AAK1‐Mediated μ2 Phosphorylation is Stimulated by Assembled Clathrin

Abstract

AAK1, the adaptor-associated kinase 1, phosphorylates the m2 subunit of AP2 and regulates the recruitment of AP2 to tyrosine-based internalization motifs found on membrane-bound receptors. AAK1 overexpression specifically inhibits the AP2-dependent internalization of transferrin receptor and LDL-receptor related protein by functionally sequestering AP2 (Conner and Schmid. J Cell Biol 2003; 162: 773). However, while AAK1 stably associates with AP2 and specifically targets the m2 subunit in vitro, m2 phosphorylation in vivo was not altered by overexpression of either wild-type or kinase-inactive AAK1. These results suggested that AAK1 might be tightly regulated in the cell. Here, we report that AAK1 is an atypical kinase that is rate limited by its stable association with AP2 and that clathrin stimulates m2 phosphorylation by AAK1. Efficient stimulation of AAK1 by clathrin involves multiple interactions between several domains on AAK1 and both heavy and light chains on clathrin. Importantly, incubation of AAK1 with clathrin cages resulted in even greater stimulation when compared to that of unassembled clathrin triskelia. Collectively, our observations indicate that clathrin function is not limited to structural and/or mechanical roles in endocytic vesicle formation: the stimulatory effects of clathrin on AAK1 activity argue that it also plays a regulatory role by modulating the activity of AP2 complexes through activation of AAK1. We suggest a model in which AAK1 is specifically activated in coated pits to enhance cargo recruitment and efficient internalization. Clathrin-dependent endocytosis is a robust and ubiquitous internalization pathway in eukaryotic cells that is mediated by the action of two oligomeric protein complexes, clathrin and the adaptor protein complex, AP2 (1). Clathrin is a heterohexameric structure composed of three heavy chains of $180 kDa and three tightly associated light chains of $30 kDa that together form three-legged structures called triskelia. While clathrin self-assembly into cages occurs under nonphysiologic conditions (2,3), the specific recruitment and assembly of clathrin into a polyhedral lattice at the plasma membrane, as well as its coupling to endocytic cargo, are coordinated by adaptor proteins, such as AP2. AP2 consists of four subunits: two large (a and b2, $105-115 kDa), one medium (m2, $50 kDa) and one small (s2, $17 kDa). Plasma membrane targeting (4,5), interaction with clathrin (6), and the recruitment of several accessory proteins important for endocytosis are supported by the large AP2 subunits (7,8). A critical step in endocytosis is the coupling of the endocytic machinery to cargo. This is coordinated through the m2 subunit of AP2 that directly interacts with the cytoplasmic tail of membrane-bound receptors marked for internalization by tyrosine-based sorting signals (9). The function of the small subunit has not been specifically elucidated, but structural studies implicate its role in stabilizing the AP2 core (10).Accumulating evidence suggests that the spatial...

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

“…The AAK1 kinase activity does not seem to be required for ⌬E monoubiquitination, but a transfected kinase dead mutant interacts less efficiently with the monoubiquitinated form of ⌬E and leads to reduced Notch activity. 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, 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).…”

YRRL motifs in the cytoplasmic domain of the thrombopoietin receptor regulate receptor internalization and degradation

“…26,27 PI(4,5)P2 interacts via 2 binding sites on AP2, one on the ␣-subunit to dock the complex at the plasma membrane, 19 and the other on the -subunit. 41 AAK-1 increases the affinity of the -subunit to bind its target cargo by phosphorylation of 156 Thr.…”

“…22,23 To ensure that AP2 specifically associates with membrane-bound proteins, phosphorylation of 156 Thr in the 2 subunit results in a conformational change in AP2, dramatically increasing the affinity of 2 for YXX⌽ motifs. 24,25 156 Thr is phosphorylated by adaptor-associated kinase 1 (AAK1), 26 the activity of which is maximized by its association with clathrin, 27,28 ensuring that AP2-cargo protein interactions are initiated only at the plasma membrane. In addition to being an endocytic signal, YXX⌽ motifs located between 6 to 9 amino acids from the transmembrane domain mediate targeting of cargo protein to the lysosome and lysosome-like organelles via interactions with AP3.…”

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.…”