Hao W, Luo Z, Zheng L, Prasad K, Lafer E M
Department of Molecular Medicine, Institute of Biotechnology, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78245, USA.
J Biol Chem. 1999 Aug 6;274(32):22785-94. doi: 10.1074/jbc.274.32.22785.
Clathrin-coated vesicles are involved in protein and lipid trafficking between intracellular compartments in eukaryotic cells. AP-2 and AP180 are the resident coat proteins of clathrin-coated vesicles in nerve terminals, and interactions between these proteins could be important in vesicle dynamics. AP180 and AP-2 each assemble clathrin efficiently under acidic conditions, but neither protein will assemble clathrin efficiently at physiological pH. We find that there is a direct, clathrin-independent interaction between AP180 and AP-2 and that the AP180-AP-2 complex is more efficient at assembling clathrin under physiological conditions than is either protein alone. AP180 is phosphorylated in vivo, and in crude vesicle extracts its phosphorylation is enhanced by stimulation of casein kinase II, which is known to be present in coated vesicles. We find that recombinant AP180 is a substrate for casein kinase II in vitro and that its phosphorylation weakens both the binding of AP-2 by AP180 and the cooperative clathrin assembly activity of these proteins. We have localized the binding site for AP-2 to amino acids 623-680 of AP180. The AP180/AP-2 interaction can be disrupted by a recombinant AP180 fragment containing the AP-2 binding site, and this fragment also disrupts the cooperative clathrin assembly activity of the AP180-AP-2 complex. These results indicate that AP180 and AP-2 interact directly to form a complex that assembles clathrin more efficiently than either protein alone. Phosphorylation of AP180, by modulating the affinity of AP180 for AP-2, may contribute to the regulation of clathrin assembly in vivo.
网格蛋白包被小泡参与真核细胞内细胞区室之间的蛋白质和脂质运输。AP - 2和AP180是神经末梢中网格蛋白包被小泡的驻留包被蛋白，这些蛋白之间的相互作用可能在小泡动力学中起重要作用。AP180和AP - 2在酸性条件下均能有效地组装网格蛋白，但在生理pH值下，这两种蛋白都不能有效地组装网格蛋白。我们发现AP180和AP - 2之间存在直接的、不依赖网格蛋白的相互作用，并且AP180 - AP - 2复合物在生理条件下比单独的任何一种蛋白更有效地组装网格蛋白。AP180在体内被磷酸化，在粗制小泡提取物中，酪蛋白激酶II的刺激会增强其磷酸化，已知酪蛋白激酶II存在于包被小泡中。我们发现重组AP180在体外是酪蛋白激酶II的底物，其磷酸化会削弱AP180与AP - 2的结合以及这些蛋白的协同网格蛋白组装活性。我们已将AP - 2的结合位点定位到AP180的623 - 680氨基酸处。含有AP - 2结合位点的重组AP180片段可破坏AP180/AP - 2相互作用，并且该片段也会破坏AP180 - AP - 2复合物的协同网格蛋白组装活性。这些结果表明，AP180和AP - 2直接相互作用形成一种复合物，该复合物比单独的任何一种蛋白更有效地组装网格蛋白。AP180的磷酸化通过调节AP180对AP - 2的亲和力，可能有助于体内网格蛋白组装的调控。
