Human Cdc7-related kinase complex -: In vitro phosphorylation of MCM by concerted actions of Cdks and Cdc7 and that of a critical threonine residue of Cdc7 by Cdks
Human Cdc7-related kinase complex -: In vitro phosphorylation of MCM by concerted actions of Cdks and Cdc7 and that of a critical threonine residue of Cdc7 by Cdks
复制标题
DOI：
10.1074/jbc.m002713200
复制
发表时间：
2000-09-15
影响因子：
4.8
通讯作者：
Arai, K
中科院分区：
文献类型：
--
作者：
Masai, H;Matsui, E;Arai, K
huCdc 7编码人酿酒酵母Cdc 7相关激酶复合物的催化亚基。表达受细胞周期调节的ASK以细胞周期依赖性方式结合并激活huCdc 7激酶（Kumagai，H.，佐藤，N.，山田，M.，Mahony，D.，Seghezzi，W.，Lees，E.，阿赖，K.，和Masai，H. 04 The Dog（1999）Cell. 19，5083-5095）。我们利用昆虫细胞表达系统表达了与ASK调节亚基复合的huCdc 7。为了促进激酶复合物的纯化，将谷胱甘肽S-转移酶（GST）与huCdc 7融合，并纯化GST-huCdc 7-ASK复合物。GST-huCdc 7蛋白本身作为激酶是惰性的，并且磷酸化完全依赖于ASK亚基的存在。它在体外使两个亚基自磷酸化，并使许多复制蛋白不同程度地磷酸化。其中，MCM 2蛋白，无论是以游离形式还是以MCM 2 -4-6-7复合物的形式，在体外都充当huCdc 7-ASK激酶复合物的优良底物。MCM 4和MCM 6也被huCdc 7磷酸化，尽管程度较低。MCM 2 -4-6-7复合物中的MCM 2和-4也被Cdks磷酸化，并且Cdks对MCM 2 -4-6-7复合物的预先磷酸化促进huCdc 7对MCM 2的磷酸化，表明Cdks和Cdc 7在MCM磷酸化以启动S期中的协作。huCdc 7和ASK蛋白也可以在体外被Cdks磷酸化。在huCdc 7的四个可能的Cdk磷酸化位点中，用丙氨酸（T376 A突变体）替换Thr-376（对应于Cdk的活化苏氨酸）显著降低激酶活性，指示通过该残基的磷酸化的激酶活化。在体外，Cdk 2-Cyclin E、Cdk 2-Cyclin A和Cdc 2-Cyclin B，而不是Cdk 4-Cyclin D1，磷酸化huCdc 7的Thr-376残基，表明Cdk可能调节huCdc 7。
huCdc7 encodes a catalytic subunit for Saccharomyces cerevisae Cdc7-related kinase complex of human. ASK, whose expression is cell cycle-regulated, binds and activates huCdc7 kinase in a cell cycle-dependent manner (Kumagai, H., Sato, N., Yamada, M., Mahony, D., Seghezzi, W., Lees, E., Arai, K., and Masai, H. (1999) Mel. Cell. Biol. 19, 5083-5095). We have expressed huCdc7 complexed with ASK regulatory subunit using the insect cell expression system. To facilitate purification of the kinase complex, glutathione S-transferase (GST) was fused to huCdc7 and GST-huCdc7-ASK complex was purified. GST-huCdc7 protein is inert as a kinase on its own, and phosphorylation absolutely depends on the presence of the ASK subunit, It autophosphorylates both subunits in vitro and phosphorylates a number of replication proteins to different extents. Among them, MCM2 protein, either in a free form or in a MCM2-4-6-7 complex, serves as an excellent substrate for huCdc7-ASK kinase complex in vitro. MCM4 and MCM6 are also phosphorylated by huCdc7 albeit to less extent. MCM2 and -4 in the MCM2-4-6-7 complex are phosphorylated by Cdks as well, and prior phosphorylation of the MCM2-4-6-7 complex by Cdks facilitates phosphorylation of MCM2 by huCdc7, suggesting collaboration between Cdks and Cdc7 in phosphorylation of MCM for initiation of S phase. huCdc7 and ASK proteins can also be phosphorylated by Cdks in vitro. Among four possible Cdk phosphorylation sites of huCdc7, replacement of Thr-376, corresponding to the activating threonine of Cdk, with alanine (T376A mutant) dramatically reduces kinase activity, indicative of kinase activation by phosphorylation of this residue. In vitro, Cdk2-Cyclin E, Cdk2-Cyclin A, and Cdc2-Cyclin B, but not Cdk4-Cyclin D1, phosphorylates the Thr-376 residue of huCdc7, suggesting possible regulation of huCdc7 by Cdks.