CYCLIN A-ASSOCIATED KINASE-ACTIVITY IS RATE-LIMITING FOR ENTRANCE INTO S-PHASE AND IS NEGATIVELY REGULATED IN G(1) BY P27(KIP1)

We have created fibroblast cell lines that express cyclin A under the control of a tetracycline-repressible promoter. When stimulated to ree nter the cell cycle after serum withdrawal, these cells were advanced prematurely into S phase by induction of cyclin A. In an asynchronous population, induction of cyclin A caused a decrease in the percentage of cells in G(1). These results demonstrate that expression of cyclin A is rate limiting for the G(1)-to-S transition and suggest that cycli n A can function as a G(1) cyclin. Although the level of exogenous cyc lin A was constant throughout the cell cycle, its associated kinase ac tivity increased as cells approached S phase. Low kinase activity in e arly G(1) was found to correlate with the presence of p27/((Kip1)) in cyclin A-associated complexes, while high kinase activity in late G(1) was correlated with its absence. These results suggest that a functio n of p27/((Kip1)) in G(1) is to prevent premature activation of cyclin A-associated kinase. Cyclin A expression in early G(1) led to phospho rylation of the product of the retinoblastoma susceptibility gene (pRb ). Thus, cyclin A expression can be rate limiting for pRb phosphorylat ion implicating pRb as a physiological substrate of the cyclin A-depen dent kinase. Taken together, these results demonstrate that deregulate d expression of cyclin A can perturb the normal regulation of the G(1) -to-S transition.