p70S6 kinase-dependent phosphorylation of the μ2 subunit of the AP2 adaptor complex is needed for clathrin-mediated endocytosis

Clathrin-mediated endocytosis (CME) is a process in which ligands and their corresponding receptors at the cell surface are internalized via clathrin-coated invaginations of the plasma membrane. Both clathrin and endocytic cargo are recruited to the clathrin-coated pit by the adaptor protein complex AP2. AP2 resides in the cytoplasm in the closed conformation and opens upon interacting with the plasma membrane. Effective CME requires both pools of AP2, open and closed, and effective transition between these states but the mechanisms regulating this transition are only partially understood. Here, we report that serine 45 (S45) of the µ2 subunit of the AP2 complex is phosphorylated in a p70S6 kinase-dependent manner in HeLa cells both with hyperactivated mTOR signaling and under basal conditions. We demonstrate that the loss of S45-µ2 phosphorylation results in decreased internalization of canonical CME cargo such as transferrin and PDGF receptors. In Caenorhabditis elegans , the absence of S45-µ2 phosphorylation produces the dumpy phenotype characteristic of the loss of function of AP2. Our live imaging experiments further suggest that these phenotypes might arise because S45-μ2 phosphorylation is needed for conformational changes of the AP2 complex. These findings uncover a mechanism central to CME control and extend our knowledge on the role of post-translational modifications of AP2 components in regulating the function of this complex.