Predominant interaction of both Ikaros and Helios with the NuRD complex in immature thymocytes
Predominant interaction of both Ikaros and Helios with the NuRD complex in immature thymocytes
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DOI：
10.1074/jbc.m702541200
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发表时间：
2007-10-12
影响因子：
4.8
通讯作者：
Smale, Stephen T.
中科院分区：
文献类型：
--
作者：
Sridharan, Rupa;Smale, Stephen T.
Ikaros是C2 H2锌指DNA结合蛋白小家族的创始成员，该家族在淋巴细胞发育期间执行关键功能。虽然已经报道了Ikaros和各种蛋白质之间的相互作用，但含有Ikaros的复合物尚未被纯化以确定其组成并鉴定主要的相互作用伴侣。在这项研究中，串联亲和纯化-质谱分析策略被开发用于分离由Ikaros和Helios形成的复合物，Helios是Ikaros家族的T细胞限制性成员，在很大程度上尚未表征。这种策略，这似乎是非常适合在哺乳动物细胞中的一般使用，依赖于N-末端多肽含有双FLAG表位，其次是烟草蚀纹病毒蛋白酶切割位点和钙调蛋白结合肽。在小鼠胸腺细胞系的提取物中，Ikaros和Helios在中等严格条件下仅与Ikaros家族的其他成员相关。然而，在低严格条件下，两种标记的蛋白质组装成更高分子量的复合物。质谱分析表明，这两种蛋白质主要与亚基的NuRD，ATP依赖性核小体重塑复合物涉及转录抑制和激活，以前报道与Ikaros。亲和纯化的Ikaros的进一步分析表明，几个丝氨酸和苏氨酸在胸腺细胞系中被磷酸化，在胸腺细胞成熟时有明显的变化。这些结果支持了这一假设，即NuRD复合物对Ikaros和Helios的功能做出了重大贡献，并且这些蛋白质的活性可能部分由磷酸化的变化来调节。
Ikaros is the founding member of a small family of C2H2 zinc-finger DNA-binding proteins that carry out critical functions during lymphocyte development. Although interactions between Ikaros and various proteins have been reported, Ikaros-containing complexes have not been purified to determine their composition and identify the predominant interacting partners. In this study, a tandem affinity purification-mass spectrometry strategy was developed for the isolation of complexes formed by Ikaros and by Helios, a T-cell-restricted member of the Ikaros family that remains largely uncharacterized. This strategy, which appears to be well suited for general use in mammalian cells, relies on an N-terminal polypeptide containing a double FLAG epitope, followed by a tobacco etch virus protease cleavage site and calmodulin binding peptide. In extracts from a murine thymocyte line, Ikaros and Helios associated under moderate stringency conditions only with other members of the Ikaros family. However, under low stringency conditions, both tagged proteins assembled into higher molecular weight complexes. Mass spectrometry revealed that both proteins associated predominantly with subunits of NuRD, an ATP-dependent nucleosome remodeling complex implicated in transcriptional repression and activation and previously reported to associate with Ikaros. Further analysis of the affinity-purified Ikaros revealed that several serines and threonines are phosphorylated in the thymocyte line, with apparent changes upon thymocyte maturation. These results support the hypothesis that the NuRD complex makes major contributions to the functions of both Ikaros and Helios and that the activities of these proteins may be regulated in part by changes in phosphorylation.