Differential requirements for AP-2 in clathrin-mediated endocytosis.
Differential requirements for AP-2 in clathrin-mediated endocytosis.
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DOI：
10.1083/jcb.200304069
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发表时间：
2003-09-01
影响因子：
7.8
通讯作者：
Schmid, Sandra L
中科院分区：
文献类型：
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作者：
Conner, Sean D;Schmid, Sandra L
AP-2复合体是网状蛋白介导的内吞作用(CME)的关键成分。它们触发网状蛋白组装，直接与货物分子相互作用，并招募许多内吞辅助因子。适配器相关蛋白1是AP-2的结合伙伴，通过磷酸化其μ2亚基来调节AP-2的功能。在这里，我们检测了腺病毒介导的WT AAK1、KK-DEAD和截断突变体在HeLa细胞中过表达的影响，并表明AAK1在体内也调节AP-2的功能。WT AAK1过表达选择性阻断转铁蛋白(TFN)受体和LRP内吞作用。抑制是不依赖于激酶的，但需要全长AAK1作为截断突变体而不是抑制。虽然没有检测到μ2的磷酸化变化，但AAK1过表达显著降低了大的适配素亚基的磷酸化，正常点状的AP-2分布分散，提示AAK1过表达通过功能性地隔离AP-2而抑制了TFN胞吞作用。令人惊讶的是，AAK1的过度表达并没有影响笼蛋白的分布和EGF的摄取。因此，AP-2可能不是涂层组装所需的化学计量比，并且在CME中可能具有比先前认为的更多的货物选择功能。
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.