title: COMMD1 interacts with the COOH terminus of NKCC1 in Calu-3 airway epithelial cells to modulate NKCC1 ubiquitination.
abstract: 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.
authors.affiliation: Willard Alan Bernbaum, Center for Cystic Fibrosis Research, Departments of Pediatrics at Rainbow Babies and Children Hospital and Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio, USA.
authors.forename: Laura
authors.initials: L
authors.lastname: Smith
authors.forename: Paul
authors.initials: P
authors.lastname: Litman
authors.forename: Carole M
authors.initials: CM
authors.lastname: Liedtke
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D003300:Copper / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000502:physiology; D006801:Humans; D017136:Ion Transport; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D028021:Sodium-Potassium-Chloride Symporters / Q000235:genetics / Q000378:metabolism*; D064506:Solute Carrier Family 12, Member 2; D020798:Two-Hybrid System Techniques
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C480519:COMMD1 protein, human; D011994:Recombinant Proteins; C578877:SLC12A2 protein, human; C578878:Slc12a2 protein, mouse; D028021:Sodium-Potassium-Chloride Symporters; D064506:Solute Carrier Family 12, Member 2; D003300:Copper
keywords: Na-K-2Cl cotransport; binding assays; biotinylation; membrane expression; silencing RNA; truncation; yeast two hybrid