Clathrin promotes incorporation of cargo into coated pits by activation of the AP2 adaptor μ2 kinase

Abstract

Endocytic cargo such as the transferrin receptor is incorporated into clathrin-coated pits by associating, via tyrosine-based motifs, with the AP2 complex. Cargo–AP2 interactions occur via the μ2 subunit of AP2, which needs to be phosphorylated for endocytosis to occur. The most likely role for μ2 phosphorylation is in cargo recruitment because μ2 phosphorylation enhances its binding to internalization motifs. Here, we investigate the control of μ2 phosphorylation. We identify clathrin as a specific activator of the μ2 kinase and, in permeabilized cells, we show that ligand sequestration, driven by exogenous clathrin, results in elevated levels of μ2 phosphorylation. Furthermore, we show that AP2 containing phospho-μ2 is mainly associated with assembled clathrin in vivo, and that the level of phospho-μ2 is strongly reduced in a chicken B cell line depleted of clathrin heavy chain. Our results imply a central role for clathrin in the regulation of cargo selection via the modulation of phospho-μ2 levels.

The Adaptor-associated Kinase 1, AAK1, Is a Positive Regulator of the Notch Pathway

“…A conformational switch between the active and the inactive forms of the kinase may explain these results, as the inactive kinase may not enable interaction with other endocytic adaptors (28) and consequent stabilization of monoubiquitinated Notch. Clathrin binding is known to stimulate AAK1 kinase activity and promote cargo recruitment into coated pits (28,29). AAK1 also exhibits multiple binding sites for accessory endocytic components such as AP2 and EH-containing proteins (21,22) and thus acts as a scaffolding hub for the recruitment of several endocytic partners.…”

“…AAK1, via phosphorylation of Thr-156 of the 2 subunit, increases the affinity of AP2 for membranes and induces a conformational change enabling cargo recruitment by this adaptor (26,27). Clathrin assembly stimulates AAK1 activity thus suggesting a role in clathrin-mediated cargo uptake (28,29). Besides its function at the internalization step, AAK1 was recently shown to act at the level of early/sorting endosome and to colocalize with EEA1 (30).…”

Flat clathrin coats on endosomes mediate degradative protein sorting by scaffolding Hrs in dynamic microdomains

“…In either of these models, the exact role of clathrin is not clear. Clathrin could either function as a scaffolding protein that restricts Hrs and, thereby, ubiquitylated 2002), serve as a mechanical barrier to inhibit membrane fusion (Sun et al, 2003), act as an allosteric regulator of Hrs or other proteins involved in the sorting process (Jackson et al, 2003), or mediate budding of vesicles from vacuolar endosomes (although such buds have not been detected by electron microscopy). It thus seems pertinent to design experiments that can address the function of clathrin on vacuolar endosomes.…”

A Novel AAK1 Splice Variant Functions at Multiple Steps of the Endocytic Pathway

“…Thus, 2 phosphorylation likely results in a conformational change that exposes the Yxx⌽ binding site and promotes AP-2 recruitment to receptors. Interestingly, AAK1 kinase activity toward 2 is dramatically stimulated by assembled clathrin (Conner et al, 2003;Jackson et al, 2003), suggesting that AAK1 is specifically activated in clathrin-coated pits to enhance cargo uptake.The Ark1/Prk1 family is characterized by a high degree of identity within the amino terminal kinase domain of member proteins (Cope et al, 1999). However, little similarity outside the kinase domain exists between family members, arguing that they may have overlapping and divergent functions.…”