The E3 Ubiquitin Ligase- and Protein Phosphatase 2A (PP2A)-binding Domains of the Alpha4 Protein Are Both Required for Alpha4 to Inhibit PP2A Degradation
The E3 Ubiquitin Ligase- and Protein Phosphatase 2A (PP2A)-binding Domains of the Alpha4 Protein Are Both Required for Alpha4 to Inhibit PP2A Degradation
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DOI：
10.1074/jbc.m111.222414
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发表时间：
2011-05-20
影响因子：
4.8
通讯作者：
Spiller, Benjamin W.
中科院分区：
文献类型：
--
作者：
LeNoue-Newton, Michele;Watkins, Guy R.;Spiller, Benjamin W.
蛋白磷酸酶2A(PP2A)通过多种机制进行调节，包括翻译后修饰和与调节蛋白的结合。Alpha4是一种这样的调节蛋白，它结合PP2A催化亚单位(PP2Ac)，并保护它免受多泛素化和降解的影响。Alpha4是一个多结构域蛋白，其C-末端结构域与MID1结合，一个可能的E3泛素连接酶，以及一个包含PP2Ac结合位点的N-末端结构域。在这项工作中，我们提出了哺乳动物Alpha4的N-末端结构域的结构，并用双电子-电子共振光谱证明它是一种柔性的四肽重复样蛋白。在结构上，Alpha4与其酵母同系物Tap42有两个重要的不同之处：1)含有PP2Ac结合残基的螺旋位置更开放，在这个区域显示出灵活性；2)Alpha4包含一个泛素相互作用的基序。通过串联泛素结合实体沉淀和放线菌亚胺追赶实验，研究了野生型和突变型Alpha4对哺乳动物细胞中PP2Ac泛素化和稳定性的影响。我们的结果表明，C端MID1结合结构域和PP2Ac结合决定簇都是Alpha4介导的保护PP2Ac免受多泛素化和降解所必需的。
Protein phosphatase 2A (PP2A) is regulated through a variety of mechanisms, including post-translational modifications and association with regulatory proteins. Alpha4 is one such regulatory protein that binds the PP2A catalytic subunit (PP2Ac) and protects it from polyubiquitination and degradation. Alpha4 is a multidomain protein with a C-terminal domain that binds Mid1, a putative E3 ubiquitin ligase, and an N-terminal domain containing the PP2Ac-binding site. In this work, we present the structure of the N-terminal domain of mammalian Alpha4 determined by x-ray crystallography and use double electron-electron resonance spectroscopy to show that it is a flexible tetratricopeptide repeat-like protein. Structurally, Alpha4 differs from its yeast homolog, Tap42, in two important ways: 1) the position of the helix containing the PP2Ac-binding residues is in a more open conformation, showing flexibility in this region; and 2) Alpha4 contains a ubiquitin-interacting motif. The effects of wild-type and mutant Alpha4 on PP2Ac ubiquitination and stability were examined in mammalian cells by performing tandem ubiquitin-binding entity precipitations and cycloheximide chase experiments. Our results reveal that both the C-terminal Mid1-binding domain and the PP2Ac-binding determinants are required for Alpha4-mediated protection of PP2Ac from polyubiquitination and degradation.