Polycystin-1 activates the Calcineurin/NFAT (nuclear factor of activated T-cells) signaling pathway
Polycystin-1 activates the Calcineurin/NFAT (nuclear factor of activated T-cells) signaling pathway
复制标题
DOI：
10.1074/jbc.m402905200
复制
发表时间：
2004-12-31
影响因子：
4.8
通讯作者：
Calvet, JP
中科院分区：
文献类型：
--
作者：
Puri, S;Magenheimer, BS;Calvet, JP
细胞内Ca 2+动员的调节与PKD 1和PKD 2基因的蛋白产物多囊蛋白-1（PC 1）和多囊蛋白-2（PC 2）的功能相关。我们现在已经证明，PC 1可以通过Ga（q）介导的磷脂酶C（PLC）的活化来活化钙调磷酸酶/NFAT（活化T细胞的核因子）信号通路。用NFAT启动子-荧光素酶报告基因瞬时转染HEK 293 T细胞证明，含有C-末端尾的膜近端区域（其包括异源三聚体G蛋白结合和激活结构域）的膜靶向PC 1构建体可以刺激NFAT荧光素酶活性。通过LiCl处理抑制糖原合成酶激酶-3 β进一步增加了PC 1介导的NFAT活性。PC 1介导的NFAT激活被钙调磷酸酶抑制剂环孢菌素A完全抑制。表达Ga α（q）亚基的构建体的共转染增强了PC 1介导的NFAT活性，而PLC（U 73122）和三磷酸肌醇和ryanodine受体（xestospongin和2-aminophenylborate）的抑制剂和非特异性Ca 2+通道阻滞剂（钆）减少了PC 1介导的NFAT活性。PC 2无法激活NFAT。NFAT-绿色荧光蛋白核定位分析表明，仅含有C-尾或含有整个11-跨膜区加C-尾的PC 1构建体诱导NFAT-绿色荧光蛋白核转位。通过逆转录-PCR和免疫定位，在M-1小鼠皮质集合管细胞系以及胚胎和成年小鼠肾脏中证实了NFAT表达。这些数据表明了一种模型，其中PC 1信号传导导致细胞内Ca 2+的持续升高，其由PC 1激活Ga（q）介导，随后是PLC激活，从细胞内储存释放Ca 2+，并激活储存操作的Ca 2+进入，从而激活钙调磷酸酶和NFAT。
Regulation of intracellular Ca2+ mobilization has been associated with the functions of polycystin-1 (PC1) and polycystin-2 (PC2), the protein products of the PKD1 and PKD2 genes. We have now demonstrated that PC1 can activate the calcineurin/NFAT ( nuclear factor of activated T-cells) signaling pathway through Galpha(q)-mediated activation of phospholipase C ( PLC). Transient transfection of HEK293T cells with an NFAT promoter-luciferase reporter demonstrated that membrane-targeted PC1 constructs containing the membrane proximal region of the C-terminal tail, which includes the heterotrimeric G protein binding and activation domain, can stimulate NFAT luciferase activity. Inhibition of glycogen synthase kinase-3beta by LiCl treatment further increased PC1-mediated NFAT activity. PC1-mediated activation of NFAT was completely inhibited by the calcineurin inhibitor, cyclosporin A. Cotransfection of a construct expressing the Galpha(q) subunit augmented PC1-mediated NFAT activity, whereas the inhibitors of PLC (U73122) and the inositol trisphosphate and ryanodine receptors (xestospongin and 2-aminophenylborate) and a nonspecific Ca2+ channel blocker (gadolinium) diminished PC1-mediated NFAT activity. PC2 was not able to activate NFAT. An NFAT-green fluorescent protein nuclear localization assay demonstrated that PC1 constructs containing the C-tail only or the entire 11-transmembrane spanning region plus C-tail induced NFAT-green fluorescent protein nuclear translocation. NFAT expression was demonstrated in the M-1 mouse cortical collecting duct cell line and in embryonic and adult mouse kidneys by reverse transcriptase-PCR and immunolocalization. These data suggest a model in which PC1 signaling leads to a sustained elevation of intracellular Ca2+ mediated by PC1 activation of Galpha(q) followed by PLC activation, release of Ca2+ from intracellular stores, and activation of store-operated Ca2+ entry, thus activating calcineurin and NFAT.