• 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.

• Biomolecules & Therapeutics
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• v.2 no.1
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• pp.39-46
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• 1994

The present study was undertaken to demonstrate whether or not bradykinin activates a phospholipase D in rabbit kidney proximal tubule cells. By measuring the formation of [$^3$H]phosphatidic acid and [$^3$H]phosphatidylethanol we could elucidate the direct stimulation of phospholipase D by bradykinin. Bradykinin leads to a rapid increase in [$^3$H]phosphatidic acid and [$^3$H]diacylglycerol, and [$^3$H]phosphatidic acid formation preceded the formation of [$^3$H]diacylglycerol. This result suggests that some phosphatidic acid seems to be formed directly from phosphatidylcholine by the action of phospholipase D, not from diacylglycerol by the action of diacylglycerol kinase. In addition, the other mechanisms by which phospholipase D is activated was examined. We have found that phospholipase D was activated and regulated by extracellular calcium ion and pertussis toxin-insensitive G protein, respectively. It has also been shown that bradykinin may activate phospholipase D through protein kinase C-dependent pathway. In conclusion, we are now, for the first time, strongly suggesting that bradykinin-induced activation of phospholipase D in the rabbit kidney proximal tubule cells is mediated by a pertussis toxin-insensitive G protein and is dependent of protein kinase C.

• The Korean Journal of Pharmacology
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• v.27 no.2
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• pp.135-144
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• 1991

Using T-lymphocytes obtained from rat peripheral blood, we found that the 44kD/pI6.8 protein was the major phosphoprotein of T-lymphocytes under basal condition, and that the 44kD/pI6.3 protein was a new phosphoprotein appeared in T-lymphocytes stimulated with ${\beta}-agonist$. The phosphorylation of the 44kD/pI6.3 protein was also induced by forskolin but inhibited by H-8 pretreatment. To clarify the character of the 44kD/pI6.3 protein, we used Con-A and kinase inhibitors, H-7 and W-7. Con-A stimulation induced phosphorylation of 44kD/pI 6.3 protein but that was inhibited by W-7 pretreatment. The phosphorytation of 44kD/pI6.3 protein was not induced by the PKC activator, PMA. Instead, the phosphorylation of 44kD/pI6.8 protein was reduced by H-7, a PKC inhibitor. From the above results,it can be concluded that the 44kD/pI6.3 protein can be a common substrate for A-kinase and CaM kinase. The two dimensional tryptic peptide mapping revealed that the 44kD/pI6.8 and 44kD/pI6.3 proteins are different.

• Journal of Life Science
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• v.25 no.5
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• pp.507-514
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• 2015

D-Xylulose is phosphorylated to D-xylulose-5-phosphate by D-xylulose kinase before it enters glycolysis via the nonoxidative pentose phosphate pathway. A gene encoding a novel D-xylulose kinase (XK) from K. gwangalliensis strain SJ2 was sequenced and expressed in E. coli. The sequence of the isolated XK gene was 1,419 bp, encoding 472 amino acids. The XK protein was more closely related to the Arthrobacter phenanthrenivorans XK than to the Bifidobacterium catenulatum one, as reflected in the sequence identity (54.9% vs. 38.7%). The XK gene was subcloned into the pCold-II expression vector. The resulting plasmid was transformed into E. coli strain BL21 (DE3) cells and the expression of the recombinant XK protein was induced by the addition of IPTG. The resulting protein was expressed as a fusion protein of approximately 48 kDa containing a N-terminal six-histidine extension that was derived from the expression vector. The expressed protein was homogenized by affinity chromatography and showed enzymatic activity corresponding to D-xylulose kinase. XK enzyme kinetic studies with D-xylulose and ATP showed a Km of 250±20 μM and 1,300±50 μM, respectively. The results obtained from this study will provide a wider knowledge base for the characterization of D-xylulose kinase at the molecular level.

• Journal of Korean Physical Therapy Science
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• v.11 no.1
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• pp.20-27
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• 2004

It is widely accepted that smooth muscle contraction is triggered by intracellular $Ca^{2+}$ ($[Ca^{2+}]_i$) released from intracellular $Ca^{2+}$ stores such as sarcoplasmic reticulum (SR) and from the extracellular space, The increased $[Ca^{2+}]_i$ can phosphorylate the 20-kDa myosin light chain ($MLC_{20}$) by activating MLC kinase (MLCK), and this initiates smooth muscle contraction. In addition to the $[Ca^{2+}]_i$-MLCK-tension pathway, a number of intracellular signal molecules, including mitogen-activated protein kinase (MAPK), protein kinase C (PKC), phosphatidylinositol 3-kinase (PI3K), and Rho-associated coiled coil-forming protein kinase (ROCK), play important roles in the regulation of smooth muscle contraction. However, the mechanisms regulating contraction of caldesmon (CaD), actin-binding protein, are not entirely elucidated in the presence of $Ca^{2+}$. It is known that CaD tightly interacts with actin and inhibits actomyosin ATPase activity. Therefore, the purpose of the present study was to investigate the roles of $Ca^{2+}$-dependent CaD in smooth muscle contraction. Endothelin-1 (ET-1), G-protein coupled receptor agonist and vasoconstrictor, increased both vascular smooth contraction and phosphorylation of CaD in the presence of $Ca^{2+}$. These results suggest that ET-1 induces contraction and phosphorylation of CaD in rat aortic smooth muscle, which may he mediated by the increase of $[Ca^{2+}]_i$.

• BMB Reports
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• v.28 no.4
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• pp.281-289
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• 1995

Homogeneous human deoxycytidine kinase was purified in one step from a variety of spontaneous human leukemic cells (T-ALL, B-ALL, B-CLL, AML, CML), and from cultured T-lymphoblast cells (MOLT-4) using the newly developed affinity medium, $dCp_4$-Sepharose. Starting with an ammonium sulfate fraction, purification was achieved in one step with the kinase being eluted from a column by the end product inhibitor, dCTP. The purified deoxycytidine kinase from T-ALL cells phosphorylated deoxyadenosine and deoxyguanosine, as well as deoxycytidine. The enzyme purified from T-ALL and B-CLL cells yielded one major band with a molecular weight of 52 kDa determined by SDS-polyacrylamide gel electrophoresis. AML and CML cells yielded one 52 kDa band and an extra band of 30 kDa molecular weight. On the other hand, B-ALL and MOLT-4 cells showed a low molecular weight band of 30 kDa only. However, the electrophoretic mobilities of enzymatic activity in 12% non-denaturing gels were identical for the dCyd kinase from all different kinds of leukemic cell lines, except that the B-ALL, B-CLL, and MOLT-4 cell preparations had an extra minor peak, all at the same position. dAdo and dCyd phosphorylating activities comigrated indicating that these activities are all associated with the same protein. Two new methods, a disk implantation method and a nitrocellulose powder method were used with a small amount of enzyme protein to raise polyclonal antibodies against dCyd kinase purified from T-ALL cells.

• Plant Biotechnology Reports
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• v.2 no.4
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• pp.227-231
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• 2008

D-class cyclins play important roles in controlling the cell cycle in development and in response to external signals by forming the regulatory subunit of cyclin-dependent kinase (CDK) complexes. To evaluate the effects of D-class cyclins in transgenic rice plants, Arabidopsis cyclin D2 gene (CycD2) was linked to the maize ubiquitin1 promoter (Ubi1) and introduced into rice by the Agrobacterium-mediated transformation method. Genomic deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and Western blot hybridizations of the Ubi1:-CycD2 plants revealed copy number of transgene and its increased expression in leaf and callus cells at messenger RNA (mRNA) and/or protein levels. The H1 kinase assay using the immunoprecipitates of protein extracts from the Ubi1:CycD2 plants and nontransgenic controls demonstrated that the introduced Arabidopsis CycD2 forms a functional CycD2/CDK complex with an unidentified CDK of rice. Shoot and root growth was enhanced in the Ubi1:CycD2 seedlings compared with nontransgenic controls, together, suggesting that Arabidopsis cyclin D2 interacts with a rice cyclin-dependent kinase, consequently enhancing seedling growth.

• Tuberculosis and Respiratory Diseases
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• v.43 no.2
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• pp.123-127
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• 1996

Secretion of surfactant phospholipid can be stimulated by a variety of agonists acting via at least three different signal transduction mechanisms. These include the adenylate cyclase system with activation of cAMP-dependent protein kinase; activation of protein kinase C either directly or subsequent to activation of phosphoinositide-specific phospholipase C and generation of diacylglycerols and inositol trisphosphate; and a third mechanism that involves incresed $Ca^{2+}$ levels and a calmodulin-dependent step. ATP stimulates secretion via all three mechanisms. The protein kinase C pathway is also coupled to phopholipase D which, acting on relatively abundant cellular phospholipids, generates diacylglycerols that further activate protein kinase C. Sustained protein kinase C activation can maintain phosphatidylcholine secretion for a prolonged period of time. It is likely that interactions between the different signaling pathways have an important role in the overall physiological regulation of surfactant secretion.

• BMB Reports
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• v.40 no.3
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• pp.432-438
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• 2007

V(D)J recombination, a site-specific gene rearrangement process occurring during the lymphocyte development, begins with DNA double strand breaks by two recombination activating gene products (RAG1/2) and finishes with the repair process by several proteins including DNA-dependent protein kinase (DNA-PK). In this report, we found that RAG2 was specifically phosphorylated by DNA-PK at the $365^{th}$ serine residue, and this phosphorylated RAG2 affected the V(D)J recombination activity in cells in the GFP expression-based assay. While the V(D)J recombination activity between wild-type RAG2 and mutant S365A RAG2 in the assay using a signal joint substrate was undistinguishable in DNA-PK deficient cells (M059J), the activity with wild-type RAG2 was largely increased in DNA-PK proficient cells (M059K) in comparison with mutant RAG2, suggesting that RAG2 phosphorylation by DNA-PK plays a crucial role in the signal joint formation during V(D)J recombination.

• Journal of Life Science
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• v.13 no.6
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• pp.903-910
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• 2003

Phospholipase D (PLD) plays an important role as a signaling molecule in the activation of neutrophils. In this study, effect of nitric oxide (NO) and cGMP on the activation of PLD in human neutrophils was investigated. Sodium nitroprusside (SNP), an agent to produce NO spontaneously in cells, alone increased PLD activity and the maximal activation was obtained with 0.5 mM SNP. Dibutyryl-cAMP, an agent to increase an intracellular cAMP concentration inhibited formyl-Met-Leu-Phe (fMLP)-stimulated PLD activity but 8-bromo-cGMP (300 $\mu$M), an agent to increase an intracellular cGMP concentration did not affect basal and fMLP-stimulated PLD activity. NO-induced activation of PLD was not blocked by KT 5823, an inhibitor of cGMP-dependent protein kinase (PKG), suggesting that NO-induced PLD activation is not mediated by cGMP. NO also stimulated p38 mitogen activated protein kinase (MAPK) in human neutrophils, indicated by increased phosphorylation of p38 MAPK in Western blotting. NO-induced phosphorylation of p38 MAPK was not inhibited by KT 5823 or n-butanol. RhoA, an regulatory factor of PLD activation was trans-located from cytosolic fraction to plasma membranes by fMLP or phorbol ester, and fMLP-stimulated but not phorbol ester-stimulated translocation of RhoA was inhibited by cGMP. These results suggest that NO stimulates PLD activity through other unidentified facto.(s) than cGMP even though cGMP inhibits the artivation of RhoA.