Conner Sean D, Schmid Sandra L
The Scripps Research Institute, La Jolla, CA 92037, USA.
J Cell Biol. 2002 Mar 4;156(5):921-9. doi: 10.1083/jcb.200108123.
The mu 2 subunit of the AP2 complex is known to be phosphorylated in vitro by a copurifying kinase, and it has been demonstrated recently that mu 2 phosphorylation is required for transferrin endocytosis (Olusanya, O., P.D. Andrews, J.R. Swedlow, and E. Smythe. 2001. Curr. Biol. 11:896-900). However, the identity of the endogenous kinase responsible for this phosphorylation is unknown. Here we identify and characterize a novel member of the Prk/Ark family of serine/threonine kinases, adaptor-associated kinase (AAK)1. We find that AAK1 copurifies with adaptor protein (AP)2 and that it directly binds the ear domain of alpha-adaptin in vivo and in vitro. In neuronal cells, AAK1 is enriched at presynaptic terminals, whereas in nonneuronal cells it colocalizes with clathrin and AP2 in clathrin-coated pits and at the leading edge of migrating cells. AAK1 specifically phosphorylates the mu subunit in vitro, and stage-specific assays for endocytosis show that mu phosphorylation by AAK1 results in a decrease in AP2-stimulated transferrin internalization. Together, these results provide strong evidence that AAK1 is the endogenous mu 2 kinase and plays a regulatory role in clathrin-mediated endocytosis. These results also lend support to the idea that clathrin-mediated endocytosis is controlled by cycles of phosphorylation/desphosphorylation.
已知AP2复合物的μ2亚基在体外可被一种共纯化的激酶磷酸化，并且最近已证明μ2磷酸化是转铁蛋白内吞作用所必需的（Olusanya, O., P.D. Andrews, J.R. Swedlow, and E. Smythe. 2001. Curr. Biol. 11:896 - 900）。然而，负责这种磷酸化的内源性激酶的身份尚不清楚。在这里，我们鉴定并表征了丝氨酸/苏氨酸激酶Prk/Ark家族的一个新成员，衔接蛋白相关激酶（AAK）1。我们发现AAK1与衔接蛋白（AP）2共纯化，并且它在体内和体外都直接结合α - 衔接蛋白的耳结构域。在神经元细胞中，AAK1在突触前末端富集，而在非神经元细胞中，它与网格蛋白和AP2在网格蛋白包被小窝以及迁移细胞的前缘共定位。AAK1在体外特异性地磷酸化μ亚基，并且内吞作用的阶段特异性分析表明，AAK1介导的μ磷酸化导致AP2刺激的转铁蛋白内化减少。总之，这些结果提供了强有力的证据，表明AAK1是内源性μ2激酶，并且在网格蛋白介导的内吞作用中起调节作用。这些结果也支持了网格蛋白介导的内吞作用受磷酸化/去磷酸化循环控制的观点。
