COMMD1 interacts with the COOH terminus of NKCC1 in Calu-3 airway epithelial cells to modulate NKCC1 ubiquitination.
COMMD1 interacts with the COOH terminus of NKCC1 in Calu-3 airway epithelial cells to modulate NKCC1 ubiquitination.
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COMMD1 与 Calu-3 气道上皮细胞中 NKCC1 的 COOH 末端相互作用，调节 NKCC1 泛素化。
DOI：
10.1152/ajpcell.00394.2012
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发表时间：
2013
期刊：
影响因子：
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通讯作者：
Liedtke,CaroleM
中科院分区：
文献类型：
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作者：
Smith,Laura;Litman,Paul;Liedtke,CaroleM
通过靶向破坏涉及羧基末端（CT-NKCC1）但不涉及氨基末端的编码NKCC1的基因，产生了Na-K-2Cl协同转运蛋白（NKCC1）缺陷小鼠。我们假设，由此产生的生理缺陷是由于与CT-NKCC1相互作用的蛋白质的丢失。使用酵母双杂交方法，发现衔接蛋白COMMD 1与CT-NKCC 1（aa 1，040 - 1，212）结合。使用GST-COMMD 1和myc-CT-NKCC 1在酵母非依赖性系统中验证结合。在结合测定中使用截短的COMMD 1和CT-NKCC 1肽以鉴定相互作用的位点。结果表明COMMD 1（aa 1 - 47）与CT-NKCC 1（aa 1，040 - 1，134）的结合呈浓度依赖性。使用重组FLAG-CT-NKCC 1在下拉中检测到内源性COMMD 1;该共下拉被COMMD 1（aa 1 - 47）阻断。CT-NKCC 1（aa 1，040 - 1，137）降低NKCC 1的基底外侧膜表达，COMMD 1（aa 1 - 47）增加NKCC 1膜表达。使用沉默（si）RNA下调COMMD 1导致内源性COMMD 1的瞬时丢失，但不影响高渗蔗糖对NKCC 1的激活。高渗透压引起NKCC 1膜表达的瞬时增加，表明NKCC 1的调节运输;使用siRNA下调COMMD 1降低基线（未刺激的）NKCC 1表达，并减弱由高渗透压引起的NKCC 1膜表达的瞬时升高。在HT29工程细胞中COMMD 1的组成性下调表现出COMMD 1的丢失和NKCC 1膜表达的降低，而对NKCC 1的活化没有影响。在Calu-3细胞和HT29细胞中COMMD 1的缺失导致泛素化的NKCC 1减少。结果表明COMMD 1在NKCC 1膜表达和泛素化的调节中起作用。
Mice deficient in Na-K-2Cl cotransporter (NKCC1) have been generated by targeted disruption of the gene encoding NKCC1 involving the carboxy terminus (CT-NKCC1) but not the amino terminus. We hypothesize that the resulting physiological defects are due to loss of proteins interacting with CT-NKCC1. Using a yeast two-hybrid approach, adaptor protein COMMD1 was found to bind to CT-NKCC1 (aa 1,040–1,212). Binding was verified in a yeast-independent system using GST-COMMD1 and myc-CT-NKCC1. Truncated COMMD1 and CT-NKCC1 peptides were used in binding assays to identify the site of interaction. The results demonstrate concentration-dependent binding of COMMD1 (aa 1–47) to CT-NKCC1 (aa 1,040–1,134). Endogenous COMMD1 was detected in pull downs using recombinant FLAG-CT-NKCC1; this co-pull down was blocked by COMMD1 (aa 1–47). CT-NKCC1 (aa 1,040–1,137) decreased basolateral membrane expression of NKCC1, and COMMD1 (aa 1–47) increased NKCC1 membrane expression. Downregulation of COMMD1 using silencing (si)RNA led to a transient loss of endogenous COMMD1 but did not affect activation of NKCC1 by hyperosmotic sucrose. Hyperosmolarity caused a transient increase in NKCC1 membrane expression, indicating regulated trafficking of NKCC1; downregulation of COMMD1 using siRNA reduced baseline (unstimulated) NKCC1 expression and blunted a transient elevation in NKCC1 membrane expression caused by hyperosmolarity. Constitutive downregulation of COMMD1 in HT29 engineered cells exhibited loss of COMMD1 and decreased NKCC1 membrane expression with no effect on activation of NKCC1. Loss of COMMD1 in Calu-3 cells and in HT29 cells led to reduced ubiquitinated NKCC1. The results indicate a role for COMMD1 in the regulation of NKCC1 membrane expression and ubiquitination.