MBD3 and HDAC1, two components of the NuRD complex, are localized at aurora-A-positive centrosomes in M phase
MBD3 and HDAC1, two components of the NuRD complex, are localized at aurora-A-positive centrosomes in M phase
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DOI：
10.1074/jbc.m208461200
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发表时间：
2002-12-13
影响因子：
4.8
通讯作者：
Ishikawa, F
中科院分区：
文献类型：
--
作者：
Sakai, H;Urano, T;Ishikawa, F
MBD3 是组蛋白脱乙酰酶 NuRD 复合物的一个组成部分，包含甲基 CpG 结合结构域 (MBD)，但不具有明显的 mCpG 特异性结合活性。 NuRD 复合物中 MBD3 的功能意义仍然是个谜，部分原因是分析 MBD3 的生化方法（例如免疫沉淀）的可用性有限。在本研究中，我们在 HeLa 细胞中稳定表达 FIAG 标记版本的 MBD3。我们发现MBD3-FLAG被整合到NuRD复合物中，并且含有MBD3-FLAG的NuRD复合物能够被抗FLAG抗体有效地免疫沉淀。通过利用该系统，我们发现 MBD3 在 G 晚期和 M 早期在体内被磷酸化。此外，我们发现 Aurora-A 是一种在 G(2) 晚期和 M 早期特异活跃的丝氨酸/苏氨酸激酶，在体外磷酸化 MBD3，在体内与 MBD3 物理结合，并在 M 早期与 MBD3 共定位于中心体。有趣的是，HDAC1 以类似于 MBD3 的方式分布在中心体。这些结果表明，M 期 NuRD 复合物的时间和空间分布以及生化修饰具有高度动态性，可能是通过与激酶（包括 Aurora-A）的相互作用实现的。这些观察结果将极大地有助于阐明 NuRD 复合物尚未表征的细胞周期控制功能。
MBD3, a component of the histone deacetylase NuRD complex, contains the methyl-CpG-binding domain (MBD), yet does not possess appreciable mCpG-specific binding activity. The functional significance of MBD3 in the NuRD complex remains enigmatic, partly because of the limited availability of biochemical approaches, such as immunoprecipitation, to analyze MBD3. In this study, we stably expressed the FIAG-tagged version of MBD3 in HeLa cells. We found that MBD3-FLAG was incorporated into the NuRD complex, and the MBD3-FLAG-containing NuRD complex was efficiently immunoprecipitated by anti-FLAG antibodies. By exploiting this system, we found that MBD3 is phosphorylated in vivo in the late G, and early M phases. Moreover, we found that Aurora-A, a serine/threonine kinase active specifically in the late G(2) and early M phases, phosphorylates MBD3 in vitro, physically associates with MBD3 in vivo, and co-localizes with MBD3 at the centrosomes in the early M phase. Interestingly, HDAC1 is distributed at the centrosomes in a manner similar to MBD3. These results suggest the highly dynamic nature of the temporal and spatial distributions, as well as the biochemical modification, of the NuRD complex in M phase, probably through an interaction with kinases, including Aurora-A. These observations will contribute significantly to the elucidation of the yet-uncharacterized cell cycle-controlled functions of the NuRD complex.