Cyclin E/Cdk2 activity is controlled by different mechanisms in the G₀ and G₁ phases of the cell cycle

The experiments described in this report were undertaken to define the parameters that regulate cyclin E/cyclin-dependent kinase 2 (Cdk2) kinase activity in mitotically quiescent, serum-starved fibroblastic cells and in cells that had been stimulated to enter the cell cycle and progress through G₁ into S phase. We have analyzed the expression of cyclin E and Cdk2, the extent to which these two proteins form complexes, and the enzymatic activity of cyclin E/Cdk2 kinase. Particular attention was focused upon subcellular localization and the effect of compartmentalization on the association between cyclin E and Cdk2. In addition, we have examined the interaction of cyclin E/Cdk2 complexes with two well-characterized inhibitors of Cdk2 kinase activity, Cip1 and Kip1. This represents the first report in which all of these parameters have been measured simultaneously in a single, normal diploid cell line. In G₀ cells, there is abundant cyclin E and Cdk2, yet there is little or no detectable Cdk2-dependent histone H1 kinase activity. After serum stimulation, there is a rapid increase in the amount of cyclin E that is bound to Cdk2, although there is no significant change in the abundance of either the cyclin or the Cdk. Immunocytochemical data indicate that cyclin E, Cip1, and Kip1 are located within the nuclei of cells in G₀, but very little Cdk2 is observed within the nuclei of serum-starved cells. Cdk2 rapidly enters the nucleus upon serum stimulation. The abundance of the cyclin E/Cdk2 complex increases to the extent that the binding capacity of Cip1 is exceeded about 8-12 h after serum stimulation. The abundance of Kip1 decreases at the same time that the Cip1 threshold is exceeded, so that cyclin E/Kip1-containing complexes decrease by 90% within 8-12 h. Cyclin E/Cdk2 kinase activity begins to increase rapidly thereafter, reaching a maximum level about 16 h after serum stimulation. We have been unable to detect histone H1 kinase activity in complexes that contain cyclin E bound to Kip1 or Cip1. We conclude that compartmentalization is the predominant barrier to activation of cyclin E-dependent kinases in quiescent cells. Cip1 and Kip1 serve to prevent premature activation of cyclin E/Cdk2 complexes that form during G₀ or early G₁.