• Biomedical Science Letters
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• v.2 no.1
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• pp.21-30
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• 1996

MAP kinases are a family of serine/threonine specific protein kinases becoming activated in response to different proliferative stimuli by phosphorylation at both threonine and tyrosine residue. Present study shows that MAP kinase was purified from P388 murine leukema cells by SP sephadex C-50, phenyl superose and Mono Q column chromatography and identified with anti-ERKl antibody by western blotting. Immnublotting analysis to the crude extract of P388 cell lysate shows 44 kD and other minor bands but partial purified fraction eluted from phenyl supherose column have 44kD and 66 kD isoform. Subcloned GST-fusion protein from N-terminal of $p56^{kk}$ was tested as a substrate for MAP kinase phosphorylation. It was showed that the wild type and mutant forms(S42A) were fully phosporylated by purified MAP kinase fraction as com-pare with the other mutant form(S59A). This finding suggest that those GST-fusion proteins may be used as substrate for the in vitro test of MAP kinase.

• YAKHAK HOEJI
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• v.45 no.3
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• pp.287-292
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• 2001

To investigate whether phospholipase $A_2$pathway is involved in histamine release of rat peritoneal mast cells, we measured histamine release in the presence of various enzyme inhibitors involved in eicosanoid pathway, such as phospholipase $A_2$, cyclooxygenase and lipoxygenase. Phospholipase $A_2$inhibitors, manoalide and OPC, significantly inhibited histamine release induced by 100 $\mu$M ATP and 1$\mu$g/ml compound 48/80. Cyclooxygenase inhibitors, ibuprofen and indomethacin, significantly inhibited ATP-induced histamine release and lipoxygenase inhibitors, baicalein and caffeic acid, also significantly inhibited. To investigate the involvement of protein kinase in ATP- and compound 48/80-induced histamine release, we observed effects of protein kinase inhibitors on histamine release. Bisindolmaleimide (protein kinase C antagonist) dose-dependently inhibited both ATP and compound 48/80-induced histamine release. Tyrosine kinase inhibitors (methyl 2,5-dihydroxy cinnamate and genistein) dose-dependently inhibited ATP and compound 48/80-induced histamine release. Protein kinase C and tyrosine kinase seem to be involved in histamine release induced by ATP and compound 48/80. These results suggest that phospholipase $A_2$pathway as well as protein kinase C and tyrosine kinase are involved in histamine release of rat peritoneal mast cells by ATP and compound 48/80.

• The Korean Journal of Pharmacology
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• v.32 no.3
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• pp.417-424
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• 1996

The present study was done to examine the involvement of protein kinase C and protein tyrosine kinase in intracellular $Ca^{2+}$ mobilization in C5a-stimulated neutrophils. Although protein kinase C inhibitors, staurosporine and H-7 inhibited intracellular $Ca^{2+}$ release in C5a-stimulated neutrophils, they did not affect $Ca^{2+}$ influx across the plasma membrane and elevation of $[Ca^{2+}]_i$ C5a-induced intracellular $Ca^{2+}$ release and $Ca^{2+}$ influx were inhibited by protein tyrosine kinase inhibitors, genistein and methyl-2,5-dihydroxycinnamate. ADP-evoked elevation of $[Ca^{2+}]_i$ was inhibited by genistein and methyl-2,5-dihydroxycinnamate but was not affectd by staurosporine and H-7. Genistein and methyl-2,5-dihydroxycinnamate reduced the store-regulated $Ca^{2+}$ influx in thapsigargin-treated neutrophils, while the effect of staurosporine and H-7 was not detected. When neutrophils were preincubated wih phorbol 12-myristate 13-acetate, the stimulatory effect of C5a on the elevation of $[Ca^{2+}]_i$ was reduced. These results suggest that protein tyrosine kinase may be involved in control of intracellular $Ca^{2+}$ release and $Ca^{2+}$ influx across the plasma membrane in C5a-activated neutrophils.

• Archives of Pharmacal Research
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• v.16 no.2
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• pp.114-117
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• 1993

Ginsenosides present in the roots of panax ginseng C.A. Meyer were shown to induce a stimulatory effect on the overexpressed cellular chicken c-src protein tyrossine kinase in NH3T3 cells. Among 4 ginsenosides studied $(G-Rb_2,\;G-Rc,\;G-Re\;and\;G-Rg_1),\;G-Rg_1$ showed the most stimulatory effect at $16.7\mu{g/ml}$ ginsenoside concentration increasing the activity by 2-4 times. Inhibitors of either protein synthesis or RNA synthesis blocked the activation of c-src proein tyrosine kinase. These results suggest that the csrc kinase activation apprars to involve an increase in the amount of protein of the kinase by transcriptional control mechanism rather than an increase in the kinase activity.

• YAKHAK HOEJI
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• v.44 no.2
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• pp.162-168
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• 2000

To investigate the effects of protein kinases on endothelin-1-induced phospholipase C activation and $Ca^{2+}$ mobilization in Rat-2 fibroblast, we measured the formation of inositol phosphates and intracellular $Ca^{2+}$ concentration with [$^3$H]inositol and Fura-2/AM, respectively. Endothelin-1 dose-dependently activated phospholipase C and increased intracellular $Ca^{2+}$ concentration. Protein kinase C activator PMA, significantly inhibited both phospholipase C activity and $Ca^{2+}$ mobilization induced by endothelin-1. Tyrosine kinase inhibitor, genistein, inhibited both. On the other hand, cyclic nucleotide (cAMP and cGMP) did not have any influence on the signaling pathway of phospholipase C-Ca$^{2+}$ mobilization induced by endothelin-1. These results suggest that protein kinase C and tyrosine kinase counteract on the signaling pathway of phospholipase C-Ca$^{2+}$ mobilization induced by endothelin-1 in Rat-2 fibroblast. fibroblast.

• Molecules and Cells
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• v.38 no.7
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• pp.651-656
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• 2015

Plant growth and development are coordinately orchestrated by environmental cues and phytohormones. Light acts as a key environmental factor for fundamental plant growth and physiology through photosensory phytochromes and underlying molecular mechanisms. Although phytochromes are known to possess serine/threonine protein kinase activities, whether they trigger a signal transduction pathway via an intracellular protein kinase network remains unknown. In analyses of mitogen-activated protein kinase kinase (MAPKK, also called MKK) mutants, the mkk3 mutant has shown both a hypersensitive response in plant hormone gibberellin (GA) and a less sensitive response in red light signaling. Surprisingly, light-induced MAPK activation in wild-type (WT) seedlings and constitutive MAPK phosphorylation in dark-grown mkk3 mutant seedlings have also been found, respectively. Therefore, this study suggests that MKK3 acts in negative regulation in darkness and in light-induced MAPK activation during dark-light transition.

• Animal cells and systems
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• v.9 no.3
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• pp.173-179
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• 2005

Cyclic-AMP-dependent protein kinase (PKA) seems to function as a negative regulator of the c-Jun $NH_2-terminal$ kinase (JNK) signaling pathway. We demonstrate here that the activity of the PKA catalytic subunit (PKAc) is reduced in apoptotic PC12 pheochromocytoma cells. Apoptotic progress was inhibited by dibutyryl cyclic AMP (dbcAMP), an analog of cAMP. The rescue by dbcAMP was attributable to inhibition of the JNK but not of the p38 signaling pathway, due to the induction of PKA activity. JNK was present in immunocomplexes of PKAc, and PKAc phosphorylated JNK in vitro. Presence of p38 kinase, however, was not prominent in immunocomplexes of PKAc. Our data suggest that JNK is a target point of negative regulation by PKAc in the JNK signaling pathway.

• Journal of Life Science
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• v.17 no.10
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• pp.1361-1367
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• 2007

I investigated the effect of KT5720, an inhibitor of protein kinase A, on the vesicular stomatitis virus (VSV) infection in BHK-21cell cultures. The virus inducted cytopathic effect (CPE) was almost completely suppressed by KT5720 at 5uM. The inhibitor, however, did not affect replication of the virus nor the synthesis of viral macromolecules. KT5720, did not block the cytoskeletal disruption, while the cell rounding was suppressed. And, the KT5720-sensitive function may be involved in developing the VSV-induced CPE, but not essential for the virus replications.

• 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.

• BMB Reports
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• v.30 no.2
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• pp.89-94
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• 1997

In this study, during the activation of neutrophil responses by sodium fluoride. involvement of protein tyrosine kinase was studied. Respiratory burst lysosomal enzyme release and elevation of $[Ca^{2+}]_i$stimulated by sodium fluoride in neutrophils were inhibited by protein kinase inhibitors, genistein and tyrphostin. The inhibitory effect of genistein and tyrphostin on superoxide and $H_{2}O_{2}$ production was less than that of protein kinase C inhibitors, staurosporine and H-7. Staurosporine and H-7 had little or no effect on the release of myeloperoxidase and acid phosphatase stimulated by sodium fluoride. EGTA and verapamil inhibited the elevation of $[Ca^{2+}]_i$ evoked by sodium fluoride. The inhibitory effect of staurosporine on the elevation of $[Ca^{2+}]_i$ was less than that of genistein. Phorbol 12-myristate 13-acetate (PMA)-stimulated superoxide production, which is sensitive to staurosporine, was further enhanced by genistein, whereas the stimulatory action of PMA on myeloperoxidase release was inhibited by genistein. A pretreatment of neutrophils with PMA signifcantly attenuated sodium fluoride-evoked elevation of $[Ca^{2+}]_i$ These results suggest that protein tyrosine kinase may be involved in the activation process of neutrophil responses due to direct stimulation of guanine nucleotide regulatory proteins. In neutrophil responses, PMA-stimulated neutrophils appear to show a different type of inhibition of protein tyrosine kinase.