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

Abstract

Phosphorylation is a critical step in regulating receptor transport through the endocytic pathway. AAK1 is a serine/ threonine kinase that is thought to coordinate the recruitment of AP-2 to receptors containing tyrosine-based internalization motifs by phosphorylating the 2 subunit. Here we have identified a long form of AAK1 (AAK1L) that contains an extended C-terminus that encodes an additional clathrin-binding domain (CBD2) consisting of multiple low-affinity interaction motifs. Protein interaction studies demonstrate that AAK1L CBD2 directly binds clathrin. However, in vitro kinase assays reveal little difference between AAK1 isoforms in their basal or clathrin-stimulated kinase activity toward the AP-2 2 subunit. However, overexpression of AAK1L CBD2 impairs transferrin endocytosis, confirming an endocytic role for AAK1. Surprisingly, CBD2 overexpression or AAK1 depletion by RNA interference significantly impairs transferrin recycling from the early/sorting endosome. These observations suggest that AAK1 functions at multiple steps of the endosomal pathway by regulating transferrin internalization and its rapid recycling back to the plasma membrane from early/sorting endosome. INTRODUCTIONThe adaptor-associated kinase 1 (AAK1) is a member of the Ark1/Prk1 family of serine/threonine kinases that are thought to regulate endocytosis by phosphorylating various endocytic accessory components (Smythe and Ayscough, 2003). AAK1 was originally identified as an interacting partner of the ␣-adaptin subunit of AP-2 and phosphorylates the 2 subunit . The 2 subunit directly binds tyrosine-based (Yxx⌽) internalization motifs found on the cytoplasmic tail of some receptors and mediates cargo recruitment to clathrin-coated vesicles (Ohno et al., 1995). Phosphorylation is thought to be a key regulatory step in cargo recognition as AAK1-mediated 2 phosphorylation increases the affinity of AP-2 for Yxx⌽ internalization motif-containing receptors (Ricotta et al., 2002). Although recent observations demonstrate that 2 phosphorylation is not obligatory for receptor uptake, it is critical for maximizing internalization efficiency (Motley et al., 2006;Olusanya et al., 2001). The resolved crystal structure of the AP-2 "core" has provided an attractive explanation for the molecular basis of the increased receptor affinity of AP-2 after phosphorylation-the Yxx⌽ binding site within 2 is buried within the AP-2 core when not phosphorylated (Collins et al., 2002). 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 b...

αvβ3 integrin-mediated adhesion is regulated through an AAK1L- and EHD3-dependent rapid recycling pathway

“…7A). Our previous findings indicate that the C-terminal clathrin binding domain (CBD2) is critical for AAK1L function (Henderson and Conner, 2007). Consistent with this, cell adhesion defects were not rescued following adenovirus-mediated expression of the short AAK1 isoform (AAK1s), which lacks CBD2.…”

“…2) and associate with EEA1-positive EE/SEs (Henderson and Conner, 2007;Naslavsky et al, 2006). However, near the cell surface, EHD3-positive endosomes did not appear to align with actin stress fibers like that of AAK1L (Fig.…”

“…We initiated our studies by targeting two early endosomal sorting factors: 1) AAK1L (Henderson and Conner, 2007) and 2) EHD3 (Naslavsky et al, 2006;Naslavsky et al, 2009). Depletion of either AAK1L or EHD3 in tTA HeLa cells resulted in an ,50% reduction in cell adhesion to tissue culture dishes after a 72 h knockdown (Fig.…”

“…1A). In addition to regulating rapid recycling of the transferrin receptor (Henderson and Conner, 2007), AAK1L has also been implicated in regulating receptor internalization (Conner and Schmid, 2002). This raised the possibility that the observed cell adhesion defects might result from impaired integrin endocytosis.…”

“…To extend our understanding of how transport through the early endosome influences integrin function, we investigated the role of two EE/SE-associated factors: (1) AAK1L (adaptor associated kinase 1 long form; Henderson and Conner, 2007), given the growing importance of kinases in integrin recycling (Roberts et al, 2004;Woods et al, 2004) and (2) EHD3, which belongs to the Eps15 homology domain (EHD) family of proteins, which serve key roles in endosomal sorting decisions (Naslavsky and Caplan, 2011;Naslavsky et al, 2006;Naslavsky et al, 2009). We employed a live cell imaging strategy to visualize the impact of AAK1L or EHD3 depletion on integrin transport in adherent cells and provide evidence demonstrating that avb3-mediated cell adhesion is maintained by integrin recycling through an AAK1L-and EHD3-dependent recycling pathway.…”