Department of Chemistry, Vanderbilt Institute for Chemical Biology (VICB), Vanderbilt University, Nashville, Tennessee 37235, USA.
J Proteome Res. 2010 Mar 5;9(3):1541-8. doi: 10.1021/pr901043e.
APPL1 is a membrane-associated adaptor protein implicated in various cellular processes, including apoptosis, proliferation, and survival. Although there is increasing interest in the biological roles as well as the protein and membrane interactions of APPL1, a comprehensive phosphorylation profile has not been generated. In this study, we use mass spectrometry (MS) to identify 13 phosphorylated residues within APPL1. By using multiple proteases (trypsin, chymotrypsin, and Glu C) and replicate experiments of linear ion trap (LTQ) MS and LTQ-Orbitrap-MS, a combined sequence coverage of 99.6% is achieved. Four of the identified sites are located in important functional domains, suggesting a potential role in regulating APPL1. One of these sites is within the BAR domain, two cluster near the edge of the PH domain, and one is located within the PTB domain. These phosphorylation sites may control APPL1 function by regulating the ability of APPL1 domains to interact with other proteins and membranes.
APPL1 是一种与膜相关的衔接蛋白，参与多种细胞过程，包括细胞凋亡、增殖和存活。尽管人们对 APPL1 的生物学作用以及蛋白质和膜相互作用越来越感兴趣，但尚未生成全面的磷酸化谱。在这项研究中，我们使用质谱 (MS) 鉴定了 APPL1 内的 13 个磷酸化残基。通过使用多种蛋白酶（胰蛋白酶、糜蛋白酶和 Glu C）以及线性离子阱 (LTQ) MS 和 LTQ-Orbitrap-MS 的重复实验，实现了 99.6%的组合序列覆盖率。鉴定出的四个位点位于重要的功能域内，这表明其在调节 APPL1 方面可能发挥作用。其中一个位点位于 BAR 结构域内，两个位点聚集在 PH 结构域的边缘附近，一个位于 PTB 结构域内。这些磷酸化位点可能通过调节 APPL1 结构域与其他蛋白质和膜相互作用的能力来控制 APPL1 的功能。
