Identification of two residues in MCM5 critical for the assembly of minichromosome maintenance complexes and signal transducer and activator of transcription-mediated transcription activation in response to IFN-γ
Identification of two residues in MCM5 critical for the assembly of minichromosome maintenance complexes and signal transducer and activator of transcription-mediated transcription activation in response to IFN-γ
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DOI：
10.1073/pnas.061487598
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发表时间：
2001-03-13
影响因子：
11.1
通讯作者：
Zhang, JJ
中科院分区：
文献类型：
--
作者：
DaFonseca, CJ;Shu, F;Zhang, JJ
作为对干扰素-γ的反应，潜在的细胞质STAT1(信号转导和转录激活子)蛋白移位到细胞核并激活转录。我们以前已经证明，STAT1招募了一组核蛋白，其中包括MCM5(微小染色体维护)和MCM3，用于转录激活。MCM5直接与STAT1的转录激活域(TAD)相互作用，增强STAT1介导的转录激活。在这份报告中，我们在MCM5中发现了两个特异性残基(R732，K734)，这两个残基是STAT1和MCM5在体外和体内直接相互作用所必需的。含有R732/K734突变的MCM5对干扰素-γ不能增强STAT1介导的转录激活作用。此外，它在体内也不能与其他MCM蛋白形成复合体，这表明这两个残基可能对MCM5中的相互作用结构域起重要作用。此外，携带ATPase和解旋酶结构域突变的MCM5并不能增强STAT1的活性。体外结合分析表明，MCM3不直接与STAT1相互作用，提示MCM3在与Stat1TAD相互作用的蛋白质组中的存在是由于MCMS与MCM5的结合。最后，对经INF-γ处理的细胞的核抽提物进行凝胶过滤分析表明，存在与STAT1洗脱的MCM5/3亚复合体。综上所述，这些结果有力地表明，在干扰素-γ诱导的基因激活过程中，STAT1通过与MCM5直接相互作用招募到一个MCM5/3亚复合体。
In response to IFN-gamma the latent cytoplasmic Stat1 (signal transducer and activator of transcription) proteins translocate into the nucleus and activate transcription. We showed previously that Stat1 recruits a group of nuclear proteins, among them MCM5 (minichromosome maintenance) and MCM3, for transcription activation. MCM5 directly interacts with the transcription activation domain (TAD) of Stat1 and enhances Stat1-mediated transcription activation. In this report, we identified two specific residues (R732, K734) in MCM5 that are required for the direct interaction between Stat1 and MCM5 both in vitro and in vivo. MCM5 containing mutations of R732/K734 did not enhance Stat1-mediated transcription activation in response to IFN-gamma. In addition, it also failed to form complexes with other MCM proteins in vivo, suggesting that these two residues may be important for an interaction domain in MCM5. Furthermore, MCM5 bearing mutations in its ATPase and helicase domains did not enhance Stat1 activity. In vitro binding assays indicate that MCM3 does not interact directly with Stat1, suggesting that the presence of MCM3 in the group of Stat1TAD-interacting proteins is due to the association of MCMS with MCM5. Finally, gel filtration analyses of nuclear extracts from INF-gamma -treated cells demonstrate that there is a MCM5/3 subcomplex coeluting with Stat1. Together, these results strongly suggest that Stat1 recruits a MCM5/3 subcomplex through direct interaction with MCM5 in the process of INF-gamma -induced gene activation.