In this study, we provide evidence that phosphorylation of the μ2 isoform of AP-2 is necessary for GPCR-induced endocytosis of Na+,K+-ATPase molecules. Our results demonstrate that other intracellular signals, such as hypoxia-induced ROS, also required μ2 phosphorylation to promote Na+,K+-ATPase endocytosis in lung alveolar epithelial cells.
Clathrin-dependent endocytosis of membrane proteins is initiated by adaptor proteins (adaptins) that simultaneously bind to cargo proteins, recruit clathrin, and promote the formation of clathrin-coated pits (8
). AP-2 phosphorylation is a key event that determines the binding affinity of the μ2 subunit to cargo proteins (18
). In the present study, DA increased the state of AP-2 μ2 subunit phosphorylation in a time-dependent manner that coincided with maximal association of AP-2 with the Na+
-ATPase and preceded its appearance in clathrin vesicles (1
). The transition between phospho- and dephospoAP-2 μ2 facilitates its association with the cargo molecules, as demonstrated by several in vitro
). In our studies, the presence of negatively charged residues (T156D and T156E) also prevented AP-2 μ2 binding to the α-subunit and Na+
-ATPase endocytosis (not shown).
In intact cells, the AP-2 μ2 subunit could be targeted by myriad protein kinases. A new kinase (AAK1) responsible for phosphorylating the AP-2 μ2 subunit has been described (21
). A detailed in vitro
study revealed that AAK1 (21
) could be inhibited by several chemical inhibitors at doses that overlap their actions on other kinases. In this study, when we used several inhibitors of PKC and dominant-negative mutants of the PKC-ζ isoform, it was possible to block AP-2 μ2 phosphorylation. Coincidentally, this isoform of PKC is also responsible for phosphorylating the Na+
-ATPase α-subunit at the ser-18 residue (a necessary step for Na+
-ATPase subunit endocytosis). Staurosporine, at a concentration that would not affect the activation of PKC-ζ, does inhibit the activation of AAK1 (21
). Therefore, it is possible that the PKC-ζ isoform would form a complex within the endocytic network and thereby express a dual effect (i.e., phosphorylating the Na+
-ATPase and facilitating AP2 phosphorylation), which together would be responsible for Na+
-ATPase internalization. Proven specific inhibitors of AAK1 in intact cells are not yet available.
The generation of ROS by lung alveolar epithelial cells during hypoxia results in a series of events leading to Na+
-ATPase internalization via a clathrin-coated–dependent mechanism (3
). Endocytosis is initiated by phosphorylation of the Na+
-ATPase α-subunit via a PKC-ζ–dependent mechanism, a process that leads to activation of phosphatidylinositol 3-kinase. The lipids generated by this kinase at the plasma membrane interface favor the association of AP-2 with the cargo. Similarly to renal epithelial cells (9
), association of AP-2 to the Na+
-ATPase requires the binding of the μ2 subunit (within the AP-2 complex) to a tyrosine-based motif (Y537) located in the α-subunit. Moreover, internalization of Na+
-ATPase molecules in response to hypoxia or exogenously administrated H2
was abolished in cells expressing the AP-2 μ2 lacking the phosphorylation site (T156).
The effects of AP-2 μ2 phosphorylation have been extensively studied mostly in reconstituted systems and during basal endocytosis in intact cells exposed to changes in temperature, such as transferrin receptor trafficking (18
). In this report, we provide the first evidence that endocytosis of an integral membrane protein (the Na+
-ATPase) in response to a physiological condition (activation of GPCR by dopamine in renal epithelial cells) or a pathophysiologic condition (e.g., hypoxia in lung alveolar epithelial cells) requires phosphorylation of AP-2 μ2 subunit (). Additionally, the results from this study suggest that during endocytosis the Na+
-ATPase may represent its own scaffold system, organizing the signaling network independently of whether it is a GPCR or a ROS signal that triggers the process, thus making the activation of PKC-ζ a possible converging point during endocytosis in kidney or lung epithelial cells.
Figure 5. Model of Na+,K+-ATPase endocytosis in response to GPCR signals and those generated by hypoxia. PKC-ζ and possibly AAK1 are the cellular targets where the GPCR or hypoxia signals converge. In the case of GPCR (dopamine), activation (more ...)