Conner Sean D, Schmid Sandra L
The Scripps Research Institute, La Jolla, CA 92037, USA.
J Cell Biol. 2003 Sep 1;162(5):773-9. doi: 10.1083/jcb.200304069.
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.
衔接蛋白2复合物是网格蛋白介导的内吞作用（CME）的关键组成部分。它们触发网格蛋白组装，直接与货物分子相互作用，并招募多种内吞辅助因子。衔接蛋白相关激酶（AAK1）是一种与衔接蛋白2结合的蛋白，通过磷酸化其μ2亚基来调节衔接蛋白2的功能。在此，我们检测了腺病毒介导的野生型AAK1、激酶失活型和截短突变体在HeLa细胞中过表达的影响，并表明AAK1在体内也调节衔接蛋白2的功能。野生型AAK1过表达选择性地阻断转铁蛋白（Tfn）受体和低密度脂蛋白受体相关蛋白（LRP）的内吞作用。抑制作用不依赖激酶，但需要全长AAK1，因为截短突变体没有抑制作用。尽管未检测到μ2磷酸化的变化，但AAK1过表达显著降低了大衔接蛋白亚基的磷酸化，并且正常的点状衔接蛋白2分布变得分散，这表明AAK1过表达通过功能性隔离衔接蛋白2来抑制Tfn内吞作用。令人惊讶的是，网格蛋白分布和表皮生长因子（EGF）摄取不受AAK1过表达的影响。因此，在包被组装过程中，衔接蛋白2可能不是化学计量学上必需的，并且在CME中可能具有比以前认为的更具货物选择性的功能。
