E2F-1 but not E2F-4 can overcome p16-induced G1 cell-cycle arrest

Background: The transition from G1 to S phase is the key regulatory step in the mammalian cell cycle, This transition is regulated positively by G1-specific cyclin-dependent kinases (cdks) and negatively by the product of the retinoblastoma tumour suppressor gene, pRb. Hypophosphorylated pRb binds to and inactivates the E2F transcription factor, which controls the expression of genes required for S-phase progression. Hyperphosphorylation of pRb in late G1 phase results in the accumulation of active E2F, a critical event in the progression to S phase. The E2F factor is not a single entity, but rather represents a family of highly related molecules, all of which bind to pRb or the pRb-related proteins p107 and p130. Results: In this study, we have used specific inhibitors of cdks to explore the requirements for cell-cycle progression from G1 to S phase. Expression of p16(lnk4), which specifically inhibits cyclin D-directed cdks, blocks cells in G1 phase; this block can be overcome by expression of the viral proteins that inactivate pRb or by E2F-1. Importantly however, the G1 arrest is not overcome by overexpression of E2F-4. By using chimeric E2F proteins, containing amino-acid sequences from E2F-1 and E2F-4, we have shown that their differential abilities to overcome a p16-imposed arrest is determined by their respective amino-terminal regions. We also demonstrate that E2F-1 can promote entry into S phase without concomitant phosphorylation of pRb. In contrast to the p16-mediated G1 block, G1 arrest mediated by the cdk inhibitors p21(Cip1) or p27(Kip1) cannot be bypassed either by inactivation of pRb or overexpression of E2F family members. Conclusions: These data demonstrate that the role of the cyclin D-directed cdks in promoting the progression of cells from G1 into S phase is wholly to activate an E2F-1-like activity through phosphorylation, thus preventing the formation of the E2F-pRb complex. The cyclin E-cdk2 complex is also required for the G1/S transition but has a different and as yet undefined role. We also provide evidence for a functional difference between E2F-1 and E2F-4, dependant upon the region that contains the DNA-binding and dimerization domains. These results indicate that these two E2F family members are likely to regulate the expression of different subsets of E2F-responsive promoters.