Proceedings of The Physiological Society
University College London (2011) Proc Physiol Soc 24, C19 and PC19
Dependence of CFTR Trafficking on LMTK2 kinase activity in Human Colonic Epithelial and Mouse Primary Airway Epithelial Cells
N. A. Bradbury1, Y. Jia1, R. J. Bridges1
1. Physiology and Biophysics, Chicago Medical School, North Chicago, Illinois, United States.
The magnitude of transepithelial chloride transport depends on both the activation status (i.e. phosphorylation) and number of CFTR channels present in the apical membrane. In polarized epithelia, CFTR undergoes rapid and efficient endocytosis with subsequent recycling back to the plasma membrane. The molecular mechanisms underlying CFTR recycling are poorly understood, yet this critical process is required for the appropriate channel copy number at the apical membrane, and is defective in the common CFTR mutant ΔF508. Here we examined the role of the novel kinase LMTK2 in regulating the trafficking of CFTR in a human colonic epithelial cell line that endogenously expresses both CFTR and LMTK2 (T84), as well as in primary mouse tracheal epithelial cells derived from wt (LMTK2+/+) and LMTK2-/- knockout mice. RNAi-mediated knockdown of LMTK2 had no effect on CFTR endocytosis in T84 cells, but markedly inhibited recycling of internalized channels, leading to a net reduction in cell surface protein and a concomitant reduction in cAMP activated chloride current. In contrast, over expression of LMTK2 increased CFTR recycling, with an attendant increase in CFTR copy number at the apical membrane and increased transepithelial chloride secretion. Expression of a kinase-dead LMTK2 mutant acted as a partial dominant-negative construct, implicating a phosphorylation event as a key element in the LMTK2 dependent recycling of CFTR in human polarized epithelia. Indeed LMTK2 is able to phosphorylate Ser737 in CFTR’s Regulatory domain. To validate further BREK as a regulator of CFTR trafficking, we utilized MTE cells obtained from wild-type and LMTK2 knockout mice as an animal model. Loss of LMTK2 activity was not associated with any loss of transepithelial resistance, indicating that the integrity of the epithelial monolayer was not compromised. However, forskolin stimulated short circuit currents were markedly lower in MTE from LMTK2-/- animals compared to those derived from wild-type animals, essentially recapitulating the observations derived from T84 cells. Peptide array analysis argues that CFTR is the primary substrate for LMTK2 dependent phosphorylation, indicating the unique position of LMTK2 as a novel drugable target to enhance the recycling of mutant CFTR back to the cell surface.
Where applicable, experiments conform with Society ethical requirements