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Src kinase가 Ouabain에 의한 Reactive Oxygen Species (ROS) 생성에 미치는 영향 연구

Src kinase가 Ouabain에 의한 Reactive Oxygen Species (ROS) 생성에 미치는 영향 연구
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Studies on the effects of Src kinase on ROS generation by ouabain
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대학원 분자생명과학부
이화여자대학교 대학원
Na,K-ATPase는 세포내 다양한 구획 (intracellular compartments)으로 신호를 전달하는 multiple protein complexes를 형성한다. Ouabain이 signaling Na,K-ATPase에 결합함으로서 cytoplamic tyrosine kinase인 Src를 활성화시키고, 그 결과 다른 단백질들을 인산화시키고, 다른 signaling modules로 소집시키는 활성화된 “binary receptor”를 형성하게 된다. 이것이 세포 종류에 따라 특이적인 방식으로 mitogen-activated protein kinase와 protein kinase C isozyme들을 포함하는 multiple protein kinase cascades를 순차적으로 활성화시킨다. 또한 ouabain과 Na,K-ATPase의 결합은 미토콘드리아로부터 ROS (Reactive Oxygen Species)의 생성을 증가시키며, 세포내 Ca2+의 농도도 조절한다는 것이 알려져 있다. 본 실험실에서는 yeast two-hybrid screening을 통해 cofilin이 Na,K-ATPase α subunit의 3번째 cytoplamic domain과 상호작용하여 Na,K-ATPase의 효소활성을 증가시킨다는 사실을 밝혔다. 본 논문에서는 이러한 연구 결과를 토대로, microplate reader를 이용하여 ouabain에 의해 ROS (Reactive Oxygen Species)가 생성됨을 확인하였으며, N-acetylcysteine (NAC)과 같은 항산화제 처리에 의해 ROS 생성이 소멸됨을 확인하였다. 또한 ouabain에 의한 ROS 생성이 활성형 cofilin을 탈인산화된 형태로 전환시킴을 western blotting assay를 통해 실험적으로 확인하였다. 이러한 실험 결과를 통해 ouabain에 의한 cofilin의 탈인산화에 ROS가 intracellular second messenger로 작용할 가능성을 확인하였다. 이어서 ouabain에 의해 생성된 ROS의 upstream으로 예상되는 Na,K-ATPase에 의해 매개되는 신호 전달 체계와 관련된 여러 inhibitor를 적용하여 ROS에 의한 fluorescence를 측정한 결과 Src kinase가 down stream에 존재하는 EGFR와 그 외에 또 다른 세포막 단백질과 상호작용하여 인산화시킴으로서 ROS를 생성한다는 것을 알 수 있었다. 이러한 Src kinase가 ROS의 source임을 재확인하고, 이것이 cofilin의 탈인산화에도 관여함을 SYF cell line과 SYF+c-Src cell line system을 이용하여 보여주었다. 또한 Ras의 dominant negative mutant인 RasN17을 transfection시킨 HeLa cell을 이용한 실험으로 Ras 역시 ouabain에 의한 ROS 생성에 관여하며, 이로 인해 cofilin의 탈인산화에 영향을 미친다는 것을 관찰하였다.;Na,K-ATPase is involved in the assembly of multiple protein complexes that transmit signals to different intracellular compartments. Binding of ouabain to the signaling Na,K-ATPase activates the cytoplasmic tyrosine kinase Src, resulting in the formation of an active “binary receptor” that phosphorylates and assembles other proteins into different signaling modules. This in turn activates multiple protein kinase cascades including mitogen-activated protein kinases and protein kinase C isozymes in a cell-specific manner. It also increases mitochondrial production of reactive oxygen species (ROS) and regulates intracellular calcium concentration. It was found in our laboratory that phosphorylated cofilin activates Na,K-ATPase activity by binding to the the third cytoplasmic domain of the α subunit of Na,K-ATPase and cofilin is dephosphorylated by ouabain. In this study, I tried to identify the signaling pathway that cause the dephosphorylation of cofilin by ouabain. I confirmed that ROS (Reactive Oxygen Species) were generated by ouabain using H₂O₂-sensitive fluorescence of CM-H2DCFDA. These effects were blocked by anti-oxidant NAC (N-acetylcysteine). It was found that cofilin was dephosphorylated by ouabain using western blotting assay. The results showed that ROS generation could play a role as an intracellular messenger in cofilin dephosphorylation by ouabain. The Na,K-ATPase-mediated signaling cascade-related inhibitors were tested to see if they could block the generation of ROS. As a result, Src kinase was thought to be the source of ouabain-induced ROS. I also employed SYF (deficient for Src, Yes, and Fyn) and SYF+c-Src cell line system to show the effects of Src kinase on ouabain-induced dephosphorylation of cofilin. The data indicates that Src plays a role in cofilin dephosphorylation by ouabain as well as ROS generation. I performed transfection of RasN17 (dominant negative Ras) into HeLa cells and western blotting assay using transfected HeLa cell extracts. I found that Ras was also involved in ROS generation by ouabain and cofilin dephosphorylation by ouabain. In conclusion, I showed that Src and Ras activation by ouabain resulted in ROS generation and thereby cofilin dephosphorylation.
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