Kobayashi H, Stewart E, Poon R, Adamczewski J P, Gannon J, Hunt T
ICRF Clare Hall Laboratories, South Mimms, Herts, England.
Mol Biol Cell. 1992 Nov;3(11):1279-94. doi: 10.1091/mbc.3.11.1279.
The binding of cyclin A to p34cdc2 and p32cdk2 and the protein kinase activity of the complexes has been measured by cell-free translation of the corresponding mRNA in extracts of frog eggs, followed by immunoprecipitation. A variety of mutant cyclin A molecules have been constructed and tested in this assay. Small deletions and point mutations of highly conserved residues in the 100-residue "cyclin box" abolish binding and activation of both p34cdc2 and p32cdk2. By contrast, large deletions at the N-terminus have no effect on kinase binding and activation, until they remove residues beyond 161, where the first conserved amino acids are found in all known examples of cyclin A. At the C-terminus, removal of 14 or more amino acids abolishes activity. We also demonstrate that deletion of, or point mutations, in the cyclin A homologue of the 10-residue "destruction box," previously described in cyclin B (Glotzer et al., 1991) abolish cyclin proteolysis at the transition from M-phase to interphase.
通过在蛙卵提取物中对相应mRNA进行无细胞翻译，随后进行免疫沉淀，来检测细胞周期蛋白A与p34cdc2和p32cdk2的结合以及复合物的蛋白激酶活性。已经构建了多种突变型细胞周期蛋白A分子并在此检测中进行了测试。在100个氨基酸的“细胞周期蛋白框”中高度保守残基的小缺失和点突变会消除p34cdc2和p32cdk2的结合及激活。相比之下，N端的大缺失对激酶结合和激活没有影响，直到它们去除了161位以外的残基，在所有已知的细胞周期蛋白A实例中，第一个保守氨基酸都位于161位。在C端，去除14个或更多氨基酸会消除活性。我们还证明，细胞周期蛋白A中与之前在细胞周期蛋白B中描述的10个氨基酸的“破坏框”同源的区域发生缺失或点突变，会消除从M期到间期转变时细胞周期蛋白的蛋白水解作用。
