Division of Biological Sciences, University of California, San Diego, La Jolla, California, USA.
Mol Cell Biol. 2012 May;32(9):1671-82. doi: 10.1128/MCB.06452-11. Epub 2012 Feb 21.
Numerous in vitro as well as genetic studies have demonstrated that the activities of the E2A proteins are regulated at multiple levels, including modulation of DNA binding by the Id proteins, association with the transcriptional modulators p300 and ETO, and posttranslational modifications. Here, we use affinity purification of tagged E47 combined with mass spectrometry in order to show that E47 interacts with the entire ensemble of Id proteins, namely, Id1, Id2, Id3, and Id4. Furthermore, we find that the lysine-specific histone demethylase 1 (LSD1), the protein arginine N-methyltransferase 5 (PRMT5), the corepressor CoREST, and the chaperones of the 14-3-3 family associate with affinity-purified E47. We also identify a spectrum of amino acid residues in E47 that are phosphorylated, including an AKT substrate site. We did, however, find that mutation of the identified AKT substrate site by itself did not perturb B cell development. In sum, these studies show that the entire ensemble of Id proteins has the ability to interact with E47, identify factors that associate with E47, and reveal a spectrum of phosphorylated residues in E47, including an AKT substrate site.
大量的体外和遗传研究表明，E2A 蛋白的活性在多个层次上受到调节，包括 Id 蛋白对 DNA 结合的调节、与转录调节剂 p300 和 ETO 的结合以及翻译后修饰。在这里，我们使用标记的 E47 的亲和纯化结合质谱法，以证明 E47 与整个 Id 蛋白复合物相互作用，即 Id1、Id2、Id3 和 Id4。此外，我们发现赖氨酸特异性组蛋白去甲基酶 1（LSD1）、精氨酸 N-甲基转移酶 5（PRMT5）、核心抑制因子 CoREST 和 14-3-3 家族的伴侣与亲和纯化的 E47 结合。我们还鉴定了 E47 中被磷酸化的氨基酸残基谱，包括 AKT 底物位点。然而，我们发现单独突变鉴定出的 AKT 底物位点本身并不会干扰 B 细胞发育。总之，这些研究表明，整个 Id 蛋白复合物都有能力与 E47 相互作用，鉴定与 E47 相关的因子，并揭示 E47 中存在一系列磷酸化残基，包括 AKT 底物位点。
