Ras activity late in G(1) phase required for p27(kip1) downregulation, passage through the restriction point, and entry into S phase in growth factor-stimulated NIH 3T3 fibroblasts

It is well documented that nas functions as a molecular switch for reentry into the cell cycle at the border between G(0) and G(1) by transducing extracellular growth stimuli into early G(1) mitogenic signals, In the present study, we investigated the role of Ras duping the late stage of the G(1) phase by using NIH 3T3 (M17) fibroblasts in which the expression of a dominant negative Res mutant, p21(Ha-Ras(Asn17)), is induced in response to dexamethasone treatment. We found that delaying the expression of Ras(Asn17) until late in the G(1) phase bg introducing dexamethasone 3 ir after the addition of epidermal growth factor (EGF) abolished the downregulation of the p27(kip1) cyclin-dependent kinase (CDK) inhibitor which normally occurred during this period, with resultant suppression of cyclin Ds/CDK4 and cyclin E/CDK2 and G(1) arrest. The immunodepletion of p27(kip1) completely eliminated the CDK inhibitor activity from EGF-stimulated, dexamethasone-treated cell lysate. The failure of p27(kip1) downregulation and G(1) arrest was also observed in cells in which Ras(Asn17) was induced after growth stimulation with a phorbol eater or alpha-thrombin and was mimicked br the addition late in the G(1) phase of inhibitors for phosphatidylinositol-3-kinase. Ras-mediated downregulation of p27(kip1) involved both the suppression of synthesis and the stimulation of the degradation of the protein, Unlike the earlier expression of nas (Asn17) at the border between G(0) and G(1), its delayed expression did not compromise the EGF-stimulated transient activation of extracellular signal-regulated kinases or inhibit the stimulated expression of a principal D-type cyclin, cyclin D1, until close to the border between G(1) and S. We conclude that Ras plays temporally distinct, phase-specific roles throughout the G(1) phase and that Ras function late in G(1) is required for p27(kip1) downregulation and passage through the restriction point, a prerequisite for entry into the S phase.