Hengst L, Göpfert U, Lashuel H A, Reed S I
Max-Planck-Institut für Biochemie, D-82152 Martinsried, Germany.
Genes Dev. 1998 Dec 15;12(24):3882-8. doi: 10.1101/gad.12.24.3882.
Cell-cycle phase transitions are controlled by cyclin-dependent kinases (Cdks). Key to the regulation of these kinase activities are Cdk inhibitors, proteins that are induced in response to various antiproliferative signals but that can also oscillate during cell-cycle progression, leading to Cdk inactivation. A current dogma is that kinase complexes containing the prototype Cdk inhibitor p21 transit between active and inactive states, in that Cdk complexes associated with one p21 molecule remain active until they associate with additional p21 molecules. However, using a number of different techniques including analytical ultracentrifugation of purified p21/cyclin A/Cdk2 complexes we demonstrate unambiguously that a single p21 molecule is sufficient for kinase inhibition and that p21-saturated complexes contain only one stably bound inhibitor molecule. Even phosphorylated forms of p21 remain efficient inhibitors of Cdk activities. Therefore the level of Cdk inactivation by p21 is determined by the fraction of kinase complexed with the inhibitor and not by the stoichiometry of inhibitor bound to the kinase or the phosphorylation state of the Cdk inhibitor.
细胞周期阶段转换由细胞周期蛋白依赖性激酶（Cdks）控制。这些激酶活性调节的关键是Cdk抑制剂，这类蛋白在响应各种抗增殖信号时被诱导产生，但也能在细胞周期进程中振荡，导致Cdk失活。当前的一个教条是，含有原型Cdk抑制剂p21的激酶复合物在活性和非活性状态之间转变，即与一个p21分子结合的Cdk复合物保持活性，直到它们与额外的p21分子结合。然而，我们使用了多种不同技术，包括对纯化的p21/细胞周期蛋白A/Cdk2复合物进行分析超速离心，明确证明单个p21分子就足以抑制激酶活性，并且p21饱和复合物仅包含一个稳定结合的抑制剂分子。即使是磷酸化形式的p21仍然是Cdk活性的有效抑制剂。因此，p21导致Cdk失活的程度取决于与抑制剂结合的激酶比例，而不是与激酶结合的抑制剂化学计量比或Cdk抑制剂的磷酸化状态。
