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.
Department of Medicine, Membrane Signaling Networks, Karolinska Institutet, Karolinska Hospital, Stockholm, Sweden.
Zongpei
Z
Chen
Rafael T
RT
Krmar
Laura
L
Dada
Riad
R
Efendiev
Ingo B
IB
Leibiger
Carlos H
CH
Pedemonte
Adrian I
AI
Katz
Jacob I
JI
Sznajder
Alejandro M
AM
Bertorello
D033962:Adaptor Protein Complex 2 / Q000378:metabolism*; D033983:Adaptor Protein Complex mu Subunits / Q000378:metabolism*; D000818:Animals; D015687:Cell Hypoxia / Q000187:drug effects; D002478:Cells, Cultured; D004298:Dopamine / Q000494:pharmacology; D004705:Endocytosis / Q000187:drug effects*; D006801:Humans; D008954:Models, Biological; D009154:Mutation / Q000235:genetics; D009893:Opossums; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding; D017382:Reactive Oxygen Species / Q000494:pharmacology*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism
D033962:Adaptor Protein Complex 2; D033983:Adaptor Protein Complex mu Subunits; D017382:Reactive Oxygen Species; D043562:Receptors, G-Protein-Coupled; C438886:adaptor protein complex 2, mu 2 subunit; D014443:Tyrosine; D000254:Sodium-Potassium-Exchanging ATPase; D004298:Dopamine