c-Cbl Facilitates Endocytosis and Lysosomal Degradation of Cystic Fibrosis Transmembrane Conductance Regulator in Human Airway Epithelial Cells
c-Cbl Facilitates Endocytosis and Lysosomal Degradation of Cystic Fibrosis Transmembrane Conductance Regulator in Human Airway Epithelial Cells
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DOI：
10.1074/jbc.m110.139881
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发表时间：
2010-08-27
影响因子：
4.8
通讯作者：
Swiatecka-Urban, Agnieszka
中科院分区：
文献类型：
--
作者：
Ye, Siying;Cihil, Kristine;Swiatecka-Urban, Agnieszka
囊性纤维化跨膜传导调节因子（CFTR）是一种cAMP激活的Cl-通道，表达于液体转运上皮细胞的顶膜。CFTR通道的顶膜密度部分地由CFTR的内吞作用和溶酶体降解或再循环至质膜的内吞后分选来确定。虽然以前的研究表明，泛素化在CFTR的胞内后分选中起作用，但具体的泛素连接酶尚不清楚。c-Cbl是具有泛素连接酶活性和蛋白衔接子功能的多功能分子。c-Cbl与CFTR在原代分化的人支气管上皮细胞和培养的人气道细胞中共免疫沉淀。小干扰RNA介导的c-Cbl沉默通过抑制CFTR内吞作用和增加CFTR介导的Cl-电流来增加CFTR在质膜中的表达。沉默c-Cbl没有改变质膜CFTR的泛素化部分的表达。此外，具有受损的泛素连接酶活性的c-Cbl突变体（FLAG-70 Z-Cbl）不影响CFTR的质膜表达或内吞作用。与此相反，c-Cbl突变体与截短的C-末端区域（FLAG-Cbl-480），负责蛋白衔接功能，有一个显性干扰CFTR的内吞作用和质膜表达的影响。此外，CFTR和c-Cbl在早期内体中共定位和共免疫沉淀，并且沉默c-Cbl减少了早期内体中泛素化CFTR的量。总之，我们的数据表明，在人气道上皮细胞，c-Cbl调节CFTR通过两种机制：首先作为一个衔接蛋白，促进CFTR内吞的泛素不依赖的机制，其次通过泛素化CFTR在早期内体，从而促进CFTR的溶酶体降解。
Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated Cl- channel expressed in the apical membrane of fluid-transporting epithelia. The apical membrane density of CFTR channels is determined, in part, by endocytosis and the postendocytic sorting of CFTR for lysosomal degradation or recycling to the plasma membrane. Although previous studies suggested that ubiquitination plays a role in the postendocytic sorting of CFTR, the specific ubiquitin ligases are unknown. c-Cbl is a multifunctional molecule with ubiquitin ligase activity and a protein adaptor function. c-Cbl co-immunoprecipitated with CFTR in primary differentiated human bronchial epithelial cells and in cultured human airway cells. Small interfering RNA-mediated silencing of c-Cbl increased CFTR expression in the plasma membrane by inhibiting CFTR endocytosis and increased CFTR-mediated Cl- currents. Silencing c-Cbl did not change the expression of the ubiquitinated fraction of plasma membrane CFTR. Moreover, the c-Cbl mutant with impaired ubiquitin ligase activity (FLAG-70Z-Cbl) did not affect the plasma membrane expression or the endocytosis of CFTR. In contrast, the c-Cbl mutant with the truncated C-terminal region (FLAG-Cbl-480), responsible for protein adaptor function, had a dominant interfering effect on the endocytosis and plasma membrane expression of CFTR. Moreover, CFTR and c-Cbl co-localized and co-immunoprecipitated in early endosomes, and silencing c-Cbl reduced the amount of ubiquitinated CFTR in early endosomes. In summary, our data demonstrate that in human airway epithelial cells, c-Cbl regulates CFTR by two mechanisms: first by acting as an adaptor protein and facilitating CFTR endocytosis by a ubiquitin-independent mechanism, and second by ubiquitinating CFTR in early endosomes and thereby facilitating the lysosomal degradation of CFTR.