TY - JOUR
T1 - Differential requirements for AP-2 in clathrin-mediated endocytosis
AU - Conner, Sean D.
AU - Schmid, Sandra L.
PY - 2003/9/1
Y1 - 2003/9/1
N2 - 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.
AB - 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.
KW - AAK1
KW - AP-2
KW - Clathrin
KW - Endocytosis
KW - Kinase
UR - http://www.scopus.com/inward/record.url?scp=0042733637&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0042733637&partnerID=8YFLogxK
U2 - 10.1083/jcb.200304069
DO - 10.1083/jcb.200304069
M3 - Article
C2 - 12952931
AN - SCOPUS:0042733637
SN - 0021-9525
VL - 162
SP - 773
EP - 779
JO - Journal of Cell Biology
JF - Journal of Cell Biology
IS - 5
ER -