CYCLIN E-INDUCED S-PHASE WITHOUT ACTIVATION OF THE PRB E2F PATHWAY/

In cells of higher eukaryotes, cyclin D-dependent kinases Cdk4 and Cdk 6 and, possibly, cyclin E-dependent Cdk2 positively regulate the G(1)- to S-phase transition, by phosphorylating the retinoblastoma protein (pRb), thereby releasing E2F transcription factors that control S-phas e genes. Here we performed microinjection and transfection experiments using rat R12 fibroblasts, their derivatives conditionally overexpres sing cyclins D1 or E, and human U-2-OS cells, to explore the action of G(1) cyclins and the relationship of E2F and cyclin E in S-phase indu ction. We demonstrate that ectopic expression of cyclin E, but not cyc lin D1, can override G(1) arrest imposed by either the p16(INK4a) Cdk inhibitor specific for Cdk4 and Cdk6 or a novel phosphorylation-defici ent mutant pRb. Several complementary approaches to assess E2F activat ion, including quantitative reporter assays in live cells, showed that the cyclin E-induced S phase and completion of the cell division cycl e can occur in the absence of E2F-mediated transactivation. Together w ith the ability of cyclin E to overcome a G(1) block induced by expres sion of dominant-negative mutant DP-1, a heterodimeric partner of E2Fs , these results provide evidence for a cyclin E-controlled S phase-pro moting event in somatic cells downstream of or parallel to phosphoryla tion of pRb and independent of E2F activation. They furthermore indica te that a lack of E2F-mediated transactivation can be compensated by h yperactivation of this cyclin E-controlled event.