CFTR Regulation in Human Airway Epithelial Cells Requires Integrity of the Actin Cytoskeleton and Compartmentalized cAMP and PKA Activity.

ΔF508CFTR rescued to the apical membrane exhibits regulatory defects suggesting that the intracellular milieu may affect its ability to respond to cAMP regulation. We recently reported that NHERF1 overexpression in CFBE41o- (CFBE) cells rescues ΔF508CFTR functional expression, by promoting F- actin organization and formation of the NHERF1-ezrin-actin complex (1). The hypothesis that the rescue of ΔF508CFTR activity induced by NHERF1 overexpression may involve recruitment of PKA to the membrane region was supported by immunocytochemical analysis of PKA localization demonstrating that PKA localization shifted from the cytosol to the sub-cortical region in CFBE cells stably overexpressing NHERF1 (CFBE/sNHERF1). Importantly, when we analyzed PKA activity by a FRET-based reporter targeted to the plasma membrane (2), we found that PKA-mediated phosphorylation at the plasma membrane was lower in CFBE cells than that found in CFBE/sNHERF1 cells. By contrast, using an untargeted, cytosolic version of the PKA activity FRET reporter (2), we found that PKA- dependent phosphorylation was significantly higher in the cytosol of CFBE than in CFBE/sNHERF1 cells. To assess if the higher PKA activity detected in CFBE cytosol could be due to a higher concentration of cAMP in this compartment, we measured cAMP changes in cells expressing FRET-based reporters for cAMP levels either untargeted (3) or targeted to the plasma membrane (4). In keeping with the observed difference in PKA activity, CFBE cells showed a significantly larger cAMP response in the bulk cytosol and a lower cAMP accumulation in the sub-plasma membrane compartment with respect to CFBE/sNHERF1. A possible explanation for the observed differences in the cAMP compartmentalization in the two cell lines is that the restoration of cortical actin cytoskeletal organization induced by NHERF1 overexpression in CFBE cells (1) may contribute to the accumulation of cAMP in the sub-plasma membrane compartment by acting as a physical barrier to cAMP diffusion. In support of this hypothesis, we found that F-actin depolymerization induced by Latrunculin B, had no effect in CFBE cells while significantly increasing both cAMP accumulation and PKA activity in the cytosol of CFBE/sNHERF1 cells at the expense of their levels and activity in sub-cortical region. Altogether these findings suggest that the organized sub-cortical cytoskeleton constitutes an efficient barrier to cAMP diffusion promoting compartmentalized cAMP/PKA signals. This work was supported by the Italian Cystic Fibrosis Research Foundation (grant FFC#1/2009) with the contribution of the “Delegazione FFC della Valdadige”; “Nicola Petruzzi e Del FFC di Molfetta”; “Gli amici di Thomas con la Delegazione FFC di Vibo Valentia”; “Delegazione FFC “La Bottega delle Donne” di Montebelluna Treviso”. 1. Favia M et al. Mol Biol Cell. 2010;21(1):73-86. 2. Allen MD, Zhang J. Biochem Biophys Res Commun. 2006;348(2):716-21. 3. Ponsioen B et al. EMBO Rep. 2004;5(12):1176-80. 4. Terrin A et al. J Cell Biol. 2006;175(3):441-51.