Cyclin E-CDK2 regulates p27(Kip1) by promoting its phosphorylation and subsequent degradation, allowing cell cycle progression. The study shows that cyclin E-CDK2 can directly phosphorylate p27 on T187, leading to its elimination from the cell and enabling transition from G1 to S phase. Mutation of T187 in p27 prevents phosphorylation and results in a G1 block resistant to cyclin E. The interaction between p27 and cyclin E-CDK2 can result in either phosphorylation and release of p27 or tight binding and inhibition of cyclin E-CDK2. ATP levels determine which state predominates, with low ATP favoring p27 as a CDK inhibitor and high ATP favoring it as a substrate. The study identifies p27 as a biologically relevant cyclin E-CDK2 substrate, demonstrating the physiological consequences of p27 phosphorylation and developing a kinetic model explaining how p27 can be both an inhibitor and a substrate of cyclin E-CDK2. The findings suggest that cyclin E-CDK2 inactivates CDK inhibitors to promote cell cycle progression. The results indicate that cyclin E-CDK2 phosphorylates p27 on T187, leading to its degradation and cell cycle progression. Cyclin D1-CDK4 does not phosphorylate p27 or modulate its abundance. Kinetic analysis shows that p27 binds cyclin E-CDK2 in two ways: as a substrate (loose binding) and as an inhibitor (tight binding). Increasing ATP concentration competes with inhibition, favoring p27 phosphorylation and release. The study also shows that p27 can be a CDK2 inhibitor or substrate depending on ATP levels. The findings highlight the complex regulation of p27 by cyclin E-CDK2, with implications for cell cycle control and cancer.Cyclin E-CDK2 regulates p27(Kip1) by promoting its phosphorylation and subsequent degradation, allowing cell cycle progression. The study shows that cyclin E-CDK2 can directly phosphorylate p27 on T187, leading to its elimination from the cell and enabling transition from G1 to S phase. Mutation of T187 in p27 prevents phosphorylation and results in a G1 block resistant to cyclin E. The interaction between p27 and cyclin E-CDK2 can result in either phosphorylation and release of p27 or tight binding and inhibition of cyclin E-CDK2. ATP levels determine which state predominates, with low ATP favoring p27 as a CDK inhibitor and high ATP favoring it as a substrate. The study identifies p27 as a biologically relevant cyclin E-CDK2 substrate, demonstrating the physiological consequences of p27 phosphorylation and developing a kinetic model explaining how p27 can be both an inhibitor and a substrate of cyclin E-CDK2. The findings suggest that cyclin E-CDK2 inactivates CDK inhibitors to promote cell cycle progression. The results indicate that cyclin E-CDK2 phosphorylates p27 on T187, leading to its degradation and cell cycle progression. Cyclin D1-CDK4 does not phosphorylate p27 or modulate its abundance. Kinetic analysis shows that p27 binds cyclin E-CDK2 in two ways: as a substrate (loose binding) and as an inhibitor (tight binding). Increasing ATP concentration competes with inhibition, favoring p27 phosphorylation and release. The study also shows that p27 can be a CDK2 inhibitor or substrate depending on ATP levels. The findings highlight the complex regulation of p27 by cyclin E-CDK2, with implications for cell cycle control and cancer.