• Molecules and Cells
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• 제40권9호
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• pp.632-642
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• 2017

The DevSR (DosSR) two-component system, which is a major regulatory system involved in oxygen sensing in mycobacteria, plays an important role in hypoxic induction of many genes in mycobacteria. We demonstrated that overexpression of the kinase domain of Mycobacterium tuberculosis (Mtb) PknB inhibited transcriptional activity of the DevR response regulator in Mycobacterium smegmatis and that this inhibitory effect was exerted through phosphorylation of DevR on Thr180 within its DNA-binding domain. Moreover, the purified kinase domain of Mtb PknB significantly phosphorylated RegX3, NarL, KdpE, TrcR, DosR, and MtrA response regulators of Mtb that contain the Thr residues corresponding to Thr180 of DevR in their DNA-binding domains, implying that transcriptional activities of these response regulators might also be inhibited when the kinase domain of PknB is overexpressed.

• Animal cells and systems
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• 제1권1호
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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.

• 대한방사성의약품학회지
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• 제5권2호
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• pp.152-157
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• 2019

Polo-like kinase 1 (Plk1) is crucial regulator of cell cycle progression during mitosis. It is known to highly overexpress in many different tumor types, and has been implicated as a potential antimitotic cancer target. The phosphopeptide, Pro-Leu-His-Ser-p-Thr (PLHSpT), was shown a high level of affinity and specificity for the polo-box domain (PBD) of Plk1. However, the peptide has the limitation of cell permeability. We designed the derivatives to enhance the limitation of PLHSpT using drug delivery system. In addition, we synthesized and evaluated its radiotracer for tumor diagnosis. This review discusses the derivative and radiotracer that are suitable for tumor treatment and diagnosis for PBD of Plk1.

• BMB Reports
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• 제39권5호
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• pp.607-617
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• 2006

A novel calcium-dependent protein kinase gene (designated as IiCPK2) was cloned from tetraploid Isatis indigotica. The full-length cDNA of IiCPK2 was 2585 bp long with an open reading frame (ORF) of 1878 bp encoding a polypeptide of 625 amino acid residues. The predicted IiCPK2 polypeptide included three domains: a kinase domain, a junction domain (or autoinhibitory region), and a C-terminal calmodulin-like domain (or calcium-binding domain), which presented a typical structure of plant CDPKs. Further analysis of IiCPK2 genomic DNA revealed that it contained 7 exons, 6 introns and the length of most exons was highly conserved. Semi-quantitative RT-PCR revealed that the expression of IiCPK2 in root, stem and leaf were much higher in tetraploid sample than that in diploid progenitor. Further expression analysis revealed that gibberellin ($GA_3$), NaCl and cold treatments could up-regulate the IiCPK2 transcription. All our findings suggest that IiCPK2 might participate in the cold, high salinity and GA3 responsive pathways.

• Molecules and Cells
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• 제37권4호
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• pp.286-294
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• 2014

Mammalian polo-like kinase 1 (Plk1) has been studied intensively as a key regulator of various cell cycle events that are critical for proper M-phase progression. The polobox domain (PBD) present in Plk1's C-terminal noncatalytic region has been shown to play a central role in targeting the N-terminal kinase domain of Plk1 to specific subcellular locations. Subsequent studies reveal that PBD binds to a phosphorylated motif generated by one of the two mechanisms - self-priming by Plk1 itself or non-self-priming by a Pro-directed kinase, such as Cdc2. Here, we comparatively review the differences in the biochemical steps of these mechanisms and discuss their physiological significance. Considering the diverse functions of Plk1 during the cell cycle, a better understanding of how the catalytic activity of Plk1 functions in concert with its cisacting PBD and how this coordinated process is intricately regulated to promote Plk1 functions will be important for providing new insights into different mechanisms underlying various Plk1-mediated biological events that occur at the multiple stages of the cell cycle.

• The Plant Pathology Journal
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• 제33권2호
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• pp.163-169
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• 2017

$SIMAPKKK{\alpha}$, a tomato (Solanum lycopersicum) mitogen-activated protein kinase kinase kinase, is a positive regulator of Pto-mediated effector-triggered immunity, which elicits programmed cell death (PCD) in plants. In this study, we examined whether putative phosphorylation sites in the conserved activation segment of the $SIMAPKKK{\alpha}$ kinase domain are critical for eliciting PCD. Three amino acids, $threonine^{353}$, $serine^{360}$ ($Ser^{360}$), or $serine^{364}$ ($Ser^{364}$), in the conserved activation segment of $SIMAPKKK{\alpha}$ kinase domain were substituted to alanine (T353A, S360A, or S364A), and these variants were transiently expressed in tomato and Nicotiana benthamiana plants. Two alanine substitutions, S360A and S364A, completely abolished $SIMAPKKK{\alpha}$ PCD-eliciting activity in both plants, while T353A substitution did not affect its PCD-eliciting activity. $SIMAPKKK{\alpha}$ wild type and variant proteins accumulated to similar levels in plant leaves. However, $SIMAPKKK{\alpha}$ protein with the largest size was missed when either S360A or S364A substitutions were expressed, whereas proteins with the smaller masses were more accumulated than those of full-length of $SIMAPKKK{\alpha}$ and T353A. These results suggest that phosphorylation of $SIMAPKKK{\alpha}$ at $Ser^{360}$ and $Ser^{364}$ is critical for PCD elicitation in plants.

• Molecules and Cells
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• 제37권4호
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• pp.322-329
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• 2014

N-acetylglucosamine kinase (GlcNAc kinase or NAGK; EC 2.7.1.59) is highly expressed and plays a critical role in the development of dendrites in brain neurons. In this study, the authors conducted structure-function analysis to verify the previously proposed 3D model structure of GlcNAc/ATP-bound NAGK. Three point NAGK mutants with different substrate binding capacities and reaction velocities were produced. Wild-type (WT) NAGK showed strong substrate preference for GlcNAc. Conversion of Cys143, which does not make direct hydrogen bonds with GlcNAc, to Ser (i.e., C143S) had the least affect on the enzymatic activity of NAGK. Conversion of Asn36, which plays a role in domain closure by making a hydrogen bond with GlcNAc, to Ala (i.e., N36A) mildly reduced NAGK enzyme activity. Conversion of Asp107, which makes hydrogen bonds with GlcNAc and would act as a proton acceptor during nucleophilic attack on the ${\gamma}$-phosphate of ATP, to Ala (i.e., D107A), caused a total loss in enzyme activity. The overexpression of EGFP-tagged WT or any of the mutant NAGKs in rat hippocampal neurons (DIV 5-9) increased dendritic architectural complexity. Finally, the overexpression of the small, but not of the large, domain of NAGK resulted in dendrite degeneration. Our data show the effect of structure on the functional aspects of NAGK, and in particular, that the small domain of NAGK, and not its NAGK kinase activity, plays a critical role in the upregulation of dendritogenesis.

• Molecules and Cells
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• 제25권1호
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• pp.43-49
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• 2008

Cancer Osaka thyroid, also known as Tpl-2 (Cot) is a member of the MAP3K kinase family and plays a key role in the regulation of the immune response to pro-inflammatory stimuli such as lipopolysaccharide (LPS) and tumour necrosis $factor-{\alpha}$ ($TNF-{\alpha}$). A series of Cot constructs with an N-terminal 6xHis tag were transiently expressed in HEK293 cells: $Cot_{130-399}$ (kinase domain), $Cot_{1-388}$ (N-terminal and kinase do-mains), $Cot_{1-413}$, $Cot_{1-438}$ (containing a putative PEST sequence), $Cot_{1-457}$ (containing both PEST and degron sequences) and $Cot_{1-467}$ (full-length protein). These Cot proteins were pulled down using an anti-6xHis antibody and separated by 2D electrophoresis. The gels were silver-stained and 21 proteins were detected that did not appear, or had substantially reduced intensity, in the control sample. Three of these were identified by MS and MS/MS analysis as Hsp90, Hsp70 and Grp78. Hsp90 appeared to bind to the kinase domain of Cot and this interaction was further investigated using co-immuno-precipitation with both overexpressed Cot in HEK293 cells and endogenous Cot in Hela cells.

• Journal of Plant Biotechnology
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• 제45권4호
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• pp.315-321
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• 2018

Molecular and functional characterization of proteins and their levels is of great interest in understanding the mechanism of diverse cellular processes. In this study, we report on the convenient Escherichia coli-based protein expression system that allows recombinant of soluble proteins expression and cytosolic domain of membrane-localised kinases, followed by the detection of autophosphorylation activity in protein kinases. This approach is applied to regulatory proteins of Arabidopsis thaliana, including 14-3-3, calmodulin, calcium-dependent protein kinase, TERMINAL FLOWER 1(TFL1), FLOWERING LOCUS T (FT), receptor-like cytoplasmic kinase and cytoplasmic domain of leucine-rich repeat-receptor like kinase proteins. Our Western blot analysis which uses phospho-specific antibodies showed that five putative LRR-RLKs and two putative RLCKs have autophosphorylation activity in vitro on threonine and/or tyrosine residue(s), suggesting their potential role in signal transduction pathways. Our findings were also discussed in the broader context of recombinant expression and biochemical analysis of soluble and membrane-localised receptor kinases in microbial systems.

• Molecules and Cells
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• 제27권1호
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• pp.39-45
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• 2009

Ligand-dependent or independent oligomerization of receptor protein tyrosine kinase (RPTK) is often an essential step for receptor activation and intracellular signaling. The novel oncogene with kinase-domain (NOK) is a unique RPTK that almost completely lacks an ectodomain, expresses intracellularly and activates constitutively. However, it is unknown whether NOK can form oligomer or what function oligomerization would have. In this study, two NOK deletion mutants were generated by either removing the ectodomain ($NOK{\Delta}ECD$) or including the endodomain (NOK-ICD). Co-immunoprecipitation demonstrated that the transmembrane (TM) domain of NOK was essential for its intermolecular interaction. The results further showed that NOK aggregated more closely as lower order oligomers (the dimer- and trimer-sized) than either deletion mutant did since NOK could be crosslinked by both Sulfo-EGS and formaldehyde, whereas either deletion mutant was only sensitive to Sulfo-EGS. Removing the NOK TM domain (NOK-ICD) not only markedly promoted higher order oligomerization, but also altered the subcellular localization of NOK and dramatically elevated the NOK-mediated constitutive activation of extracellular signal-regulated kinase (ERK). Moreover, NOK-ICD but not NOK or $NOK{\Delta}ECD$ was co-localized with the upstream signaling molecule RAS on cell membrane. Thus, TM-mediated intermolecular contacting may be mainly responsible for the constitutive activation of NOK and contribute to the autoinhibitory effect on RAS/MAPK signaling.