Yoon Hye-Young, Jacques Kerry, Nealon Beth, Stauffer Stacey, Premont Richard T, Randazzo Paul A
Laboratory of Cellular Oncology, Center for Cancer Research, National Cancer Institute, Building 37, Room 4118, National Institutes of Health, Bethesda, MD 20892, USA.
Cell Signal. 2004 Sep;16(9):1033-44. doi: 10.1016/j.cellsig.2004.02.008.
The Arf GAPs are a structurally diverse group of proteins that catalyze the hydrolysis of GTP bound to Arf1. Here, we directly compare the role of amino acids 2-17 of Arf1, a GTP- and phospholipid-sensitive switch, for interaction with three Arf GAPs: Arf GAP1, AGAP1 and ASAP1. Sequestration of amino acids 2-17 with an antibody inhibited interaction with the three tested Arf GAPs. Examination of Arf1 mutants also indicated that [2-17]Arf1 is a critical structural determinant of interaction with all three Arf GAPs; however, the effect of specific mutations differed among the GAPs. Compared to wild-type Arf1, Arf1 with the amino terminal 13 ([Delta13]Arf1) and 17 amino acids ([Delta17]Arf1) deleted had 200- and 4000-fold reduced interaction with ASAP1 and 150-fold reduced interaction with AGAP1. In contrast, deletion of the amino terminus of Arf reduced interaction with Arf GAP1 by 5-fold. By analysis of point mutants, we found that lysines 15 and 16 had a greater contribution to productive interaction between Arf1, ASAP1 and AGAP1 than between Arf1 and Arf GAP1. Leucine 8 contributed to the interaction with Arf GAP1 but not with ASAP1 and AGAP1. Amino acids 2-17 of Arf1, isolated from the protein, inhibited GAP activity of Arf GAP1, ASAP1 and AGAP1 and bound directly to ASAP1. Taken together, our results indicate that (i) Arf GAPs interact with amino acids 2-17 of Arf1 and (ii) each subgroup of Arf GAPs has a unique interface with Arf1.
Arf GAP蛋白是一类结构多样的蛋白质，可催化与Arf1结合的GTP水解。在此，我们直接比较了Arf1的2 - 17位氨基酸（一个对GTP和磷脂敏感的开关）与三种Arf GAP蛋白（Arf GAP1、AGAP1和ASAP1）相互作用的作用。用抗体隔离2 - 17位氨基酸会抑制与三种测试的Arf GAP蛋白的相互作用。对Arf1突变体的检测还表明，[2 - 17]Arf1是与所有三种Arf GAP蛋白相互作用的关键结构决定因素；然而，特定突变的影响在不同的GAP蛋白之间有所不同。与野生型Arf1相比，缺失氨基末端13个氨基酸（[Δ13]Arf1）和17个氨基酸（[Δ17]Arf1）的Arf1与ASAP1的相互作用降低了200倍和4000倍，与AGAP1的相互作用降低了150倍。相比之下，Arf氨基末端的缺失使与Arf GAP1的相互作用降低了5倍。通过对点突变体的分析，我们发现赖氨酸15和16对Arf1与ASAP1和AGAP1之间的有效相互作用的贡献大于Arf1与Arf GAP1之间的贡献。亮氨酸8有助于与Arf GAP1的相互作用，但与ASAP1和AGAP1无关。从蛋白质中分离出的Arf1的2 - 17位氨基酸可抑制Arf GAP1、ASAP1和AGAP1的GAP活性，并直接与ASAP1结合。综上所述，我们的结果表明：（i）Arf GAP蛋白与Arf1的2 - 17位氨基酸相互作用；（ii）Arf GAP蛋白的每个亚组与Arf1都有独特的界面。
