• 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.

• 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.

• 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.

• 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.

• 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.

• Microbiology and Biotechnology Letters
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• v.37 no.2
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• pp.105-109
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• 2009

Among signal transduction systems by protein phosphorylation Akt/PKB protein kinase which is one of serine/threonine kinases, is known to regulate the survival and death of the cell and glucose metabolism. Thus, Akt/PKB protein kinase has been used as one of the target proteins to find anti-cancer agents from natural products. In this study, human Akt/PKB protein kinase was expressed in Escherichia coli expression system for the mass production. Human Akt/PKB protein kinase expressed in E. coli formed inclusion body under the general condition. However, most of the expressed protein was solubilized under the culture temperature at $27^{\circ}C$ and 0.01-0.09 mM of IPTG for induction of the protein expression. The expressed protein was purified using $Ni^{2+}$-NTA agarose column and confirmed by using anti-Akt antibody. Subsequently, the purified human Akt/PKB protein kinase was activated by in vitro phosphorylation using cellular extract containing kinases. The activated protein was confirmed to phosphorylate the specific fluorescent peptide specially designed as the artificial substrate for Akt/PKB protein kinase.

• 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.

• Molecules and Cells
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• v.44 no.7
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• pp.444-457
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• 2021

Although the mechanism of chronic myeloid leukemia (CML) initiation through BCR/ABL oncogene has been well characterized, CML cell differentiation into erythroid lineage cells remains poorly understood. Using CRISPR-Cas9 screening, we identify Chromobox 8 (CBX8) as a negative regulator of K562 cell differentiation into erythrocytes. CBX8 is degraded via proteasomal pathway during K562 cell differentiation, which activates the expression of erythroid differentiation-related genes that are repressed by CBX8 in the complex of PRC1. During the differentiation process, the serine/threonine-protein kinase PIM1 phosphorylates serine 196 on CBX8, which contributes to CBX8 reduction. When CD235A expression levels are analyzed, the result reveals that the knockdown of PIM1 inhibits K562 cell differentiation. We also identify TRIM28 as another interaction partner of CBX8 by proteomic analysis. Intriguingly, TRIM28 maintains protein stability of CBX8 and TRIM28 loss significantly induces proteasomal degradation of CBX8, resulting in an acceleration of erythroid differentiation. Here, we demonstrate the involvement of the CBX8-TRIM28 axis during CML cell differentiation, suggesting that CBX8 and TRIM28 are promising novel targets for CML research.

• Journal of the Korean Magnetic Resonance Society
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• v.19 no.2
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• pp.88-94
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• 2015

Bub1 is one of the spindle checkpoint proteins and plays a role in recruitment of the related proteins to kinetochore. Here, we studied the structural characteristic of the evolutionarily conserved 160 amino acid region in the N-terminus (hBub1 CD1), using Circular Dichroism (CD) and NMR. Our CD results showed that hBub1 CD1 is a highly helical protein and its structure was affected by pH: as pH was elevated to basic pH, the helical propensity increased. This could be related to the surface charge of the hBub1 CD1. However, the structural change did not largely depend on the salt concentration, though the thermal stability a little increased. The previous NMR analysis revealed that the hBub1 CD1 adopts eight helices, which is consistent with the CD result. Our result would be helpful for evaluating the molecular mechanism of the hBub1 CD1 and protein-protein interactions.