AAK1 (green) colocalizes with endocytic regions (red) in neurons.

Two new studies indicate that cycles of phosphorylation and dephosphorylation may be required during clathrin-mediated endocytosis. A newly identified kinase is responsible for the required phosphorylation.

Clathrin and the adaptor protein 2 (AP2) complex are the major coat proteins of clathrin-coated vesicles (CCVs). Among components of the AP2 complex are α and β2 adaptins, which mediate AP2 membrane targeting and interactions with clathrin, and the μ2 subunit, which may recognize the endocytic cargo.

Phosphorylation and dephosphorylation have been implicated in endocytosis and at least two kinase activities copurify with CCVs. The identity of the kinase and its substrate had not been determined, however. Now, Conner and Schmid (page 921) and Ricotta et al. (page 791) identify one of the kinase activities and demonstrate its effect on cargo binding.

Conner and Schmid show that the serine/threonine kinase AAK1 directly interacts with α-adaptin in vitro and in vivo. AAK1 cofractionates with AP2 complexes and with clathrin, and localizes to regions active in endocytosis. Ricotta et al. demonstrate that AAK1 corresponds to the endogenous kinase that phosphorylates the μ2 subunit. The phosphorylation of μ2 enhances binding of AP2 to sorting signals in vitro by 25-fold.Previous in vivo experiments indicated that phosphorylation of AP2 was required for clathrin-mediated endocytosis, even though this inhibits AP2 binding to clathrin. Conner and Schmid confirm that maintenance of the μ2 subunit in its phosphorylated form inhibits endocytosis, suggesting that a protein phosphatase activity is also required to dephosphorylate μ2. Possibly, cycles of phosphorylation and dephosphorylation facilitate concentration of the cargo at the plasma membrane through multiple rounds of AP2-mediated recruitment. Perhaps only when clustered cargo inhibits continued cycling can AP2 bind to clathrin and drive vesicle formation and cargo uptake. ▪