Na,K-ATPase와 IgE-Dependent Histamine Releasing Factor의 결합에 영향을 미치는 Protein Kinase C Isotype에 관한 연구
PKC Isotype that Affects the Interaction of HRF with Na,K-ATPase
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본 실험에서는 HRF가 다량 존재하는 RBL-2H3 세포주에다양한 PKC isotype별 억제제를 처리하여 in vitro상에서Na,K-ATPase α1L3를 이용한 pull-down assay와 RBL-2H3세포를 이용한 membrane fractionation을 실시하였다. 그 결과 HRF는 in vitro에서 α1L3와 결합한다는 사실을 재확인할 수 있었고 실제 세포주 내에서 Na,K-ATPase와 결합한다는 것을 알 수 있었다. 사용한 약물로부터 PKC α, β, δ 뿐아니라 protein phosphatase 2B(PP2B)도 HRF와 α1L3의결합에 관여한다는 사실을 알 수 있었다. 또한 이들 PKC,Fig. 3. Interaction of HRF and Na,K-ATPase confirmed by immunoprecipitation in Cos-7 cells. Cos-7 cells were transfected withrecombinant Hemagglutinin tagged HRF constructs using Lipofectamine reagent. The HRF transfected Cos-7 cells were lysed in hypotonicbuffer. Whole cell extracts were incubated with anti-Hemagglutinin antibody and protein A agarose bead suspension. The bound proteinswere eluted by boiling with sample buffer and analyzed using anti-Na,K-ATPase antibody. (A) Na,K-ATPase was detected in crudeextracts. (B) Immunoprecipitates were detected with anti-Na,K-ATPase antibody. 1: purified Na,K-ATPase, 2: Marker, 3: Mock, 4: Control,5: PMA, 6: PMA (L), 7: Go6976, 8: Rott, 9: OkA, 10: CsA.Fig. 4. Histamine release assay. RBL-2H3 cells cultured in 24well plate were treated with the indicated drugs after serumstarvation. The level of histamine in supernatants of drug treatedcells were mesured. RBL-2H3 cells treated indicated drugs withoutIgE showed that their spontaneous histamine releasing wereeffected by some drugs itself.266 SOHN AND LEEPP2B에 의해 인산화된 HRF 분자는 cytosolic fraction으로이행할 수 있으며 이러한 결과는 탈인산화된 HRF가 Na,KATPase와결합하여 Na,K-ATPase의 기능을 조절한다고 추정할 수 있다. 그러나 약물자체가 histamine 분비에 영향을미칠 수 있으며 cytosolic HRF보다 exocytosis된 HRF가histamine를 더 분비하도록 할 수 있으므로, 약물을 전처리한 세포에 외부에서 HRF를 첨가하여 histamine이 유리되는정도가 어떻게 변화하는지를 HRF를 가하지 않은 결과와 비교해야 할 것이다.
Abstract
IgE-dependent histamine releasing factor (HRF), previously known as P23/P21 or translationally controlled tumor protein (TCTP), induces the degranulation of histamine in mast cell and basophil. Yeast two hybrid results showed that HRF interacts with the alpha subunit of Na,K-ATPase, suggesting that HRF is a regulator for governing the activity of Na,KATPase. In this study, we examined the interaction of HRF and Na,K-ATPase after treatments of various PKC isotype inhibitors. Membrane fractionation, pull-down assay and immunoprecipitation results showed that PKC α, PKC β, PKC δ subunits are involved in the phosphorylation of HRF. However, these results did not correlate with the results of histmamine release assay since histamine release assay results suggested that some PKC isotype inhibitors induced the histamine release in RBL-2H3 cell.
참고문헌 (17)
- "Effects of calcium antagonists on human basophilhistamine release", immu(l) (1981)
- "Downstreamsignals initiated in mast cells by Fcepsilon RI and otherreceptors", 8(l), pp.766~772 (1996)
- "Pharmacologicregulation of histamine release by the human recombinanthistamine releasing factor", 103(l), pp.937~943 (1999)
- "Nucleotide sequence of a majormessenger RNA for a 21 kilodalton polypeptide that is undertranslational control in mouse tumor cells" (1988)
- "A role for the sodium enzymein degranulation of rat basophilic leukemia cells", potassium ade(sine triphosphatase), pp.1449~26 1460 (1996)
- "aprotein identified in several nontumoral cells includingerythrocytes", pp.150~18 155 (1993)
- "Direct evidence that FK506 inhibition ofFcepsilon RI-mediated exocytosis from RBL mast cellsinvolves calcineurin", 290, pp.258~263 (1998), Kincaid
- "PMA and calcium ionophore induce myosin and F-actinrearrangement during histamine recretion from RBL-2H3cells", pp.354~29 365 (1994)
- "Studies of IgE-dependent histamine releasing factors heterogenecity of IgE", immu(l), pp.506~512 (1987)
- "Molecular identification of an IgEdependenthistamine releasing factor", 269, pp.688~269 690 (1995)
- "Pharmacology of IgE-mediated desensitization of humanbasophils Effects of protein kinase C and Src-family kinaseinhibitors", 60, pp.1717~1727 (2000)
- "Effects ofnifedipine on histamine release from human basophilleucocytes", pp.298~42 304 (1987)
- "Spontaneous release of histamine frombasophils and histamine releasing factor in patients withatopic dermatitis and food hypersensitivity", pp.228~232 (1989)
- "Calcium channel blockers in immediate hypersensitivity", pp.~25 (1983)
- "Role of sodium in mediator release from human basophils", immu(l), pp.978~986 (1992)
- "functional evidence that it does not bind tothe IgE molecule", 103(l), pp.642~648 (1999)
- "Identification of the self-interaction of rat TCTP", IgE-dependent histamine-releasing factor using yeast twohybridsystem, 2000, pp.384, 379 379~382 382