Sorensen Erika B, Conner Sean D
Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455, USA.
Traffic. 2008 Sep;9(10):1791-800. doi: 10.1111/j.1600-0854.2008.00790.x. Epub 2008 Jul 24.
Numb is an endocytic protein that is proposed to influence clathrin-coated pit assembly, although its mode of action and the mechanisms that regulate its activity are unknown. In this study, we show that Numb binds to and is phosphorylated by adaptor-associated kinase 1 (AAK1), a key endocytic kinase. We find that AAK1 redistributes Numb to perinuclear endosomes when overexpressed, while kinase depletion causes Numb to accumulate at the plasma membrane. Overexpression of a Numb point mutant (T102A) that lacks the AAK1 phosphorylation site potently disrupts transferrin and low-density lipoprotein internalization but does not impact EGF uptake. Consistent with Numb redistribution results, we find that T102A Numb no longer localizes to perinuclear endosomes. Instead, it is enriched at the plasma membrane where it shows elevated levels of colocalization with coated pit markers. Collectively, these observations demonstrate that Numb endocytic activity is regulated by AAK1 and that phosphorylation may be a critical step in promoting coated pit maturation.
Numb是一种内吞蛋白，尽管其作用方式和调节其活性的机制尚不清楚，但有人提出它会影响网格蛋白包被小窝的组装。在本研究中，我们表明Numb与衔接蛋白相关激酶1（AAK1，一种关键的内吞激酶）结合并被其磷酸化。我们发现，过表达时AAK1会将Numb重新分布到核周内体，而激酶缺失会导致Numb在质膜积累。缺乏AAK1磷酸化位点的Numb点突变体（T102A）的过表达会强烈破坏转铁蛋白和低密度脂蛋白的内化，但不影响表皮生长因子的摄取。与Numb重新分布结果一致，我们发现T102A Numb不再定位于核周内体。相反，它在质膜上富集，在那里它与包被小窝标记物的共定位水平升高。总的来说，这些观察结果表明Numb的内吞活性受AAK1调节，磷酸化可能是促进包被小窝成熟的关键步骤。
