Inhibition of DNA synthesis by RB:: effects on G1/S transition and S-phase progression

The retinoblastoma tumor suppressor protein, RB, is a negative regulator of cell proliferation. Growth inhibitory activity of RE is attenuated by phosphorylation. Mutation of a combination of phosphorylation sites leads to a constitutively active RE. In Rat-1 cells, the phosphorylation-site-mutated (PSM)-RB, but not wild-type RE, can inhibit S-phase entry. In PSiM-RB-arrested G(1) cells, normal levels of cyclin E and cyclin E-associated kinase activity were detected, but the expression of cyclin A was inhibited. The ectopic expression of cyclin E restored cyclin A expression and drove the PSM-RB expressing cells into S phase. Interestingly, Rat-1 cells coexpressing cyclin E and PSM-RB could not complete DNA replication. Microinjection of cells that have passed through the G(1) restriction point with plasmids expressing PSM-RB also led to the inhibition of DNA synthesis. The S-phase inhibitory activity of PSM-RB could be attenuated by the coinjection of SV40 T-antigen, adenovirus E1A, or a high level of E2F-1 expression plasmids. However, the S-phase inhibitory activity of PSM-RB could not be overcome by the coinjection of cyclin E or cyclin A expression plasmids. These results reveal a novel role for RE in the inhibition of S-phase progression that is distinct from the inhibition of the G(1)/S transition, and suggest that continued phosphorylation of RE beyond G(1)/S is required for the completion of DNA replication.