Distinct roles for retinoblastoma protein family members in cell cycle control

The E2F family of eukaryotic transcription factors have recently been shown to play a critical role in the regulation of the G{dollar}sb1{dollar} to S-phase transition in the cell. This family of proteins is regulated in large part through cell cycle dependent interactions with the retinoblastoma (Rb) tumor supressor protein and its related family members, p107 and p130. The overall goal of the experiments in this thesis has been to assign distinct regulatory roles to the three Rb family members, focusing primarily on p107 and p130.; The p107 protein is shown to repress E2F-dependent transcription when overexpressed in cells. This repression is dependent upon p107's ability to interact with E2F. The primary E2F-p107 complex seen in cells also contains cyclin A/E, and cdk2. This higher order complex can be observed in all phases of the cell cycle in proliferating cells. However, interaction with cyclin A/E and cdk2 is not required for p107's ability to repress E2F-dependent transcription or to arrest cell growth in G{dollar}sb1{dollar}. It is also shown that the p107 promoter is repressed in Go cells through E2F sites.; Studies of the p130 protein indicate that it is found in complexes with E2F uniquely in quiescent or terminally differentiated cells. As cells enter the cell cycle, the p130 protein is phosphorylated and then degraded. The in vivo phosphorylation of p130 is dependent upon G{dollar}sb1{dollar} cyclin kinase activity and the inhibition of this activity results in the maintenance of both E2F-p130 complexes and p130 protein levels. Loss of the p130 protein can also be prevented by an inhibitor of the 26S proteosome, N-acetyl-L-leucinyl-L-leucinal-L-norleucinal (LLnL). This suggests that p130 degradation occurs via the ubiquitin pathway. Additionally, a p130 mRNA transcript can be detected in growing cells at a time when the p130 protein is undetectable. Thus, p130 protein levels are regulated in part through post-transcriptional mechanisms. The presence of the E2F-p130 complex in quiescent cells correlates with the active repression of a class of cellular promoters containing E2F sites. It is proposed that accumulation of the E2F-p130 complex in quiescent cells results in the negative regulation of certain key target genes involved in cell growth control.