The WW domain of neural protein FE65 interacts with proline-rich motifs in Mena, the mammalian homolog of Drosophila enabled
The WW domain of neural protein FE65 interacts with proline-rich motifs in Mena, the mammalian homolog of Drosophila enabled
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DOI：
10.1074/jbc.272.52.32869
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发表时间：
1997-12-26
影响因子：
4.8
通讯作者：
Sudol, M
中科院分区：
文献类型：
--
作者：
Ermekova, KS;Zambrano, N;Sudol, M
神经蛋白FE 65包含两种蛋白质-蛋白质相互作用模块：一个WW结合结构域和两个磷酸酪氨酸结合结构域。FE 65的羧基末端磷酸酪氨酸结合结构域在体内与β-淀粉样前体蛋白相互作用，这与阿尔茨海默病有关。为了理解这种衔接蛋白的功能，我们鉴定了FE 65 WW结构域的结合伴侣。通过筛选FE 65 WW结构域配体的表达文库，回收共享脯氨酸-脯氨酸-亮氨酸-脯氨酸核心基序的富含脯氨酸的序列。分子量为60，75，80，140，和200 kDa的五种蛋白质可以从小鼠脑裂解液中纯化的FE 65 WW结构域的亲和力。我们确定了这五种蛋白质中的两种为Mena编码的80-和180-kDa同种型，Mena是果蝇使能基因的哺乳动物同源物。使用肽合成的SPOTs技术，我们鉴定了Mena中与FE 65 WW结构域相互作用的序列，并发现它们含有标志性的脯氨酸-脯氨酸-亮氨酸-脯氨酸基序。最后，我们证明了Mena结合FE 65在体内通过免疫共沉淀试验从COS细胞提取物。Mena-FE 65 WW结构域结合的特异性通过竞争测定来证实。FE 65-Mena复合物的进一步表征可以确定这些蛋白质在β-淀粉样前体蛋白生物发生中的生理作用，并可能有助于理解阿尔茨海默病的分子变化机制。
The neural protein FE65 contains two types of protein-protein interaction modules: one WW binding domain and two phosphotyrosine binding domains. The carboxyl-terminal phosphotyrosine binding domain of FE65 interacts in vivo with the beta-amyloid precursor protein, which is implicated in Alzheimer disease. To understand the function of this adapter protein, we identified binding partners for the FE65 WW domain. Proline-rich sequences sharing a proline-proline-leucine-proline core motif were recovered by screening expression libraries for ligands of the FE65 WW domain. Five proteins of molecular masses 60, 75, 80, 140, and 200 kDa could be purified from mouse brain lysates by affinity to the FE65 WW domain. We identified two of these five proteins as the 80- and 180-kDa isoforms encoded by Mena, the mammalian homolog of the Drosophila Enabled gene. Using the SPOTs technique of peptide synthesis, we identified the sequences in Mena that interact with the FE65 WW domain and found that they contain the signature proline-proline-leucine-proline motif. Finally, we demonstrated that Mena binds to FE65 in vivo by coimmunoprecipitation assay from COS cell extracts. The specificity of the Mena-FE65 WW domain association was confirmed by competition assays. Further characterization of the FE65-Mena complex may identify a physiological role for these proteins in beta-amyloid precursor protein biogenesis and may help in understanding the mechanism of molecular changes that underlie Alzheimer disease.