Abstract
Epithelial cell polarization involves several kinase signaling cascades that eventually divide the surface membrane into an apical and a basolateral part. One kinase, which is activated during the polarization process, is phosphoinositide 3-kinase (PI3K). In MDCK cells, the basolateral potassium channel Kv7.1 requires PI3K activity for surface-expression during the polarization process. Here, we demonstrate that Kv7.1 surface expression requires tonic PI3K activity as PI3K inhibition triggers endocytosis of these channels in polarized MDCK. Pharmacological inhibition of SGK1 gave similar results as PI3K inhibition, whereas overexpression of constitutively active SGK1 overruled it, suggesting that SGK1 is the primary downstream target of PI3K in this process. Furthermore, knockdown of the ubiquitin ligase Nedd4-2 overruled PI3K inhibition, whereas a Nedd4-2 interaction-deficient Kv7.1 mutant was resistant to both PI3K and SGK1 inhibition. Altogether, these data suggest that a PI3K-SGK1 pathway stabilizes Kv7.1 surface expression by inhibiting Nedd4-2-dependent endocytosis and thereby demonstrates that Nedd4-2 is a key regulator of Kv7.1 localization and turnover in epithelial cells.
Highlights
phosphoinositide 3-kinase (PI3K) regulates the surface expression of Kv7.1
We have previously reported that Kv7.1 is retained intracellularly during the early stages of epithelial cell polarization induced by the calcium switch but is redistributed to the basolateral cell surface in a PI3K-dependent manner at a later stage [17]
As PI3K activity regulates the formation of the basolateral membrane and appears to be required for its preservation [28], we speculated whether tonic PI3K activity would be required to maintain Kv7.1 expression at the basolateral plasma membrane of polarized Madin-Darby canine kidney (MDCK) cells
Summary
PI3K regulates the surface expression of Kv7.1. Results: Kv7.1 surface expression depends on PI3K activity in polarized epithelial cells. In MDCK cells, the basolateral potassium channel Kv7.1 requires PI3K activity for surface-expression during the polarization process. Knockdown of the ubiquitin ligase Nedd overruled PI3K inhibition, whereas a Nedd interaction-deficient Kv7.1 mutant was resistant to both PI3K and SGK1 inhibition These data suggest that a PI3K-SGK1 pathway stabilizes Kv7.1 surface expression by inhibiting Nedd4-2-dependent endocytosis and thereby demonstrates that Nedd is a key regulator of Kv7.1 localization and turnover in epithelial cells. Because the interaction of Nedd with both ENaC and Kv7.1 is mediated by intrinsic sequences known as PY motifs, it is possible that the interaction of Nedd with Kv7.1 is inhibited by the same phosphorylation of Nedd4-2 Overall, these observations position SGK1 and Akt as possible downstream targets of the PI3K effect upon Kv7.1 localization during the polarization process. Our data suggest that a PI3K-SGK1 pathway promotes cell surface stabilization of Kv7.1 by inhibiting Nedd4-2-dependent endocytosis and places Nedd as a key regulator of Kv7.1 surface expression and turnover
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