Yes-Associated Protein 65 Localizes P62c-Yes to the Apical Compartment of Airway Epithelia by Association with Ebp50

Abstract

We recently showed that the COOH terminus of the cystic fibrosis transmembrane conductance regulator associates with the submembranous scaffolding protein EBP50 (ERM-binding phosphoprotein 50 kD; also called Na+/H+ exchanger regulatory factor). Since EBP50 associates with ezrin, this interaction links the cystic fibrosis transmembrane conductance regulator (CFTR) to the cortical actin cytoskeleton. EBP50 has two PDZ domains, and CFTR binds with high affinity to the first PDZ domain. Here, we report that Yes-associated protein 65 (YAP65) binds with high affinity to the second EBP50 PDZ domain. YAP65 is concentrated at the apical membrane in airway epithelia and interacts with EBP50 in cells. The COOH terminus of YAP65 is necessary and sufficient to mediate association with EBP50. The EBP50–YAP65 interaction is involved in the compartmentalization of YAP65 at the apical membrane since mutant YAP65 proteins lacking the EBP50 interaction motif are mislocalized when expressed in airway epithelial cells. In addition, we show that the nonreceptor tyrosine kinase c-Yes is contained within EBP50 protein complexes by association with YAP65. Subapical EBP50 protein complexes, containing the nonreceptor tyrosine kinase c-Yes, may regulate apical signal transduction pathways leading to changes in ion transport, cytoskeletal organization, or gene expression in epithelial cells.

Characterization of Wild-Type and ΔF508 Cystic Fibrosis Transmembrane Regulator in Human Respiratory Epithelia

“…We localized ezrin and EBP50 to the apical PM of human airway epithelial cells (Short et al, 1998;Mohler et al, 1999;Ingraffea et al, 2002;Kulaksiz et al, 2002). Both proteins colocalized to the microvillus domain of epithelial cells in nasal, tracheal, bronchial, and bronchiolar superficial epithelia and were expressed at equivalent levels in CF and normal epithelia.…”

“…We focused on EBP50 and ezrin localization as these are the most widely studied ERM proteins relevant to positioning CFTR within the PM (Short et al, 1998;Mohler et al, 1999).…”

“…Rabbit polyclonal antibodies against EBP50 (Mohler et al, 1999), MUC5AC, and InsP3-receptors (Mohler et al, 2004) were gifts from Dr. S. Milgram (UNC-CH), Dr. J Sheehan (UNC-CH), and Dr. P. Mohler (Duke University), respectively; the rabbit polyclonal antibody against AQP5 (Kreda et al, 2001) and the rat monoclonal antibody (mAb) against tubulin were from Chemicon (Temecula, CA); the mAb against ezrin (Mohler et al, 1999) was from BD Transduction Laboratories (San Diego, CA); the mAb against MUC5AC was from NeoMarkers (Fremont, CA); and mAb against ␤IV-tubulin from Sigma (St. Louis, MO); all secondary antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA). Phalloidin labeled with AlexaFluor dyes to identify the actin cytoskeleton, and concanavalin A fluorescently labeled to identify the endoplasmic reticulum (Mateo et al, 2003) were from Molecular Probes (Eugene, OR).…”

“…In some experiments, reducing (sodium borohydride) or oxidizing agents (hydrogen peroxide; both from Sigma) were used after the permeabilization step for epitope retrieval. Serial tissue sections were incubated with antibodies against MUC5AC to identify goblet cells, ␤IV-tubulin or tubulin to identify ciliated cells, AQP5 to identify serous cells in gland acini (Kreda et al, 2001), and ezrin and EBP50 to stain the scaffolding complex associated with CFTR (Short et al, 1998;Mohler et al, 1999). In some experiments, CFTR mAbs were coincubated with polyclonal antibodies against AQP5 and EBP50, or a rat mAb against tubulin, followed by fluorescent anti-mouse IgG Fab fragment and AlexaFluor 633 phalloidin plus either fluorescent anti-rabbit IgG, or anti-rat IgG, diluted 1:200, 1:150, 1:200, and 1:200 respectively.…”

“…However, it has recently been suggested that scaffolding proteins of the ezrin-radixin-moesin (ERM) family must be correctly localized at the PM for proper CFTR trafficking and function (Short et al, 1998;Mohler et al, 1999;Huang et al, 2003). Epithelial inflammation and remodeling have been reported to alter ERM protein expression (Laoukili et al, 2001), which could impair localization of ⌬F508 protein to the apical PM.…”