Department of Biochemistry, University of Washington, Seattle, Washington, 98195.
Department of Biological Structure, University of Washington, Seattle, Washington, 98195.
Protein Sci. 2018 Jun;27(6):1057-1067. doi: 10.1002/pro.3413. Epub 2018 Apr 25.
Poly(ADP-ribosyl)ation (PARylation) catalyzed by the tankyrase enzymes (Tankyrase-1 and -2; a.k.a. PARP-5a and -5b) is involved in mitosis, telomere length regulation, GLUT-4 vesicle transport, and cell growth and differentiation. Together with the E3 ubiquitin ligase RNF146 (a.k.a. Iduna), tankyrases regulate the cellular levels of several important proteins including Axin, 3BP2, and angiomotins, which are key regulators of Wnt, Src and Hippo signaling, respectively. These tankyrase substrates are first PARylated and then ubiquitylated by RNF146, which is allosterically activated by binding to PAR polymer. Each tankyrase substrate is recognized by a tankyrase-binding motif (TBM). Here we show that RNF146 binds directly to tankyrases via motifs in its C-terminal region. Four of these RNF146 motifs represent novel, extended TBMs, that have one or two additional amino acids between the most conserved Arg and Gly residues. The individual RNF146 motifs display weak binding, but together mediate a strong multivalent interaction with the substrate-binding region of TNKS, forming a robust one-to-one complex. A crystal structure of the first RNF146 noncanonical TBM in complex with the second ankyrin repeat domain of TNKS shows how an extended motif can be accommodated in a peptide-binding groove on tankyrases. Overall, our work demonstrates the existence of a new class of extended TBMs that exist in previously uncharacterized tankyrase-binding proteins including those of IF4A1 and NELFE.
多聚（ADP-核糖）化（PAR 化）由 Tankyrase 酶（Tankyrase-1 和 -2；也称为 PARP-5a 和 -5b）催化，参与有丝分裂、端粒长度调节、GLUT-4 囊泡运输以及细胞生长和分化。Tankyrase 与 E3 泛素连接酶 RNF146（也称为 Iduna）一起，调节包括 Axin、3BP2 和 angiomotin 在内的几种重要蛋白质的细胞水平，它们分别是 Wnt、Src 和 Hippo 信号的关键调节剂。这些 Tankyrase 底物首先被 PAR 化，然后被 RNF146 泛素化，后者通过与 PAR 聚合物结合而变构激活。每个 Tankyrase 底物都被 Tankyrase 结合基序（TBM）识别。在这里，我们表明 RNF146 通过其 C 末端区域的基序直接与 Tankyrase 结合。RNF146 的这四个基序代表新的、扩展的 TBM，在最保守的 Arg 和 Gly 残基之间有一个或两个额外的氨基酸。单个 RNF146 基序显示出较弱的结合，但共同介导与 TNKS 底物结合区域的强多价相互作用，形成稳定的一对一复合物。第一个 RNF146 非典型 TBM 与 TNKS 的第二个锚蛋白重复结构域的复合物的晶体结构显示了扩展基序如何适应 Tankyrase 上的肽结合槽。总的来说，我们的工作证明了存在一类新的扩展 TBM，它们存在于以前未表征的 Tankyrase 结合蛋白中，包括 IF4A1 和 NELFE。
