• 한국의학물리학회지:의학물리
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• 제15권4호
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• pp.220-227
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• 2004

N형 칼슘통로의 비활성화기전에 관하여는 아직까지도 막전압의존성 기전과 칼슘의존성 기전간에 논란이 계속되고 있다. 2003년에 의학물리에 발표한 논문1)에서 본 연구자는 N형 칼슘통로의 비활성화 기전은 2가지 성분 -빠른 성분과 느린 성분을 가지고 있고 빠른 성분은 칼슘의존적이 아니며 오직 느린 성분만이 칼슘의존적일 가능성을 제시하였다. 본 논문에서는 막전압의존성 기전이 옳건 칼슘의존성 기전이 옳건 간에 세포 신호전달 체계로서 비활성화와 연계된 기전이 필요하므로 이러한 맥락에서 인산화 기전을 연구하였다. 흰쥐 경동맥 결절뉴론을 단일 세포로 얻은 후 whole cell patch clamp technique를 사용하여 N형 칼슘전류를 기록하고 대조 세포내액을 사용하였을 때와 phosphatase inhibitor인 okadaic acid를 포함한 세포내액을 사용하였을 때의 차이를 비교하였다. Okadaic acid에 의하여 비활성화정도가 증가되었고 이러한 okadaic acid 효과는 주로 N형 통로를 통하여 영향을 미침을 N형 칼슘통로 억제제인 $\omega$-conotoxin GVIA를 사용함으로써 확인하였다. Okadaic acid에 의한 비활성화 증가 효과는 protein kinase를 비특이적으로 억제하는 staurosporine에 의하여 억제되었고 또한 calmodulin dependent protein kinase의 특이적 억제제인 lavendustin C에 의하여 억제되었으므로 인산화과정이 N형 칼슘통로 비활성화와 관련되어 있고 특히 calmodulin을 통한 인산화과정이 주로 관여함을 확인하였다. 본 연구자가 발표한 선행논문1)에 의해 외부의 2가 양이온에 의해 빠른 비활성화가 진행되며, 본 논문에 의하여 인산화과정에 의해 빠른 비활성화가 촉진된다는 사실이 확인되었다. 그러나 본 연구결과만으로는 인산화과정이 비활성화 자체라고는 볼 수 없으며 단지 인산화과정에 의해 비활성화가 가속되었다고 해석할 수 밖에 없다. 인산화과정이 비활성화자 체인지 여부는 2가 양이온이 칼슘통로에 작용하는 결합부위에 관한 연구 및 인산화 부위가 칼슘통로인지 아니면 다른 조절 부위인지 여부를 확인할 수 있는 연구가 진행되어야 확실히 알 수 있을 것이다.

• 한국자기공명학회논문지
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• 제21권1호
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• pp.20-25
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• 2017

The cytoplasmic domains A and B of the mannitol transporter enzyme $II^{Mtl}$ are covalently linked in Escherichia coli, but separately expressed in Thermoanaerobacter Tengcongensis. The phosphorylation of domain B ($TtIIB^{Mtl}$) substantially increases the binding affinity to the domain A ($TtIIA^{Mtl}$) in T. Tengcongensis. To understand the structural basis of the enhanced domain-domain interaction by protein phosphorylation, we obtained NMR backbone assignments of the phospho-$TtIIB^{Mtl}$ using a standard suite of triple resonance experiments. Our results will be useful to monitor chemical shift changes at the active site of phosphorylation and the binding interfaces.

• Animal cells and systems
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• 제2권2호
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• pp.223-227
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• 1998

We isolated two soluble forms of glutamate dehydrogenase isoproteins, GDH I and GDH II, from bovine brain. The regulation of GDH I and GDH II by phosphorylation and dephosphorylation has been examined in various conditions. There were dose- and time- dependent activation of the GDH isoproteins when phosphorylated by cAMP-dependent protein kinase. The phosphorylated GDH had 1.1 mol of covalently bound phosphate/mol of subunit and a 2-fold increased specific activity. The phosphorylated amino acid was identified as serine. When treated with alkaline phosphatase, the activities of the phosphorylated GDH isoproteins were reduced in dose and time dependent manner and returned to those of unphosphorylated enzymes. There were no significant differences between GDH I and GDH II in their sensitivities to the action of phosphorylation and dephosphorylation demonstrating that the microenvironmental structures of the phosphorylation site in GDH isoproteins are similar to each other, These results results suggest that the inter-conversion between less active form of brain GDH isoproteins and more active form is regulated by phosphorylation through cAMP-dependent protein kineses.

• 약학회지
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• 제29권2호
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• pp.76-89
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• 1985

The effects of ginseng saponin on the activity, phosphorylation, [$^{3}$H] ouabain binding and light scattering (disruption) of purified $Na^{+}$ ,$K^{+}$ -ATPase isolated from the outer medulla of sheep kidney were compared to those of gypsophila saponin, sodium dodecylsulfate (SDS), and Triton X-100 on the same parameters. $Na^{+}$ , $K^{+}$ -ATPase activity, phosphorylation, and [$^{3}H$] ouabain binding were inhibited by ginseng saponin (triol>total>diol), SDS, or Triton X-100, but increased by gypsophila saponin. Low doses of ginseng saponin (3.mu.g saponin/.mu.g protein) decreased phosphorylation sites and ouabain binding site concentration (Bmax) without any change of turnover number and affinity for ouabain binding which were decreased by high dose of ginseng saponin (over 10.mu.g saponin/.mu.g protein), SDS or Triton X-100. On the other hand, gypsophila saponin increased the affinity without any change of Bmax for ouabain binding. Inhibition of $Na^{+}$ ,$K^{+}$ -ATPase activity by ginseng saponin and SDS or Triton X-100 appeared before and after decrease in light scattering, respectively. These data suggest that ginseng saponins (total, diol, triol saponin) inhibit $Na^{+}$ , $K^{+}$ -ATPase activity by specific direct and general detergent action at low and high concentrations, respectively, and this inhibitory action of ginseng sapornin to $Na^{+}$ , $K^{+}$ -ATPase is not general action of all saponins.

• Asian-Australasian Journal of Animal Sciences
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• 제21권2호
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• pp.181-189
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• 2008

The aim of this study was to evaluate the effects of $Ca^{2+}$, $HCO_3{^-}$ and BSA on the in vitro capacitation-associated protein tyrosine phosphorylation, hyperactivation and acrosome reaction in guinea pig sperm. Caudal epididymal sperm were incubated in four different groups: modified TALP (Tyrode's albumin lactate pyruvate) or TALP without one of the medium constituents ($Ca^{2+}$, $HCO_3{^-}$ and BSA). After incubation for the required time (0 h, 0.5 h, 1 h, 3 h, 5 h, and 7 h), sperm were removed for further experiment. The capacitation effect was assessed by CTC (Chlortetracycline) staining. Western blotting and indirect immunofluorescence were used to analyze the level and localization of tyrosine phosphorylation. The results showed that guinea pig sperm underwent a time-dependent increase in protein tyrosine phosphorylation during the in vitro capacitation and the percentage of protein tyrosine phosphorylated sperm increased from 36% to 92% from the beginning of incubation to 7 h incubation. Also, there was a shift in the site of phosphotyrosine-specific fluorescence from the head of sperm to both the head and the flagellum. Moreover, an absence of $Ca^{2+}$ or $HCO_3{^-}$ inhibited in vitro hyperactivation and acrosome reaction and decreased the phosphorylation of the proteins throughout the period of in vitro capacitation. However, an absence of BSA could not influence these processes if substituted by polyvinyl alcohol (PVA) in the medium.

• Biomolecules & Therapeutics
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• 제25권6호
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• pp.593-598
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• 2017

The $Na^+/H^+$ exchanger-1 (NHE-1) is a ubiquitously expressed pH-regulatory membrane protein that functions in the brain, heart, and other organs. It is increased by intracellular acidosis through the interaction of intracellular $H^+$ with an allosteric modifier site in the transport domain. In the previous study, we reported that glutamate-induced NHE-1 phosphorylation mediated by activation of protein kinase C-${\beta}$ (PKC-${\beta}$) in cultured neuron cells via extracellular signal-regulated kinases (ERK)/p90 ribosomal s6 kinases (p90RSK) pathway results in NHE-1 activation. However, whether glutamate stimulates NHE-1 activity solely by the allosteric mechanism remains elusive. Cultured primary cortical neuronal cells were subjected to intracellular acidosis by exposure to $100{\mu}M$ glutamate or 20 mM $NH_4Cl$. After the desired duration of intracellular acidosis, the phosphorylation and activation of PKC-${\beta}$, ERK1/2 and p90RSK were determined by Western blotting. We investigated whether the duration of intracellular acidosis is controlled by glutamate exposure time. The NHE-1 activation increased while intracellular acidosis sustained for >3 min. To determine if sustained intracellular acidosis induced NHE-1 phosphorylation, we examined phosphorylation of NHE-1 induced by intracellular acidosis by transient exposure to $NH_4Cl$. Sustained intracellular acidosis led to activation and phosphorylation of NHE-1. In addition, sustained intracellular acidosis also activated the PKC-${\beta}$, ERK1/2, and p90RSK in neuronal cells. We conclude that glutamate stimulates NHE-1 activity through sustained intracellular acidosis, which mediates NHE-1 phosphorylation regulated by PKC-${\beta}$/ERK1/2/p90RSK pathway in neuronal cells.

• 생명과학회지
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• 제22권3호
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• pp.428-436
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• 2012

단백질 인산화는 세포의 활동을 조절하는 보편적인 과정이다. 브라시노스테로이드(brassinostreoid)에 의해 매개되는 신호전달은 브라시노스테로이드에 의해 활성화된 세포막상의 protein kinase 로부터 인산화되어 있는 전사인자들을 탈인산화하는 연속적인 인산화/탈인산화 과정이다. 브라시노스테로이드에 의해 매개되는 신호전달의 연구는 인산화에 관여하는 kinase 기질상의 아미노산을 밝히고, 그와 관련된 돌연변이체의 표현형을 알아봄으로써 급속하게 발전하였다. BRI1과 BAK1의 자기인산화(autophosphorylation), 상호인산화(transphosphorylation), 타이로신 인산화(tyrosine phosphorylation)를 밝힘으로써 그들의 조절작용을 식물의 생리학적, 발생학적 과정을 더 이해할 수 있는 장이 열렸다. 브라시노스테로이드에 의한 인산화는 수용체에 의해 매개되는 세포 내 함입(endocytosis)과 그에 뒤따르는 수용체의 파괴현상에서도 볼 수 있다. 인산화/탈인산화 과정에 관련하여 브라시노스테로이드에 의해 매개되는 신호전달은 더 연구할 여지가 많이 남아 있다. 이 총설은 단백질의 인산화/탈인산화 과정을 통한 브라시노스테로이드의 신호전달 연구의 최근 상황을 기술하였다.

• Journal of Photoscience
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• 제9권2호
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• pp.482-484
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• 2002

UV irradiation activates various intracellular signaling pathways causing cell death in a DNA damage-dependent and an independent manner. As DNA photoproducts, major forms of DNA damage, are maximally formed by UV light at 260-nm, short wavelength UV (UVC) is more harmful than middle wavelength UV (UVB). However, the differences or similarities in responses of DNA damage-independent intracellular signaling molecules to UVB and UVC are not elucidated. We examined activation of signaling molecules towards apoptosis in normal human fibroblastic cells after irradiation with UVB or UVC at a dose generating the equal amount of DNA photoproducts. Both UVB and UVC induced transient phosphorylation of ERK and sustained phosphorylation of p38. Phosphorylation of p53 at Ser15 and at Ser392 residues were also observed, which were inhibited by a phosphoinositide 3-kinase inhibitor, wortmannin. In contrast, an antioxidant N-acetyl-cysteine and a p38 inhibitor SB203580 suppressed only Ser392 phosphorylation, suggesting that UV-induced oxidative stress and p38 activation were involved in the phosphorylation of this site. The apoptic signals such as mitochondrial cytochrome C release and annexin V binding were then observed. Overall, no difference was found in chronological responses of p53, MAPK, and apoptosis between UVB-irradiated and UVC-irradiated cells. These results suggested that DNA damage-independent intracellular signaling molecules similarly responded to UVB and UVC when the equal level of DNA photoproducts were generated.

• BMB Reports
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• 제41권1호
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• pp.41-47
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• 2008

Fe65 is characterized as an adaptor precursor (APP) through its PID2 element, as well as with the other members of the APP protein family. With the serum- and glucocorticoid-induced kinase 1 (SGK1) substrate specificity information, we found that the putative site of phosphorylation in Fe65 by SGK1 is present on its $Ser^{566}$ residue in $^{560}CRVRFLSFLA^{569}$(X60469). Thus, we demonstrated that Fe65 and the fluorescein-labeled Fe65 peptide $FITC-^{560}CRVRFLSFLA^{569}$ are phosphorylated in vitro by SGK1. Phosphorylation of the $Ser^{566}$ residue was also demonstrated using a $Ser^{566}$ phospho-specific antibody. The phospho Fe65 was found mainly in the nucleus, while Fe65 S556A mutant was localized primarily to the cytoplasm. Therefore, these data suggest that SGK1 phosphorylates the $Ser^{566}$ residue of Fe65 and that this phosphorylation promotes the migration of Fe65 to the nucleus of the cell.

• BMB Reports
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• 제40권3호
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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.