Site-specific phosphorylation of MCM4 during the cell cycle in mammalian cells
Site-specific phosphorylation of MCM4 during the cell cycle in mammalian cells
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DOI：
10.1111/j.1742-4658.2006.05146.x
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发表时间：
2006-03-01
期刊：
影响因子：
5.4
通讯作者：
Ishimi, Y
中科院分区：
文献类型：
--
作者：
Komamura-Kohno, Y;Karasawa-Shimizu, K;Ishimi, Y
MCM 4是一种假定的复制解旋酶的亚基，在细胞周期中至少部分地被细胞周期蛋白依赖性激酶（CDK）磷酸化，CDK在DNA复制的调节中起核心作用。然而，MCM 4的磷酸化的详细表征仍有待进行。我们使用抗磷酸化MCM 4血清检测了人MCM 4在Ser 3、Thr 7、Thr 19、Ser 32、Ser 54、Ser 88和Thr 110的磷酸化。HeLa细胞的蛋白质印迹分析表明，MCM 4在这7个位点的磷酸化可分为两组：（a）在G（2）和M期（Thr 7、Thr 19、Ser 32、Ser 54、Ser 88和Thr 110）中大大增强的磷酸化，和（B）在间期（Ser 3）中牢固检测到的磷酸化。我们目前的数据表明，在Thr 7，Thr 19，Ser 32，Ser 88和Thr 110在M期的磷酸化需要CDK 1，使用温度敏感的突变体的小鼠CDK 1，和磷酸化在网站3和32在间期需要CDK 2，使用显性负突变的人CDK 2。基于这些结果和那些从体外磷酸化的MCM 4与CDK 2/细胞周期蛋白A，我们讨论负责MCM 4磷酸化的激酶。使用抗磷酸血清检测到的磷酸化MCM 4表现出不同的染色质亲和力。对染色质结合的MCM 4在位点3和32处磷酸化的核定位的研究表明，它们通常不与复制DNA共定位。出乎意料的是，在位点32磷酸化的MCM 4在细胞周期中富集在核仁中。这些结果表明，MCM 4的磷酸化在哺乳动物细胞周期中MCM的功能中具有几种不同的和位点特异性的作用。
MCM4, a subunit of a putative replicative helicase, is phosphorylated during the cell cycle, at least in part by cyclin-dependent kinases (CDK), which play a central role in the regulation of DNA replication. However, detailed characterization of the phosphorylation of MCM4 remains to be performed. We examined the phosphorylation of human MCM4 at Ser3, Thr7, Thr19, Ser32, Ser54, Ser88 and Thr110 using anti-phosphoMCM4 sera. Western blot analysis of HeLa cells indicated that phosphorylation of MCM4 at these seven sites can be classified into two groups: (a) phosphorylation that is greatly enhanced in the G(2) and M phases (Thr7, Thr19, Ser32, Ser54, Ser88 and Thr110), and (b) phosphorylation that is firmly detected during interphase (Ser3). We present data indicating that phosphorylation at Thr7, Thr19, Ser32, Ser88 and Thr110 in the M phase requires CDK1, using a temperature-sensitive mutant of mouse CDK1, and phosphorylation at sites 3 and 32 during interphase requires CDK2, using a dominant-negative mutant of human CDK2. Based on these results and those from in vitro phosphorylation of MCM4 with CDK2/cyclin A, we discuss the kinases responsible for MCM4 phosphorylation. Phosphorylated MCM4 detected using anti-phospho sera exhibited different affinities for chromatin. Studies on the nuclear localization of chromatin-bound MCM4 phosphorylated at sites 3 and 32 suggested that they are not generally colocalized with replicating DNA. Unexpectedly, MCM4 phosphorylated at site 32 was enriched in the nucleolus through the cell cycle. These results suggest that phosphorylation of MCM4 has several distinct and site-specific roles in the function of MCM during the mammalian cell cycle.