Differential requirements for AP-2 in clathrin-mediated endocytosis

The Scripps Research Institute, La Jolla, CA 92037, USA.
The Journal of Cell Biology (Impact Factor: 9.83). 10/2003; 162(5):773-9. DOI: 10.1083/jcb.200304069
Source: PubMed


AP-2 complexes are key components in clathrin-mediated endocytosis (CME). They trigger clathrin assembly, interact directly with cargo molecules, and recruit a number of endocytic accessory factors. Adaptor-associated kinase (AAK1), an AP-2 binding partner, modulates AP-2 function by phosphorylating its mu2 subunit. Here, we examined the effects of adenoviral-mediated overexpression of WT AAK1, kinase-dead, and truncation mutants in HeLa cells, and show that AAK1 also regulates AP-2 function in vivo. WT AAK1 overexpression selectively blocks transferrin (Tfn) receptor and LRP endocytosis. Inhibition was kinase independent, but required the full-length AAK1 as truncation mutants were not inhibitory. Although changes in mu2 phosphorylation were not detected, AAK1 overexpression significantly decreased the phosphorylation of large adaptin subunits and the normally punctate AP-2 distribution was dispersed, suggesting that AAK1 overexpression inhibited Tfn endocytosis by functionally sequestering AP-2. Surprisingly, clathrin distribution and EGF uptake were unaffected by AAK1 overexpression. Thus, AP-2 may not be stoichiometrically required for coat assembly, and may have a more cargo-selective function in CME than previously thought.

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    • "To test if AAK1 kinase activity depends on S635 in the C-terminal AP-2 binding domain, we examined AAK1 autophosphorylation and found it was not affected by S635A mutation (Figure S5D). To test AAK1's functional role we expressed AAK1 kinase dead (AAK1-KD) K74A (Conner and Schmid, 2003), the AAK1 non-phosphorylatable mutant S635A (AAK1-SA) or the AAK1 phospho-mimetic mutant S635D (AAK1-SD) together with GFP in dissociated hippocampal neurons. A small subset of neurons with very high expression of mutant AAK1 looked unhealthy and was not included in analysis. "
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    • "Transmembrane cargos destined for internalization are recruited into clathrin-coated pits through interaction with appropriate clathrin adaptors. One such accessory factor is adaptor protein 2 (AP2), a heterotetrameric complex consisting of a, b, m, and s subunits (Conner and Schmid, 2003). AP2-dependent cargo recruitment can be regulated by reversible protein phosphorylation by actin-related kinase (Ark) family serine/threonine kinases (Smythe and Ayscough, 2003). "
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