Clathrin-mediated endocytosis (CME) is key to maintaining the transmembrane protein composition of cells' limiting membranes. During mammalian CME, a reversible phosphorylation event occurs on Thr156 of the μ2 subunit of the main endocytic clathrin adaptor, AP2. We show that this phosphorylation event starts during clathrin-coated pit (CCP) initiation and increases throughout CCP lifetime. μ2Thr156 phosphorylation favors a new, cargo-bound conformation of AP2 and simultaneously creates a binding platform for the endocytic NECAP proteins but without significantly altering AP2's cargo affinity in vitro. We describe the structural bases of both. NECAP arrival at CCPs parallels that of clathrin and increases with μ2Thr156 phosphorylation. In turn, NECAP recruits drivers of late stages of CCP formation, including SNX9, via a site distinct from where NECAP binds AP2. Disruption of the different modules of this phosphorylation-based temporal regulatory system results in CCP maturation being delayed and/or stalled, hence impairing global rates of CME.

• Keywords
• AAK1, AP2 endocytic adaptor, NECAP, NMR, Numb-associated kinases (NAK), SNX9, TIRF, clathrin-mediated endocytosis, crystallography, regulation by phosphorylation,
• MeSH
• Adaptor Protein Complex alpha Subunits genetics   MeSH
• Adaptor Protein Complex 2 genetics   metabolism   MeSH
• Endocytosis genetics   MeSH
• Phosphorylation genetics   MeSH
• Clathrin genetics   metabolism   MeSH
• Clathrin-Coated Vesicles genetics   metabolism   MeSH
• Humans MeSH
• Coated Pits, Cell-Membrane genetics   metabolism   MeSH
• Sorting Nexins genetics   MeSH
• Protein Binding genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adaptor Protein Complex alpha Subunits MeSH
• Adaptor Protein Complex 2 MeSH
• Clathrin MeSH
• NECAP1 protein, human MeSH Browser
• SNX9 protein, human MeSH Browser
• Sorting Nexins MeSH