The intermediate filament protein CK19 stabilizes CFTR at the plasma membrane

The protein interactions that govern apical membrane CFTR stability and density are not entirely elucidated. We used CFTR immunoprecipitation from cell lysates of human airway epithelial cell lines (LBE-AA, expressing wt-CFTR, and LBE-KK, expressing ΔF508CFTR; kindly provided by Dr. Scott Randell, UNC-CH) followed by mass spectrometry to probe CFTR interacting proteins and identified cytokeratin 19 (CK19) as a novel CFTR binding partner. CK19 is a type I intermediate filament protein that has been functionally associated with cellular differentiation processes and cytoskeletal organization. Co-immunoprecipitation experiments confirmed a CFTRCK19 interaction in Calu-3 cells which endogenously express CFTR. Immunofluorescence confocal microscopy revealed that CK19 co-localized with CFTR at the apical membrane domain of polarized Calu-3 cells. To test for functional roles of the interaction, we co-expressed wt-CFTR with CK19 in HEK293 cells. Under steady-state conditions, CK19 stabilized wt-CFTR at the plasma membrane. As detected by cell surface biotinylation followed by CFTR immunoprecipitation, co-expression of CK19 increased the plasma membrane expression of wt-CFTR by 1.5-fold. Consistent with these results, over-expression (via recombinant adenovirus) of CK19 in Calu-3 and HeLa cells stably expressing wt-CFTR resulted in an approximately 2- fold increase in CFTR Cl- currents/fluxes in whole-cell patch clamp and SPQ assays. Internalization assays of cell surface CFTR indicated that expression of CK19 reduced the rate of wt-CFTR endocytosis from the plasma membrane. Thus, CK19 inhibiting CFTR endocytosis appears to be a key aspect of the mechanism by which CK19 promotes apical CFTR stability. Pulse-chase experiments indicated that CK19 expression enhanced the maturation efficiency of wt-CFTR while having no significant effect in promoting the maturation of ΔF508-CFTR. Interestingly, however, over-expression of CK19 stabilized temperature rescued ΔF508-CFTR at the plasma membrane and increased the plasma membrane density of the rescued ΔF508-CFTR protein by 4.6-fold. This result was supported by SPQ assays showing that CK19 expression potentiated forskolin-stimulated anion efflux after temperature rescue of ΔF508-CFTR. Taken together, our data reveal a novel mechanism by which the intermediate filament protein, CK19, stabilizes CFTR at the plasma membrane and increases its plasma membrane density by inhibiting its endocytosis. The retention of rescued ΔF508-CFTR at the plasma membrane by CK19 may provide a therapeutic approach to CF by stabilizing mutant protein that escapes ER-associated degradation, or by facilitating the actions of ER-targeted correctors of ΔF508-CFTR. [Supported by NIH grants DK68196, DK72506 and by the CF Foundation