL3 and CCL4 production, than did M. tbc H37Rv. Specific inhibitors for mitogen-activated protein kinase (MAPK) kinase (MEK; U0126 and PD98059) significantly inhibited the expression of CCL3 and CCL4 in human monocytes. Mycobactetia-mediated expression of CCL3 and CCL4 was not inhibited by the Ras inhibitor manumycin A or the Raf-1 inhibitor GW 5074. On the other hand, phospholipase C (PLC) inhibitor (U73122) and protein kinase C (PKC)specific inhibitors ($G\ddot{o}6976$ and Ro31-8220) significantly reduced M. tbc-induced activation of ERK 1/2 and chemokine synthesis. Conclusion: These results are the first to demonstrate that the PLC-PKC-MEK-ERK, not the Ras-Raf-MEK-ERK, pathway is the major signaling pathway inducing M. tbc-mediated CCL3 and CCL4 expression in human primary monocytes.

• The Korean Journal of Physiology and Pharmacology
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• v.19 no.6
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• pp.549-555
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• 2015

We attempted to investigate molecular mechanisms underlying phenotypic change of vascular smooth muscle cells (VSMCs) by determining signaling molecules involved in chemokine production. Treatment of human aortic smooth muscle cells (HAoSMCs) with thrombin resulted not only in elevated transcription of the (C-C motif) ligand 11 (CCL11) gene but also in enhanced secretion of CCL11 protein. Co-treatment of HAoSMCs with GF109230X, an inhibitor of protein kinase C, or GW5074, an inhibitor of Raf-1 kinase, caused inhibition of ERK1/2 phosphorylation and significantly attenuated expression of CCL11 at transcriptional and protein levels induced by thrombin. Both Akt phosphorylation and CCL11 expression induced by thrombin were attenuated in the presence of pertussis toxin (PTX), an inhibitor of Gi protein-coupled receptor, or LY294002, a PI3K inhibitor. In addition, thrombin-induced production of CCL11 was significantly attenuated by pharmacological inhibition of Akt or MEK which phosphorylates ERK1/2. These results indicate that thrombin is likely to promote expression of CCL11 via PKC/Raf-1/ERK1/2 and PTX-sensitive protease-activated receptors /PI3K/Akt pathways in HAoSMCs. We propose that multiple signaling pathways are involved in change of VSMCs to a secretory phenotype.

• BMB Reports
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• v.45 no.7
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• pp.414-418
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• 2012

Triglycerides (TG) are implicated in the development of atherosclerosis through formation of foam cells and induction of macrophage cell death. In this study, we report that addition of exogenous TG induced cell death in phorbol 12-myristate 13-acetate-differentiated THP-1 human macrophages. TG treatment induced a dramatic decrease in interleukin-$1{\beta}$ (IL-$1{\beta}$) mRNA expression in a dose- and time-dependent manner. The expression of granulocyte macrophage colony-stimulating factor and platelet endothelial cell adhesion molecule remained unchanged. To identify signaling pathways involved in TG-induced downregulation of IL-$1{\beta}$, we added p38 MAPK, protein kinase C (PKC) or c-Raf1 specific inhibitors. We found that inhibition of p38 MAPK alleviated the TG-induced downregulation of IL-$1{\beta}$, whereas inhibition of PKC and c-Raf1 had no effect. This is the first report showing decreased IL-$1{\beta}$ expression during TG-induced cell death in a human macrophage line. Our results suggest that downregulation of IL-$1{\beta}$ expression by TG-treated macrophages may play a role during atherogenesis.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.20
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• pp.8539-8548
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• 2014

Mitogen-activated protein kinase (MAPK) is an important signaling pathway in living beings in response to extracellular stimuli. There are 5 main subgroups manipulating by a set of sequential actions: ERK(ERK1/ERK2), c-Jun N(JNK/SAPK), p38 MAPK($p38{\alpha}$, $p38{\beta}$, $p38{\gamma}$ and $p38{\delta}$), and ERK3/ERK4/ERK5. When stimulated, factors of upstream or downstream change, and by interacting with each other, these groups have long been recognized to be related to multiple biologic processes such as cell proliferation, differentiation, death, migration, invasion and inflammation. However, once abnormally activated, cancer may occur. Several components of the MAPK network have already been proposed as targets in cancer therapy, such as p38, JNK, ERK, MEK, RAF, RAS, and DUSP1. Among them, alteration of the RAS-RAF-MEK-ERK-MAPK(RAS-MAPK) pathway has frequently been reported in human cancer as a result of abnormal activation of receptor tyrosine kinases or gain-of-function mutations in genes. The reported roles of MAPK signaling in apoptotic cell death are controversial, so that further in-depth investigations are needed to address these controversies. Based on an extensive analysis of published data, the goal of this review is to provide an overview on recent studies about the mechanism of MAP kinases, and how it generates certain tumors, as well as related treatments.