Identification of Calcium/Calmodulin-Dependent Phosphatase as the Dephosphorylating Enzyme of IgE-Dependent Histamine-Releasing Factor in RBL-2H3

IgE-dependent histamine-releasing factor(HRF) was initially described as a secretagogue for secretion of histamine from IgE+ basophils from a subset of allergic donors. Previously, we identified that S98 residue of HRF was phosphorylated using anti-HRFpS98 antibody which specifically recognizes the phosphorylated serine residue of HRF and HRFS98A mutant construct. In vitro kinase assay, only wild type HRF was phosphorylated by PKC, and S98A HRF was not affected by PKC. In this study, we attempted to characterize the phosphatase which specifically dephosphorylates HRF by immunoprecipitation and pull-down assay. In RBL-2H3 cells, HRF interacted only with calcineurin (also called as PP2B, calcium/calmodulin-dependent phosphatase) but not with PP1 or PP2A. The results suggest that HRF is most likely dephosphory-lated by calcineurin.

RBL-2H3 세포에서 IgE-depnedent Histamine-releasing Factor의 탈인산화 효소에 관한 연구

RBL-2H3 cell lysates에 anti-protein phosphatase(PP) 1, 2A, 2B 항체를 첨가한 후 immunoprecipitation을 실시한 결과 PP2B를 가해준 샘플에서만 HRF를 확인하였다. 역으로 monoclonal anti-HRF 항체를 가한 후 immunoprecipitation을 실시한 결과 PP1, 2A는 검출되지 않았으나 PP2B의 경우는 regulatory subunit(19 kDa), catalyic subunit(60 kDa) 모두 확인할 수 있었다. Affinity chromatography를 통해서도 PP2B가 HRF의 탈인산화에 관여함을 확인하였다 즉 19kDa의 PP2B regulatory subunit과 60kDa의 catalytic subunit 모두가 확인되었으며 외부 $Ca^{2+}$이온 첨가 여부에 따른 차이는 관찰할 수 없었다. 결론적으로 RBL-2H3 cell에서 PP2B는 PP1이나 PP2A에 비해 상대적으로 그 존재량은 적으나 HRF와 상호작용하는 phosphatase로서 검출된 반면 PP1이나 PP2A는 검출되지 않았다.