Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232-6600, USA.
Biochemistry. 2010 Mar 2;49(8):1713-8. doi: 10.1021/bi901837h.
Multiple regulatory mechanisms control the activity of the protein serine/threonine phosphatase 2A catalytic subunit (PP2Ac), including post-translational modifications and its association with regulatory subunits and interacting proteins. Alpha4 is a PP2Ac-interacting protein that is hypothesized to play a role in PP2Ac ubiquitination via its interaction with the E3 ubiquitin ligase Mid1. In this report, we show that alpha4 serves as a necessary adaptor protein that provides a binding platform for both PP2Ac and Mid1. We also identify a novel ubiquitin-interacting motif (UIM) within alpha4 (amino acid residues 46-60) and analyze the interaction between alpha4 and ubiquitin using NMR. Consistent with other UIM-containing proteins, alpha4 is monoubiquitinated. Interestingly, deletion of the UIM within alpha4 enhances its association with polyubiquitinated proteins. Lastly, we demonstrate that addition of wild-type alpha4 but not an alpha4 UIM deletion mutant suppresses PP2Ac polyubiquitination. Thus, the polyubiquitination of PP2Ac is inhibited by the UIM within alpha4. These findings reveal direct regulation of PP2Ac polyubiquitination by a novel UIM within the adaptor protein alpha4.
多种调控机制控制着蛋白丝氨酸/苏氨酸磷酸酶 2A 催化亚基（PP2Ac）的活性，包括翻译后修饰及其与调节亚基和相互作用蛋白的结合。α4 是一种与 PP2Ac 相互作用的蛋白，据推测，它通过与 E3 泛素连接酶 Mid1 的相互作用，在 PP2Ac 的泛素化中发挥作用。在本报告中，我们表明 α4 作为一种必需的衔接蛋白，为 PP2Ac 和 Mid1 提供了结合平台。我们还在 α4 内鉴定了一个新的泛素相互作用基序（UIM）（氨基酸残基 46-60），并使用 NMR 分析了 α4 和泛素之间的相互作用。与其他含有 UIM 的蛋白质一致，α4 被单泛素化。有趣的是，α4 内 UIM 的缺失增强了其与多泛素化蛋白的结合。最后，我们证明添加野生型 α4 但不是 α4 UIM 缺失突变体可抑制 PP2Ac 的多泛素化。因此，α4 内的 UIM 抑制了 PP2Ac 的多泛素化。这些发现揭示了衔接蛋白 α4 内的新型 UIM 对 PP2Ac 多泛素化的直接调控。
