ACTIVATION OF HUMAN CYCLIN-DEPENDENT KINASES INVITRO
ACTIVATION OF HUMAN CYCLIN-DEPENDENT KINASES INVITRO
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DOI：
10.1091/mbc.3.5.571
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发表时间：
1992-05-01
影响因子：
3.3
通讯作者：
MORGAN, DO
中科院分区：
文献类型：
--
作者：
DESAI, D;GU, Y;MORGAN, DO
我们分析了人类周期蛋白依赖性激酶在无细胞系统中的激活。重组杆状病毒感染昆虫细胞可产生人CDC2、细胞周期蛋白依赖性激酶2 （CDK2）、细胞周期蛋白A和细胞周期蛋白B1。感染细胞裂解物中的CDC2或CDK2单体可以通过添加含有细胞周期蛋白A或B1的裂解物而被激活。细胞周期蛋白B1激活CDC2，以及细胞周期蛋白A和B1激活CDK2，都伴随着高分子量复合物的形成。相比之下，CDC2不能有效地与细胞周期蛋白a结合。我们详细研究了细胞周期蛋白B1对CDC2的激活，发现CDC2在苏氨酸161上磷酸化。CDC2与细胞周期蛋白B1的结合也发生在CDC2磷酸化被阻止的条件下，导致一个无活性复合物，然后在添加细胞提取物时可以被磷酸化和激活。高度纯化的CDC2和细胞周期蛋白B1也形成了非活性复合物，可以被粗细胞提取物中的未知成分以atp依赖的方式激活。这些数据表明，CDC2激活过程始于细胞周期蛋白结合，之后CDC2磷酸化，由一个单独的酶催化，导致激活。
We have analyzed the activation of human cyclin-dependent kinases in a cell-free system. Human CDC2, cyclin-dependent kinase 2 (CDK2), cyclin A, and cyclin B1 were produced in insect cells by infection with recombinant baculoviruses. CDC2 or CDK2 monomers in lysates of infected cells could be activated by the addition of lysates containing cyclin A or B1. CDC2 activation by cyclin B1, as well as CDK2 activation by cyclins A and B1, was accompanied by the formation of high molecular weight complexes. In contrast, CDC2 did not bind effectively to cyclin A. CDC2 activation by cyclin B1 was studied in detail and was found to be accompanied by phosphorylation of CDC2 on Threonine 161. The binding of CDC2 to cyclin B1 also occurred under conditions where CDC2 phosphorylation was prevented, resulting in an inactive complex that could then be phosphorylated and activated on addition of cell extract. Highly purified CDC2 and cyclin B1 also formed inactive complexes that could be activated in an ATP-dependent fashion by unidentified components in crude cell extracts. These data suggest that the CDC2 activation process begins with cyclin binding, after which CDC2 phosphorylation, catalyzed by a separate enzyme, leads to activation.