Fu Lianwu, Rab Andras, Tang Li ping, Bebok Zsuzsa, Rowe Steven M, Bartoszewski Rafal, Collawn James F
Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America; Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama, United States of America; Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
PLoS One. 2015 Apr 16;10(4):e0123131. doi: 10.1371/journal.pone.0123131. eCollection 2015.
The ΔF508 mutant form of the cystic fibrosis transmembrane conductance regulator (ΔF508 CFTR) that is normally degraded by the ER-associated degradative pathway can be rescued to the cell surface through low-temperature (27°C) culture or small molecular corrector treatment. However, it is unstable on the cell surface, and rapidly internalized and targeted to the lysosomal compartment for degradation. To understand the mechanism of this rapid turnover, we examined the role of two adaptor complexes (AP-2 and Dab2) and three E3 ubiquitin ligases (c-Cbl, CHIP, and Nedd4-2) on low-temperature rescued ΔF508 CFTR endocytosis and degradation in human airway epithelial cells. Our results demonstrate that siRNA depletion of either AP-2 or Dab2 inhibits ΔF508 CFTR endocytosis by 69% and 83%, respectively. AP-2 or Dab2 depletion also increases the rescued protein half-life of ΔF508 CFTR by ~18% and ~91%, respectively. In contrast, the depletion of each of the E3 ligases had no effect on ΔF508 CFTR endocytosis, whereas CHIP depletion significantly increased the surface half-life of ΔF508 CFTR. To determine where and when the ubiquitination occurs during ΔF508 CFTR turnover, we monitored the ubiquitination of rescued ΔF508 CFTR during the time course of CFTR endocytosis. Our results indicate that ubiquitination of the surface pool of ΔF508 CFTR begins to increase 15 min after internalization, suggesting that CFTR is ubiquitinated in a post-endocytic compartment. This post-endocytic ubiquination of ΔF508 CFTR could be blocked by either inhibiting endocytosis, by siRNA knockdown of CHIP, or by treating cells with the CFTR corrector, VX-809. Our results indicate that the post-endocytic ubiquitination of CFTR by CHIP is a critical step in the peripheral quality control of cell surface ΔF508 CFTR.
囊性纤维化跨膜传导调节因子的ΔF508突变体形式（ΔF508 CFTR）通常会通过内质网相关降解途径被降解，而通过低温（27°C）培养或小分子校正剂处理，它可以被挽救至细胞表面。然而，它在细胞表面不稳定，会迅速内化并靶向溶酶体区室进行降解。为了理解这种快速周转的机制，我们研究了两种衔接蛋白复合物（AP - 2和Dab2）以及三种E3泛素连接酶（c - Cbl、CHIP和Nedd4 - 2）在低温挽救的ΔF508 CFTR内吞作用和降解过程中在人气道上皮细胞中的作用。我们的结果表明，AP - 2或Dab2的siRNA耗竭分别抑制ΔF508 CFTR内吞作用69%和83%。AP - 2或Dab2的耗竭还分别使挽救的ΔF508 CFTR蛋白半衰期增加约18%和约91%。相比之下，每种E3连接酶的耗竭对ΔF508 CFTR内吞作用没有影响，而CHIP的耗竭显著增加了ΔF508 CFTR的表面半衰期。为了确定在ΔF508 CFTR周转过程中泛素化发生的位置和时间，我们在CFTR内吞作用的时间进程中监测了挽救的ΔF508 CFTR的泛素化情况。我们的结果表明，内化后15分钟，ΔF508 CFTR表面池的泛素化开始增加，这表明CFTR在内吞后区室中被泛素化。通过抑制内吞作用、CHIP的siRNA敲低或用CFTR校正剂VX - 809处理细胞，可以阻断ΔF508 CFTR的这种内吞后泛素化。我们的结果表明，CHIP介导的CFTR内吞后泛素化是细胞表面ΔF508 CFTR外周质量控制的关键步骤。
