Phosphorylation of adaptor protein-2 mu2 is essential for Na+,K+-ATPase endocytosis in response to either G protein-coupled receptor or reactive oxygen species.

Activation of G protein-coupled receptor by dopamine and hypoxia-generated reactive oxygen species promote Na+,K+-ATPase endocytosis. This effect is clathrin dependent and involves the activation of protein kinase C (PKC)-zeta and phosphorylation of the Na+,K+-ATPase alpha-subunit. Because the incorporation of cargo into clathrin vesicles requires association with adaptor proteins, we studied whether phosphorylation of adaptor protein (AP)-2 plays a role in its binding to the Na+,K+-ATPase alpha-subunit and thereby in its endocytosis. Dopamine induces a time-dependent phosphorylation of the AP-2 mu2 subunit. Using specific inhibitors and dominant-negative mutants, we establish that this effect was mediated by activation of the adaptor associated kinase 1/PKC-zeta isoform. Expression of the AP-2 mu2 bearing a mutation in its phosphorylation site (T156A) prevented Na+,K+-ATPase endocytosis and changes in activity induced by dopamine. Similarly, in lung alveolar epithelial cells, hypoxia-induced endocytosis of Na+,K+-ATPase requires the binding of AP-2 to the tyrosine-based motif (Tyr-537) located in the Na+,K+-ATPase alpha-subunit, and this effect requires phosphorylation of the AP-2 mu2 subunit. We conclude that phosphorylation of AP-2 mu2 subunit is essential for Na+,K+-ATPase endocytosis in response to a variety of signals, such as dopamine or reactive oxygen species.

The 14-3-3 protein translates the NA+,K+-ATPase {alpha}1-subunit phosphorylation signal into binding and activation of phosphoinositide 3-kinase during endocytosis.

Clathrin-dependent endocytosis of Na(+),K(+)-ATPase molecules in response to G protein-coupled receptor signals is triggered by phosphorylation of the alpha-subunit and the binding of phosphoinositide 3-kinase. In this study, we describe a molecular mechanism linking phosphorylation of Na(+),K(+)-ATPase alpha-subunit to binding and activation of phosphoinositide 3-kinase. Co-immunoprecipitation studies, as well as experiments using confocal microscopy, revealed that dopamine favored the association of 14-3-3 protein with the basolateral plasma membrane and its co-localization with the Na(+),K(+)-ATPase alpha-subunit. The functional relevance of this interaction was established in opossum kidney cells expressing a 14-3-3 dominant negative mutant, where dopamine failed to decrease Na(+),K(+)-ATPase activity and to promote its endocytosis. The phosphorylated Ser-18 residue within the alpha-subunit N terminus is critical for 14-3-3 binding. Activation of phosphoinositide 3-kinase by dopamine during Na(+),K(+)-ATPase endocytosis requires the binding of the kinase to a proline-rich domain within the alpha-subunit, and this effect was blocked by the presence of a 14-3-3 dominant negative mutant. Thus, the 14-3-3 protein represents a critical linking mechanism for recruiting phosphoinositide 3-kinase to the site of Na(+),K(+)-ATPase endocytosis.