Cai K, Dynlacht B D
Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA.
Proc Natl Acad Sci U S A. 1998 Oct 13;95(21):12254-9. doi: 10.1073/pnas.95.21.12254.
Elevated levels of the p21(WAF1) (p21) cyclin-dependent kinase inhibitor induce growth arrest. We have characterized a panel of monoclonal antibodies against human p21 in an effort to understand the dynamic regulatory interactions between this and other cellular proteins during the cell cycle. The use of these reagents has allowed us to address several important, yet unresolved, issues concerning the biological activity of p21, including the potential kinase activity of complexes that associate with this cyclin-dependent kinase inhibitor. We have found that the kinase activity of cyclin A/Cdk2 associated with p21 is significantly lower than that of cyclin A/Cdk2 free of p21, suggesting that p21 abolishes its activity in vivo, and the use of multiple antibodies has enabled us to begin the study of the molecular architecture of p21 complexes in vivo. In addition, we found that human fibroblasts released from a quiescent state display abundant amounts of p21 devoid of associated proteins ("free" p21), the levels of which decrease as cells approach S phase. Cyclin A levels increase as the amount of monomeric p21 decreases, resulting in an excess of cyclin A/Cdk2 complexes that are not bound to, or inactivated by, p21. Our data strengthen the notion that the G1-to-S phase transition in human fibroblasts occurs when the concentration of cyclin A/Cdk2 surpasses that of p21.
p21（WAF1）（p21）细胞周期蛋白依赖性激酶抑制剂水平升高会诱导生长停滞。我们已对一组抗人p21的单克隆抗体进行了特性分析，以期了解在细胞周期中它与其他细胞蛋白之间的动态调节相互作用。这些试剂的使用使我们能够解决几个有关p21生物学活性的重要但尚未解决的问题，包括与这种细胞周期蛋白依赖性激酶抑制剂相关的复合物的潜在激酶活性。我们发现，与p21相关的细胞周期蛋白A/Cdk2的激酶活性明显低于不含p21的细胞周期蛋白A/Cdk2，这表明p21在体内消除了其活性，并且多种抗体的使用使我们能够开始研究体内p21复合物的分子结构。此外，我们发现从静止状态释放的人成纤维细胞显示出大量不含相关蛋白的p21（“游离”p21），随着细胞接近S期，其水平会降低。随着单体p21数量的减少，细胞周期蛋白A水平升高，导致过量的细胞周期蛋白A/Cdk2复合物未与p21结合或未被p21灭活。我们的数据强化了这样一种观点，即当细胞周期蛋白A/Cdk2的浓度超过p21时，人成纤维细胞会发生从G1期到S期的转变。
