Stabilization of p27(Kip1) protein is an early mechanism of Rb-mediated G1 cell cycle arrest

Abstract

The tumor suppressor protein pRb acts as a negative regulator of the cell cycle in the G1-S transition. The current pRb/E2F model of cell cycle inhibition predicts that the reduction in levels of proteins of E2F target genes precedes G1 cell cycle arrest, but the temporal aspect of this model has not been examined. Careful time course experiments after controlled re-expression of pRb in pRb-deficient tumor cells have demonstrated that reduction in protein levels of important E2F targets, such as cyclin E/Cdk2 and cyclin A/Cdk2, lagged behind the onset of G1 arrest. In comparison, the kinase activities of cyclin E/Cdk2 and cyclin A/Cdk2 were inhibited with the same kinetics as the onset of G1 cell cycle arrest together with accumulation of the kinase inhibitor p27Kip1. It is well established that p27Kip1 degradation is dependent on its association with the SCFSkp2 Roc1 ubiquitin lipase. Our results show that pRb interacts with SCF Skp2 ubiquitin ligase in vivo and in vitro. Our data suggests that pRb interferes with p27Kip1 ubiquitination through a dissociation of interaction between SCFSkp2Roc1 and p27 Kip1. These results point to a novel mechanism by which pRb may function to inhibit G1-S transition before the effects of E2F repression take place.