Cytokeratin 19 stabilizes CFTR at the plasma membrane

To search for CFTR interacting partners in human airway epithelial cells, we immunoprecipitated CFTR and identified cytokeratin 19 (CK19) by mass spectrometry as a novel CFTR binding protein. CK19 is a type I intermediate filament protein that is functionally associated with cellular differentiation processes and cytoskeletal organization. Co-immunoprecipitation experiments confirmed a CFTR-CK19 interaction in Calu-3 cells endogenously expressing CFTR, and immunofluorescence confocal microscopy revealed their co-localization at the apical membrane domain of polarized Calu-3 cells. To test its functional role, we co-expressed CK19 with wt-CFTR 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 2.5-fold. In Calu-3 and HeLa cells stably expressing wt-CFTR, the over-expression of CK19 using recombinant adenovirus (AdCK19) produced ~2-fold increase in CFTR-mediated Cl- fluxes/currents, assayed by SPQ and whole-cell patch clamp. The physiological role of CK19 was examined using recombinant lentiviruses containing shRNAs targeting its expression in HEK293 cells. Knockdown of endogenous CK19 by 50% yielded an 85% reduction in wt- CFTR at the plasma membrane. Cell surface CFTR internalization assays indicated that the expression of CK19 reduced the rate of wt-CFTR endocytosis. Thus, inhibition of CFTR endocytosis is 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, the over-expression of CK19 stabilized temperature rescued ΔF508-CFTR at the plasma membrane, increasing its density by 4.6-fold. This result was supported by transepithelial current measurements across CFBE41o-ΔF508 epithelia, which showed that CK19 expression potentiated forskolin-stimulated Cl- current by 65% after temperature rescue of the mutant. Taken together, our data reveal a novel mechanism by which the intermediate filament protein, CK19, stabilizes CFTR at the plasma membrane, increasing its plasma membrane density by inhibiting CFTR 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 trafficking