• Journal of Microbiology and Biotechnology
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• v.8 no.4
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• pp.325-332
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• 1998

In vitro phosphorylation experiments with a cell extract of Streptomyces coelicolor A3(2) M130 in the presence of ${\gamma}-[^32P]$]ATP revealed the presence of multiple phosphorylated proteins, including the AfsR/AfsK kinases which control the biosynthesis of A-factor, actinorhodin, and undecylprodigiosin. Phosphorylation of AfsR by a cell extract as an AfsK source was significantly inhibited by Ser/Thr protein kinase inhibitors, staurosporine and K-252a, at concentrations giving 50% inhibition ($IC_50$) of $1{\mu}M\;and\;0.1{\mu}M$, respectively. Further in vitro experiments with the cell extracts showed that phosphorylation of multiple proteins was inhibited by various protein kinase inhibitors with different inhibitory profiles. Manganese and calcium ions in the reaction mixture also modulate phosphorylation of multiple proteins. Manganese at 10 mM greatly enhanced the phosphorylation and partially circumvented the inhibition caused by staurosporine and K-252a. A calcium-activated protein kinase(s) was little affected by these inhibitors. Herbimycin and radicicol, which are known as tyrosine kinase inhibitors, did not show any significant inhibition of AfsR phosphorylation. Consistent with the in vitro effect of the kinase inhibitors, they inhibited aerial mycelium formation and pigmented antibiotic production on solid media. On the contrary, when assayed in liquid culture, the amount of actinorhodin produced was increased by staurosporine and K-252a and greatly decreased by manganese. All of these data clearly show that the genus Streptomyces possesses several protein kinases of eukaryotic types which are involved in the regulatory network for morphogenesis and secondary metabolism.

• The Korean Journal of Physiology and Pharmacology
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• v.18 no.2
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• pp.155-161
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• 2014

Overexpression of amyloid precursor protein with the Swedish mutation causes abnormal hyperphosphorylation of the microtubule-associated protein tau. Hyperphosphorylated isoforms of tau are major components of neurofibrillary tangles, which are histopathological hallmarks of Alzheimer's disease. Protein phosphatase 2A (PP2A), a major tau protein phosphatase, consists of a structural A subunit, catalytic C subunit, and a variety of regulatory B subunits. The B subunits have been reported to modulate function of the PP2A holoenzyme by regulating substrate binding, enzyme activity, and subcellular localization. In the current study, we characterized regulatory B subunit-specific regulation of tau protein phosphorylation. We showed that the PP2A B subunit PPP2R2A mediated dephosphorylation of tau protein at Ser-199, Ser-202/Thr-205, Thr-231, Ser-262, and Ser-422. Down-regulation of PPP2R5D expression decreased tau phosphorylation at Ser-202/Thr-205, Thr-231, and Ser-422, which indicates activation of the tau kinase glycogen synthase kinase 3 beta ($GSK3{\beta}$) by PP2A with PPP2R5D subunit. The level of activating phosphorylation of the $GSK3{\beta}$ kinase Akt at Thr-308 and Ser-473 were both increased by PPP2R5D knockdown. We also characterized B subunit-specific phosphorylation sites in tau using mass spectrometric analysis. Liquid chromatography-mass spectrometry revealed that the phosphorylation status of the tau protein may be affected by PP2A, depending on the specific B subunits. These studies further our understanding of the function of various B subunits in mediating site-specific regulation of tau protein phosphorylation.

• Korean Journal of Food Science and Technology
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• v.28 no.6
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• pp.1009-1013
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• 1996

This study was carried to investigate the emulsion stability of microcrystalline chitins (MCC) prepared from chitins of different molecular sizes and different degrees of phosphorylation in a model fatty food system. Chitins of low, medium and high molecular size prepared from crabshells were phosphorylated to 30-50% to make MCC. MCC prepared from chitin of medium molecular size revealed a high emulsion stability. The best emulsion stability was observed in MCC prepared from medium size chitin with 40% phosphorylation (M-40-MCC). The fat binding capacity of MCC was not significantly different among the samples. MCC with 50% phosphorylation had more fat binding capacity, ranging from 650-690%. When 2.46% of M-40-MCC was applied to a coconutoil-water system as an emulsifier, emulsion separation was observed from 10 min after emulsification, indicating that it could not be used as a sole emulsifier. When 50% of emulsifier (Span-60 &Tween-60) in a liquid coffee creamer, selected as a model fatty foods, was replaced by M-40-MCC, emulsion stability was as good as control. Consistency of liquid creamer was decreased and L value increased as the amount of MCC repacement increased. A liquid creamer with 50% emulsifier replaced by MCC had sensory characteristics equivalent to control.

• Korean Journal of Microbiology
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• v.2 no.1
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• pp.12-16
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• 1964

Lee, Yung Nok and Chin, Pyung (Dept. of Biology, Korea University, Seoul, Korea) : Action of ascorbic acid indoleacetic acid on the oxidation of succinate and coupled phosphorylation in Chlorella mitochondria. Kor. Jour. Microbiol., Vol.2, No.1, p12-16 (1964) Mitochondria were isolated from Chlorella ellipsoidea and the action of ascorbic acid and indoleacetic acid on the succinate oxidation and coupled phosphorylation in mitochondria suspension were examined. Oxidation of succinate used as substrate, and phosphorylation coupled to oxidation were strikingly enhanced by the addition of ascorbic acid, while in case of indoleacetic acid it were a little. In a view of phosphorylative efficiency, P/O ratio resulting from the addition of ascorbic acid was decreased and it may be considered as the result of a partial oxidation of ascorbate in mitochondria.

• BMB Reports
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• v.33 no.4
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• pp.349-352
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• 2000

Proteins that are involved in cellular signal cascade experience phosphorylation and dephosphorylation cycles in their tyrosine residue(s) during cell adhesion. In order to identify the protein(s), which tyrosine desidues are specifically phosphorylated when the cells attached to the substrate, we compared the tyrosine phosphorylation level of proteins between suspension and adhered culture condition in rat fibroblast 3Yl cells. We found that a cluster of 70 kDa protein was specifically phosphorylated when the cells adhered to the substrate, but did not effect the cells held in suspension. The phosphorylated protein is identified as paxillin, a focal adhesion protein in immunoprecipitation and immunobloting analysis. These results suggest that the tyrosine phosphorylation of paxillin may play a role in cell-substrate adhesion.

• Microbiology and Biotechnology Letters
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• v.24 no.6
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• pp.641-646
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• 1996

[$\gamma$-$^{32}$P]ATP was used to test phosphorylation of membrane proteins of mating type a cells of heterobasidiomycetous yeast Rhodosporidium toruloides separated by non-denaturing electrophoresis. The phosphoprotein was observed in the membrane proteins. The phosphorylation was inhibited by the pheromone rhodotorucine A (Rh. A) secreted by mating type A of the yeast. Rh. A didn't inhibit the phosphorylation in the presence of a trigger peptidase (TPase) inhibitor, antipain. Partially digested Rh. A by trypsin maintained the phosphorylation inhibitory activity. These results show that TPase activity plays an important role in the transduction of pheromone signal in the yeast.

• BMB Reports
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• v.29 no.1
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• pp.27-31
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• 1996

Using topoisomerase II (topo II) isozyme-specific antibodies, we investigated the phosphorylation of topo $II{\alpha}$ in mitotic HeLa cells. Topo $II{\alpha}$ was specifically modified in the mitotic cells, resulting in slow migration on SDS-polyacrylamide gel electrophoresis. To characterize the nature of this modification, we treated the nuclear extracts prepared from the mitotic cells with alkaline phosphatase. After the treatment with alkaline phosphatase, the slowly migrated band disappeared and instead a normal (170 kDa) topo $II{\alpha}$ band appeared. These results indicate that human topo $II{\alpha}$ is modified at a specific site(s) in M phase by phosphorylation, supporting the possibility that M phase-specific phosphorylation of topo II is critical for mitotic chromosome condensation and segregation.

• Bulletin of the Korean Chemical Society
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• v.33 no.4
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• pp.1285-1289
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• 2012

RAP80 plays a key role in DNA damage responses by recognizing K63-linked polyubiquitin moieties through its two ubiquitin-interacting motif (UIM) domains. The linker between the two UIMs possesses a phosphorylation site, but the relationship between phosphorylation and polyubiquitin recognition remains elusive. We investigated the interaction between a phosphorylation-mimic RAP80 mutant S101E and linear polyubiquitins, structurally equivalent to the K63-linked ones, using isothermal titration calorimetry (ITC). ITC analysis revealed differential binding affinities for linear tetraubiquitin by otherwise equivalent UIMs in S101E. Mutational analysis supported such differential polyubiquitin recognition by S101E. Our results suggest a potential crosstalk between polyubiquitin recognition and phosphorylation in RAP80.

• Journal of Life Science
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• v.7 no.4
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• pp.322-328
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• 1997

Two phosphorylated proteins having molecular weights of 57kD and 72kD were detected from the slubilized membrane protein fraction of mating type a cells of Rhodosporidium toruloides which belongs to heterobasidiomycetous yeast. The phosphorylation of the protein was inhibited by a sexual pheromone, Rhodotorucine A (Rh. A), which is secreted from mating type a cells. On the other hand, counterpart mating type A cells and M-39 strain which is a styerile mutant derived from a cells, had also the same two phosphorylated proteins, However, the phosphorylation of the protein from A cells, and M-39 strain were not inhibited by the Rh. A. It suggests that inhibition of the phosphorylation reaction by the Rh. A in mating type a cells is a mating-type-specific reaction that relate to transduction mechanism of sexual pheromone signaling.

• Journal of the Korean Society of Food Science and Nutrition
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• v.26 no.6
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• pp.1246-1251
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• 1997

When the membrane protein fraction of mating type a cells of heterobasidiomycetous yeast R. toruloides was phosphorylated in vitro, two phosphorylated proteins of 72Kd and 57Kd were detected on SDS-polyacryamide gel. The phosphorylation reaction was inhibited by rhodotorucine A(Rh. A) which is a sexual pheromone secreted by mating type A cells. The inhibition of phosphorylation by Rh. A was dependent on $Ca^{2+}$, and independent on $Mg^{2+}$ or calmodulin. When adding trigger peptidase(TPase) inhibitor, antipain, no inhibition of phosphory was observed. Also, by adding the trysin-digested product of Rh. A, the phosphorylation was inhibited as the action of Rh. A. From these results, it is expected that the inhibition of membrane protein phosphorylation should be caused by the digested product of Rh. A with TPase.