Gehring Eva-Maria, Lam Rebecca S, Siraskar Gulab, Koutsouki Evgenia, Seebohm Guiscard, Ureche Oana N, Ureche Liviu, Baltaev Ravshan, Tavare Jeremy M, Lang Florian
Department of Physiology, University of Tübingen, Gmelinstrasse 5, D-72076 Tübingen, Germany.
Biochem Biophys Res Commun. 2009 Dec 18;390(3):952-7. doi: 10.1016/j.bbrc.2009.10.084. Epub 2009 Oct 21.
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated Cl(-) channel critically important in Cl(-) secreting epithelia. Mutations in the CFTR gene, such as (DeltaF508)CFTR leads to cystic fibrosis, a severe disease with defective Cl(-) secretion. CFTR is stimulated by the serum and glucocorticoid-inducible kinase SGK1. The SGK1 dependent regulation of several carriers and channels involves the phosphatidylinositol-3-phosphate-5-kinase PIKfyve, which similarly mediates the regulation of glucose carriers by PKB/Akt. The present study was thus performed to elucidate whether PKB/Akt and PIKfyve are regulators of CFTR. To this end CFTR or (DeltaF508)CFTR were expressed in Xenopus oocytes alone or together with PKB, PIKfyve or the SGK1/PKB resistant mutant (S318A)PIKfyve, and the current generated by cAMP upregulation with 10muM forskolin+1mM IBMX determined utilizing dual electrode voltage clamp. As a result, forskolin/IBMX treatment triggered a current (I(cAMP)) in CFTR-expressing Xenopus oocytes, but not in oocytes expressing (DeltaF508)CFTR. Coexpression of PKB/Akt and PIKfyve, but not of (S318A)PIKfyve, stimulated I(cAMP) in CFTR-expressing ( approximately 2- to 3-fold) but not in (DeltaF508)CFTR-expressing or water injected Xenopus oocytes. Immunohistochemistry revealed that the coexpression of PIKfyve, but not of (S318A)PIKfyve, enhanced the CFTR protein abundance but not the (DeltaF508)CFTR protein abundance in CFTR or (DeltaF508)CFTR-expressing oocytes. The present observations reveal a novel powerful regulator of intact but not of defective CFTR.
囊性纤维化跨膜传导调节因子（CFTR）是一种cAMP激活的Cl(-)通道，在Cl(-)分泌上皮细胞中至关重要。CFTR基因的突变，如（DeltaF508）CFTR，会导致囊性纤维化，这是一种Cl(-)分泌缺陷的严重疾病。CFTR受血清和糖皮质激素诱导激酶SGK1刺激。SGK1对几种载体和通道的依赖性调节涉及磷脂酰肌醇-3-磷酸-5-激酶PIKfyve，其同样介导PKB/Akt对葡萄糖载体的调节。因此，本研究旨在阐明PKB/Akt和PIKfyve是否为CFTR的调节因子。为此，CFTR或（DeltaF508）CFTR单独或与PKB、PIKfyve或SGK1/PKB抗性突变体（S318A）PIKfyve一起在非洲爪蟾卵母细胞中表达，并用双电极电压钳测定用10μM福斯可林+1mM异丁基甲基黄嘌呤上调cAMP产生的电流。结果，福斯可林/异丁基甲基黄嘌呤处理在表达CFTR的非洲爪蟾卵母细胞中引发电流（I(cAMP)），但在表达（DeltaF508）CFTR的卵母细胞中未引发。PKB/Akt和PIKfyve的共表达，但不是（S318A）PIKfyve的共表达，刺激了表达CFTR的卵母细胞中的I(cAMP)（约2至3倍），但在表达（DeltaF508）CFTR的卵母细胞或注射水的非洲爪蟾卵母细胞中未刺激。免疫组织化学显示，PIKfyve的共表达，但不是（S318A）PIKfyve的共表达，增加了表达CFTR或（DeltaF508）CFTR的卵母细胞中CFTR蛋白丰度，但没有增加（DeltaF508）CFTR蛋白丰度。本观察结果揭示了一种针对完整而非缺陷CFTR的新型强效调节因子。
