• BMB Reports
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• v.36 no.4
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• pp.421-425
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• 2003

The serine/threonine protein kinase family is a large and diverse group of enzymes that are involved in the regulation of multiple cellular pathways. Elevated kinase activity has been implicated in many diseases and frequently targeted for the development of pharmacological inhibitors. Therefore, non-radioactive antibody-based kinase assays that allow high throughput screening of compound libraries have been developed. However, they require a generation of antibodies against the phosphorylated form of a specific substrate. We report here a time-resolved fluorescence assay platform that utilizes a commercially-available generic anti-phosphothreonine antibody and permits assaying kinases that are able to phosporylate threonin residues on protein substrates. Using this approach, we developed an assay for Cdc7/Dbf4 kinase activity, determined the $K_m$ for ATP, and identified rottlerin as a non-ATP competitive inhibitor of this enzyme.

• Microbiology and Biotechnology Letters
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• v.35 no.2
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• pp.112-117
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• 2007

Protein kinases play very important role for maintaining viability in prokaryote and eukaryote. The metabolism of prokaryotic cell is generally regulated by bacterial two-component regulatory systems that are composed of histidine and asparitic acid kinases, however, some eukaryotic signal transduction system such as, serine and threonine kinases, have been also found to be involved in the regulation of morphogenesis and physiological differentiation in Streptomyces. Streptomyces griseus, a streptomycin producer, was expected to have varlous types of eukaryotic-type serine/threonine protein kinases, controlling morphogenesis. Thus, many steps of chromatographies were applied to isolate serine and threonine kinases from S. griseus IFO13350. The immunoaffinity steps using anti-phosphoserine, anti-phosphothreonine, and anti-phosphotyrosine agarose column chramatographies were successfully introduced to identify eukaryotic protein kinases from S. griseus IFO13350. Eight proteins with the expected molecular weight of 14, 29, 31, 35, 40, 52, 56, and 60 kDa, were identified on SDS-PAGE, and the their kination activity was confirmed by nonradioactive protein kination assay using FITC-labeled peptide as the substrate.

• Animal cells and systems
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• v.1 no.1
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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.

• 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.

• Proceedings of the Korean Society of Potoscience Conference
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• spring
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• pp.21-21
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• 1997

• Animal cells and systems
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• v.11 no.2
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• pp.205-213
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• 2007

Nek6 belongs to NIMA1 (never in mitosis, gene A) related kinase, which was originally identified in Aspergillus nidulans as a serine/threonine kinase critical for cell cycle progression. We noticed that the putative SUMOylation site is localized on the $K^{252}$ residue in $^{251}FKsD^{254}$ of Nek6, based on the consensus sequence ${\Phi}KxE$; where ${\Phi}$ represents L, I, V or F and x is any amino acid. We observed that the Nek6 SUMO mutant (K252R) has decreased protein kinase activity, nuclear speckle localization and protein stability, compared with that of the Nek6 wild type. However, the Nek6 SUMO mutant increased the cell survival rate of COS-1 cells as determined by FACS analysis. Therefore, our data suggest that SUMOylation on the $K^{252}$ residue of Nek6 is required for its normal functions, such as proper nuclear localization, kinase activity and protein stability, to control cell cycle.

• Genomics & Informatics
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• v.6 no.1
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• pp.44-49
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• 2008

Protein kinase C (PKC) is a family of kinases involved in the transduction of cellular signals that promote lipid hydrolysis. PKC plays a pivotal role in mediating cellular responses to extracellular stimuli involved in proliferation, differentiation and apoptosis. Comparative analysis of the PKC-${\alpha},{\beta},{\varepsilon}$ isozymes of 200 recently sequenced microbial genomes was carried out using variety of bioinformatics tools. Diversity and evolution of PKC was determined by sequence alignment. The ser/thr protein kinases of Streptomyces coelicolor A3 (2), is the only bacteria to show sequence alignment score greater than 30% with all the three PKC isotypes in the sequence alignment. S.coelicolor is the subject of our interest because it is notable for the production of pharmaceutically useful compounds including anti-tumor agents, immunosupressants and over two-thirds of all natural antibiotics currently available. The comparative analysis of three human isotypes of PKC and Serine/threonine protein kinase of S.coelicolor was carried out and possible mechanism of action of PKC was derived. Our analysis indicates that Serine/ threonine protein kinase from S. coelicolor can be a good candidate for potent anti-tumor agent. The presence of three representative isotypes of the PKC super family in this organism helps us to understand the mechanism of PKC from evolutionary perspective.

• Journal of Life Science
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• v.16 no.6
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• pp.955-959
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• 2006

A dynamic cycle of addition and removal of O-linked N-acetylglucosamine (O-GlcNAc) at serine and threonine residues is emerging as a key regulator of nuclear and cytoplasmic protein activity. In this work, immunocytochemistry was carried out to investigate the subcellular expression of GlcNAc kinase (NAGK, EC 2.7.1.59) that catalyzes the phosphorylation of GlcNAc to GlcNAc 6-phosphate. Immunostainings of cultured rat hippocampal neurons revealed patchy or punctate distribution of NAGK. When NAGK is doublestained with caveolin-1 or flotillin, markers for caveolae and lipid rafts, respectively, NAGK was co-localized with these markers. These results indicate that most, if not all, of the NAGK immunopunctae represent caveolae and lipid rafts, and suggest NAGK's role in these membrane microdomains.

• BMB Reports
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• v.40 no.5
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• pp.749-756
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• 2007

Phosphorylation on serine/threonine or tyrosine residues of target proteins is an essential and significant regulatory mechanism in signal transduction during many cellular and life processes, including spermatogenesis, oogenesis and fertilization. In the present work, we reported the isolation and characterization of mouse testis-specific serine/threonine kinase 5 (Tssk5), which contains four alternatively spliced variants including, Tssk5$\alpha$, Tssk5$\beta$, Tssk5$\gamma$ and Tssk5$\delta$. Moreover, the locus of Tssk5 is on chromosome 14qC3 and the four variants had a similar high expression in the testis and the heart; however, had a low expression in other tissues, except for Tssk5$\alpha$ which also had comparably high expression in the spleen. Each variant of Tssk5 expression began in the testis 16 days after birth. Aside from TSSK5$\alpha$, the other isoforms have an insertion of ten amino acid residues (RLTPSLSAAG) in region VIb (HRD domain) (His-Arg-Asp). Moreover, only TSSK5$\alpha$ exhibited kinase activity and consistently, a further Luciferase Reporter Assay demonstrated that TSSK5$\beta$, TSSK5$\gamma$ and TSSK5$\delta$ cannot be stimulated at the CREB/CRE responsive pathway in comparison to TSSK5$\alpha$. These findings suggest that TSSK5$\beta$, TSSK5$\gamma$, TSSK5$\delta$ may be pseudokinases due to the insertion, which may damage the structure responsible for active kinase activity. Pull-down assay experiments indicated that TSSK5$\beta$, TSSK5 $\gamma$ and TSSK5$\delta$ can directly interact with TSSK5$\alpha$. In summary, these four isoforms with similar expression patterns may be involved in spermatogenesis through a coordinative way in testis.

• 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.