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© The Rockefeller University Press, 0021-9525/2003/9/773 $5.00
The Journal of Cell Biology, Volume 162, Number 5, 773-780

Differential requirements for AP-2 in clathrin-mediated endocytosis

The Scripps Research Institute, La Jolla, CA 92037

Address correspondence to Sandra Schmid, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: (858) 784-2311. Fax: (858) 784-9126. email: slschmid{at}scripps.edu

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 µ2 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 µ2 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.

Key Words: endocytosis; AP-2; clathrin; AAK1; kinase

Abbreviations used in this paper: AAK1, adaptor-associated kinase; CCV, clathrin-coated vesicle; CME, clathrin-mediated endocytosis; siRNA, small interfering RNA; Tfn, transferrin; TfnR, Tfn receptor; tTA, tetracycline transactivator.

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