Identification of Zinc-finger BED Domain-containing 3 (Zbed3) as a Novel Axin-interacting Protein That Activates Wnt/β-Catenin Signaling
Identification of Zinc-finger BED Domain-containing 3 (Zbed3) as a Novel Axin-interacting Protein That Activates Wnt/β-Catenin Signaling
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DOI：
10.1074/jbc.m807753200
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发表时间：
2009-03-13
影响因子：
4.8
通讯作者：
Li, Lin
中科院分区：
文献类型：
--
作者：
Chen, Ting;Li, Meng;Li, Lin
Axin是Wnt/β-连环蛋白途径的关键调节剂，在磷酸化和降解细胞质β-连环蛋白中充当支架蛋白。典型的Wnt蛋白似乎通过诱导LRP 5/6与Axin的相互作用来稳定β-连环蛋白。这种相互作用需要LRP 5/6细胞内结构域PPPP（S/T）PX（T/S）基序中Ser或Thr残基的磷酸化。在这项工作中，我们确定了一个新的Axin相互作用蛋白，锌指BED结构域包含3（Zbed 3），通过酵母双杂交筛选。在哺乳动物细胞中的免疫共沉淀实验和体外下拉测定中证实了这种相互作用。此外，我们发现Zbed 3还含有PPPPSPT基序，这对它与Axin的结合至关重要。基序中的Ser和Thr残基似乎也被糖原合成酶激酶3 β（GSK 3 β）和CKI家族激酶磷酸化，因为GSK 3 β和CKI β可以增强Zbed 3与Axin的相互作用。将该基序中的Ser（SA）或Thr（TA）残基突变为Ala显著削弱了其与Axin相互作用的能力。表达Zbed 3，而不是这些突变体，导致抑制GSK 3 β介导的β-连环蛋白磷酸化，细胞质β-连环蛋白积累，并激活淋巴增强子结合因子-1依赖性报告基因转录。此外，用RNA干扰敲低Zbed 3减弱了Wnt诱导的β-连环蛋白积累、淋巴增强子结合因子-1依赖性荧光素酶报告基因活性和Wnt靶基因表达。这些结果共同表明，Zbed 3是一种新的Axin结合蛋白，参与Wnt/β-catenin信号转导调节。
Axin, a key modulator of the Wnt/beta-catenin pathway, acts as a scaffold protein in phosphorylating and degrading cytoplasmic beta-catenin. Canonical Wnt proteins appear to stabilize beta-catenin by inducing the interaction of LRP5/6 with Axin. This interaction requires the phosphorylation of the Ser or Thr residues in the PPPP(S/T)PX(T/S) motifs at the intracellular domain of LRP5/6. In this work, we identified a novel Axin-interacting protein, zinc-finger BED domain-containing 3 (Zbed3), by yeast two-hybrid screening. The interaction was confirmed in co-immunoprecipitation experiment in mammalian cells and in vitro pulldown assays. Moreover, we found Zbed3 also contains a PPPPSPT motif, which is crucial to its binding to Axin. The Ser and Thr residues in the motif appear to be also phosphorylated by glycogen synthase kinase 3 beta (GSK3 beta) and the CKI family kinases, as GSK3 beta and CKI epsilon could enhance the interaction of Zbed3 with Axin. Mutation of the Ser (SA) or Thr (TA) residue to Ala in the motif markedly impaired its ability to interact with Axin. Expressing Zbed3, but not these mutants, led to inhibition of GSK3 beta-mediated beta-catenin phosphorylation, cytoplasmic beta-catenin accumulation, and activation of lymphoid enhancer binding factor-1-dependent reporter gene transcription. Furthermore, knockdown of Zbed3 with RNA interference attenuated Wnt-induced beta-catenin accumulation, lymphoid enhancer binding factor-1-dependent luciferase reporter activity, and the Wnt target gene expression. These results together indicate that Zbed3 is a novel Axin-binding protein that is involved in Wnt/beta-catenin signaling modulation.