Phosphorylation of adaptor protein-2 μ2 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)-ζ and phosphorylation of the Na⁺,K⁺-ATPase α-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 α-subunit and thereby in its endocytosis. Dopamine induces a time-dependent phosphorylation of the AP-2 μ2 subunit. Using specific inhibitors and dominant-negative mutants, we establish that this effect was mediated by activation of the adaptor associated kinase 1 /PKC-ζ isoform. Expression of the AP-2 μ2 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 α-subunit, and this effect requires phosphorylation of the AP-2 μ2 subunit. We conclude that phosphorylation of AP-2 μ2 subunit is essential for Na ⁺,K⁺-ATPase endocytosis in response to a variety of signals, such as dopamine or reactive oxygen species.