Phosphorylation of the AP2 μ subunit by AAK1 mediates high affinity binding to membrane protein sorting signals

Abstract

During receptor-mediated endocytosis, AP2 complexes act as a bridge between the cargo membrane proteins and the clathrin coat by binding to sorting signals via the μ2 subunit and to clathrin via the β subunit. Here we show that binding of AP2 to sorting signals in vitro is regulated by phosphorylation of the μ2 subunit of AP2. Phosphorylation of μ2 enhances the binding affinity of AP2 for sorting motifs as much as 25-fold compared with dephosphorylated AP2. The recognition of sorting signals was not affected by the phosphorylation status of the α or β2 subunit, suggesting that phosphorylation of μ2 is critical for regulation of AP2 binding to sorting signals. Phosphorylation of μ2 occurs at a single threonine residue (Thr-156) and is mediated by the newly discovered adaptor-associated kinase, AAK1, which copurifies with AP2. We propose that phosphorylation of the AP2 μ2 subunit by AAK1 ensures high affinity binding of AP2 to sorting signals of cargo membrane proteins during the initial steps of receptor-mediated endocytosis.

Crystal structure of the clathrin adaptor protein 1 core

“…Phosphorylation of a threonine residue in the linker between -chain domains ( 2Thr-156, corresponding to 1Thr-154) enhances affinity for Ypp⌽ sorting signals (28,29). Coassembly of intact AP complexes with clathrin into coats in vitro likewise enhances their affinity for Ypp⌽ sorting signals, and the subunit becomes susceptible to a specific trypsin cleavage (30).…”

Molecular Structure, Function, and Dynamics of Clathrin-Mediated Membrane Traffic

“…Thus, the open state is compatible with all known membrane interactions of the adaptor. Transition to the open state is apparently favored not only by binding to PtdIns(4,5)P 2 at multiple sites but also by binding to phosphoinositides phosphorylated at the 3 0 position (Rapoport et al 1997), by the interaction with clathrin (Matsui and Kirchhausen 1990;Rapoport et al 1997), and by phosphorylation of Thr156, in the m2 linker, by the clathrin-activated, a-subunit appendage domain-binding protein kinase AAK1 (Ricotta et al 2002;Conner et al 2003). The clathrin-box motif in the b-chain hinge is the principal site of interaction between heterotetrameric adaptors and clathrin (Fig.…”

Scd5p Mediates Phosphoregulation of Actin and Endocytosis by the Type 1 Phosphatase Glc7p in Yeast

“…The adaptor-associated kinase AAK1 negatively regulates clathrin-mediated endocytosis by phosphorylating the 2 subunit of the AP2 adaptor protein complex Ricotta et al, 2002). Interestingly, AAK1 shares with Prk1p not only the extensive kinase sequence homology but also the similar recognition motif Ricotta et al, 2002).…”

“…The adaptor-associated kinase AAK1 negatively regulates clathrin-mediated endocytosis by phosphorylating the 2 subunit of the AP2 adaptor protein complex Ricotta et al, 2002). Interestingly, AAK1 shares with Prk1p not only the extensive kinase sequence homology but also the similar recognition motif Ricotta et al, 2002). Another mammalian homologue of Prk1p, GAK, also exhibits a similar specificity on the 2 subunit and plays a regulatory role in endocytosis (Zhang et al, 2005).…”