Cytokeratin 19 stabilizes CFTR at the plasma membrane
(2009) 23rd annual North American Cystic Fibrosis Conference — Location: Minneapolis, MN (15.October.2009)
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- Abstract
- 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
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Sun, F., Zhang, R., Noël, S., Gong, X., Peters, K., & Frizzell, R. (2009). Cytokeratin 19 stabilizes CFTR at the plasma membrane. Pediatric Pulmonology, 44(S32), 224. https://hdl.handle.net/2078.5/159246 (Original work published 2009)