CYCLIN E-CDK2 IS A REGULATOR OF P27(KIP1)

CDK inhibitors are thought to prevent cell proliferation By negatively regulating cyclin-CDK complexes. We propose that the opposite is also true, that cyclin-CDK complexes in mammalian cells can promote cell c ycle progression by directly down-regulating CDK inhibitors. We show t hat expression of cyclin E-CDK2 in murine fibroblasts causes phosphory lation of the CDK inhibitor p27(Kip1) On T187, and that cyclin E-CDK2 can directly phosphorylate p27 T187 in vitro. We further show that cyc lin E-CDK2-dependent phosphorylation of p27 results in elimination of p27 from the cell, allowing cells to transit from G(1) to S phase, Mor eover, mutation of T187 in p27 to alanine creates a p27 protein that c auses a G(1) block resistant to cyclin E and whose level of expression is not modulated by cyclin E. A kinetic analysis of the interaction b etween p27 and cyclin E-CDK2 explains how p27 can be regulated by the same enzyme it targets for inhibition. We show that p27 interacts with cyclin E-CDK2 in at least two distinct ways: one resulting in p27 pho sphorylation and release, the other in tight binding and cyclin E-CDK2 inhibition. The binding of ATP to the CDK governs which state predomi nates. At low ATP (<50 mu M) p27 is primarily a CDK inhibitor, but at ATP concentrations approaching physiological levels (>1 mM) p27 is mor e likely to be a substrate. Thus, we have identified p27 as a biologic ally relevant cyclin E-CDK2 substrate, demonstrated the physiological consequences of p27 phosphorylation, and developed a kinetic model to explain how p27 can be both an inhibitor and a substrate of cyclin E-C DK2.