Cyclin E-induced S phase without activation of the pRb/E2F pathway

In cells of higher eukaryotes, cyclin D-dependent kinases Cdk4 and Cdk6 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-phase genes. Here we performed microinjection and transfection experiments using rat R12 fibroblasts, their derivatives conditionally overexpressing 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 induction. We demonstrate that ectopic expression of cyclin E, but not cyclin D1, can override G ₁ arrest imposed by either the p16(INK4a) Cdk inhibitor specific for Cdk4 and Cdk6 or a novel phosphorylation-deficient mutant pRb. Several complementary approaches to assess E2F activation, including quantitative reporter assays in live cells, showed that the cyclin E-induced S phase and completion of the cell division cycle can occur in the absence of E2F-mediated transactivation. Together with the ability of cyclin E to overcome a G ₁ block induced by expression of dominant-negative mutant DP-1, a heterodimeric partner of E2Fs, these results provide evidence for a cyclin E-controlled S phase-promoting event in somatic cells downstream of or parallel to phosphorylation of pRb and independent of E2F activation. They furthermore indicate that a lack of E2F-mediated transactivation can be compensated by hyperactivation of this cyclin E-controlled event.