• Journal of Applied Biological Chemistry
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• v.42 no.1
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• pp.19-24
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• 1999

Plants have been shown to contains $Ca^{2+}$/calmodulin-stimulated GAD and NAD kinase. To test how calmodulin and calmodulin methylation affect the activation of GAD and NAD kinase, GAD and NAD kinase were partially purified from tobacco plants. GAD was also partially purified from E. coli transformed with a plasmid carrying a cloned tobacco GAD gene. We find that GAD from the transformed E. coli showed 60-fold $Ca^{2+}$/calmodulin-dependent activation. However, GAD from tobacco plants was stimulated only about 3.8-fold by the addition of calmodulin in the presence of calcium, suggesting high background activity of the enzyme was possibly due to bound endogenous tobacco calmodulin. There were no significant differences in the tobacco GAD activator properties between calmodulins. A monoclonal antibody against petunia GAD interacted strongly with both GAD from tobacco plants and GAD from cloned gene. NAD kinase from tobacco plants showed a complete $Ca^{2+}$/calmodulin dependency for activity. Unmethylated calmodulins activated GAD in a manner similar to methylated calmodulin. However, the maximum level of NAD kinase activation obtained with unmethylated calmodulins is approximately 4-fold higher than methylated calmodutins. These data suggested that endogenous tobacco calmodulin may interact more tightly with GAD than NAD kinase and that calmodulin methylation affects the activator properties of calmodulins for tobacco NAD kinase but not for GAD.

• Rapid Communication in Photoscience
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• v.2 no.2
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• pp.56-59
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• 2013

Plant phytochromes, molecular light switches that regulate various aspects of plant growth and development, are known as autophosphorylating serine/threonine kinases. Although recent studies reveal that phytochrome autophosphorylation plays an important role in the regulation of phytochrome signaling through the control of phyA protein stability, the in vivo functional roles of phytochrome kinase activity in plant light signaling are largely unknown. Thus, it is necessary to investigate the detailed function of phytochrome as a protein kinase, which might include mapping of kinase domain on the phytochrome molecule, searching for substrates that could be phosphorylated by phyA, and in vivo functional analysis of the kinase activity with phytochrome mutants displaying reduced kinase activity. Our recent studies reveal that the kinase activity of phytochrome plays a positive role in plant light signaling. Therefore, we highlight the current knowledge about the functional roles of phytochrome kinase activity in the light signal transduction of plants, based on our recent results.

• 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.

• Molecules and Cells
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• v.21 no.2
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• pp.294-301
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• 2006

We have previously reported that phosphorylation of eukaryotic elongation factor 2 (eEF2) is related to the differentiation of chick embryonic muscle cells in culture. In the present study, we found that eEF2 phosphorylation declined shortly after induction of differentiation of L6 myoblasts, when the cells prepare for terminal differentiation by withdrawing from the cell cycle. This decrease in phosphorylation was prevented by inhibitors of phosphoinositide 3-kinase (PI3-kinase) that strongly inhibit myoblast differentiation. We hypothesized that PI3-kinase plays an important role in myoblast differentiation by regulating eEF2 phosphorylation in the early stages of differentiation. To test this hypothesis, myoblasts were synchronized at in $G_2/M$ and cultured in fresh differentiation medium (DM) or growth medium (GM). In DM the released cells accumulated in $G_0$/$G_1$ while in GM they progressed to S phase. In addition, cyclin D1 was more rapidly degraded in DM than in GM, and eEF2 phosphorylation decreased more. Inhibitors of PI3-kinase increased eEF2 phosphorylation, but PI3-kinase became more activated when eEF2 phosphorylation declined. These results suggest that the regulation of L6 myoblast differentiation by PI3-kinase is related to eEF2 phosphorylation.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.32 no.2
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• pp.112-117
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• 2010

Aurora kinases represent a novel family of serine/threonine kinases crucial for cell cycle control. Aurora-2 kinase is mainly involved in centrosome function, mitotic entry, and spindle assembly. Aurora-2 kinase overexpression causes centrosome amplification and the formation of multipolar mitotic spindles, which leads to tumor aneuploidy and so it has been found to play an important role in tumorigenicity in many cancers such as colorectal cancer, breast cancer and cervical cancer. Hence, the goal of this study is to identify the correlation of clinicopathlogical factors and overexpression of Aurora-2 kinase in oral squamous cell carcinoma. We studied the immunohistochemical staining of Aurora-2 kinase in 20 specimens of 20 patients with oral squamous cell carcinoma and the relationships between Aurora-2 kinase over expression and each of the clinico-pathological parameters were analyzed by Pearson correlation analysis. Statistical significance was set at P < 0.05. The results were as follows. 1. In the immunohistochemical study of poorly differentiated and invasive oral squamous cell carcinoma, the high level staining of Aurora-2 kinase was observed. 2. The correlation between immunohistochemical Aurora-2 kinase expression and histopathological differentiation of specimens was significant. These findings suggest that overexpression of Aurora-2 kinase may play a important role in carcinogenesis of oral squamous cell carcinoma.

• Molecular & Cellular Toxicology
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• v.4 no.3
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• pp.224-229
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• 2008

Eucommia ulmoides (Duchung) is commonly used for treatment of diabetes in Korean traditional medicine. However, the exact mechanism of its anti-diabetic effect has not yet been fully elucidated. In this study, the effect of E. ulmoides extract on glucose uptake was investigated in L6 rat skeletal muscle cells. E. ulmoides extract stimulated the activity of phosphatidylinositol (PI) 3-kinase that is a major regulatory molecule in glucose uptake pathway. Protein kinase B (PKB) and protein kinase C-${\xi}$ (PKC-${\xi}$), downstream mediators of PI 3-kinase, were also activated by E. ulmoides extract. We assessed the activity of AMP-activated protein kinase (AMPK), another regulatory molecule in glucose uptake pathway. Phosphorylation level of AMPK did not change with treatment of E. ulmoides extract. Phosphorylations of p38 mitogen activated protein kinase (p38 MAPK) and acetyl-CoA carboxylase (ACC), downstream mediators of AMPK, were not significantly different. Taken together, our results suggest that E. ulmoides may stimulate glucose uptake through PI 3-kinase but not AMPK in L6 skeletal muscle cells.

• Toxicological Research
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• v.17
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• pp.251-256
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• 2001

The expression of phase II detoxifying enzymes is affected by a variety of compounds and the induction of the enzymes plays an essential role in chemoprevention. A variety of phytochemicals such as sulfur-containing chemoprotective agents (SCC) may trigger cellular signals and activate phase II gene expression through ARE activation. see induces glutathione S-transferases. Studies were conducted to investigate the role of mitogen-activated protein (MAP) kinase and phosphatidylinositol 3-kinase (PI3-kinase) in the induction of GST (e.g. rGSTA2) by sec. We also studied the MAP kinase pathway responsible for the GST expression by see and compared that with the pathway activated by oxidative stress as a result of sulfur amino acids deprivation (SAAD). see inhibited phosphorylation of ERK1/2 although the effect of see on JNK and p38 MAP kinase was minimal. Wortmannin and LY294002. PI3-kinase inhibitors. abolished the increases in rGSTA2 mRNA and protein levels by SCC. Deprivation of cystine and methionine caused oxidative stress in H4IIE cells. as evidenced by a decrease in the reduced glutathione and an increase in prooxidant production. Electrophoretic mobility shift assay revealed that the ARE complex consisting of Nrf-1/2 and Maf proteins was activated 12~48 h. The rGSTA2 mRNA and protein levels were increased by SAAD. Activation of ARE and induction of rGSTA2 were both completely inhibited by PI3-kinase inhibitors. Inhibition of p38 MAP kinase by SB203580 prevented the ARE-mediated rGSTA2 induction. The results of this study showed that PI3-kinase might play an essential role in the ARE-mediated rGSTA2 induction by see or SAAD and that the dual MAP kinase pathways were responsible for the enzyme induction.

• Asian-Australasian Journal of Animal Sciences
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• v.16 no.8
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• pp.1199-1204
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• 2003

The aim of this study was to investigate the role of phosphatidylinositol 3-kinase (PI3 kinase) and the mitogenactivating protein (MAP) kinase pathway on the differentiation of ovine preadipocytes. In order to investigate this issue, we monitored glycerol 3-phosphate dehydrogenase (GPDH) activity during differentiation with specific inhibitors of PI3 kinase and MAP kinase-Erk kinase, LY294002 and PD098059, respectively. The preadipocytes, which were obtained from ovine subcutaneous adipose tissues, were proliferated to confluence and then differentiated to adipocytes in differentiation medium with each inhibitor for 10 days. The confluent preadipocytes and differentiated adipocytes at days 3, 7 and 10 were harvested for assay of GPDH activity. LY294002 inhibited the differentiation program in dose- and day-dependent manners during 10 days of differentiation. PD098059 did not affect GPDH activity during differentiation. Furthermore, the expression of peroxisome proliferator-activated receptor ${\gamma}2$ (PPAR-${\gamma}2$), the representative early gene of differentiation, was markedly reduced by LY294002 treatment, although PD098059 did not change it. Our results demonstrated that the activation of PI3 kinase contributes to the differentiation process of ovine preadipocytes.

• Biomedical Science Letters
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• v.25 no.2
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• pp.177-184
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• 2019

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of Parkinson's disease (PD). LRRK2 contains a functional kinase and GTPase domains. A pathogenic G2019S mutation that is the most prevalent among the LRRK2 mutations and is also found in sporadic cases, increases its kinase activity. Therefore, identification of LRRK2 kinase substrates and the development of kinase inhibitors are under intensive investigation to find PD therapeutics. Several recent studies have suggested members of Rab proteins, a branch of the GTPase superfamily, as LRRK2 kinase substrates. Rab proteins are key regulators of cellular vesicle trafficking. Among more than 60 members of human Rab proteins, Rab3, Rab5, Rab8, Rab10, Rab12, Rab29, Rab35, and Rab43 have been identified as LRRK2 kinase substrates. However, most studies have used human embryonic kidney (HEK) 293T cells overexpressing LRRK2/Rab proteins or murine embryonic fibroblast (MEF) cells which are not relevant to PD, rather than neuronal cells. In this study, we tested whether Rab proteins are phosphorylated by LRRK2 in astroglia in addition to neurons. Among the various Rab substrates, we tested phosphorylation of Rab10, because of the commercial availability and credibility of the phospho-Rab10 (pRab10) antibody, in combination with a specific LRRK2 kinase inhibitor. Based on the results of specific LRRK2 kinase inhibitor treatment, we concluded that LRRK2 phosphorylates Rab10 in the tested brain cells such as primary neurons, astrocytes and BV2 microglial cells.

• BMB Reports
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• v.43 no.11
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• pp.711-719
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• 2010

Kinomics is an emerging and promising approach for deciphering kinomes. Chemical kinomics is a discipline of chemical genomics that is also referred to as "chemogenomics", which is derived from chemistry and biology. Chemical kinomics has become a powerful approach to decipher complicated phosphorylation-based cellular signaling networks with the aid of small molecules that modulate kinase functions. Moreover, chemical kinomics has played a pivotal role in the field of kinase drug discovery as it enables identification of new molecular targets of small molecule kinase modulators and/or exploitation of novel functions of known kinases and has also provided novel chemical entities as hit/lead compounds. In this short review, contemporary chemical kinomics technologies such as activity-based protein profiling, T7 kinasetagged phages, kinobeads, three-hybrid systems, fluorescenttagged kinase binding assays, and chemical genomic profiling are discussed along with a novel allosteric Bcr-Abl kinase inhibitor (GNF-2/GNF-5) as a successful application of chemical kinomics approaches.