Association with E2F-1 governs intracellular trafficking and polyubiquitination of DP-1

The cell cycle-regulated transcription factor E2F is a family of heterodime rs composed of E2F and DP protein subunits, While DP proteins stabilize DNA binding of E2F proteins, and influence the entry of E2F-4 and E2F-5 into t he nucleus, the role of DP proteins in E2F-dependent gene expression is not well understood. Using immunolocalization, immunoprecipitation, and cell f ractionation experiments, here we show association with E2F subunits govern s intracellular trafficking and ubiquitination of DP-1, In transient transf ection experiments, DP-1 polypeptides that stably bound E2F-1 entered the n ucleus. DP-1 proteins that failed to associate with E2F subunits accumulate d in the cell cytoplasm as polyubiquitinated DP-1, A Chinese hamster cell l ine that conditionally expresses HA-DP-1 was used to examine the effect of DP-1 on cell cycle progression, In serum response experiments, moderate inc reases in HA-DP-1 led to a threefold increase in E2F DNA binding activity i n vitro, a corresponding increase in dhfr gene expression during transition of G1, and higher rates of S phase entry. However, flow cytometry showed c ells expressing very high levels of HA-DP-1 failed to enter the S phase. In hibition of cell cycle progression by high levels of HA-DP-1 was associated with the accumulation of other ubiquitinated cellular proteins, including c-jun and the cyclin-dependent kinase inhibitor p21, indicating that degrad ation of ubiquitinated proteins is required for progression from G(0) to S phase even in the presence of activated E2F, Under similar conditions, expr ession of E2F-1 reduced the levels of ubiquitinated cellular proteins and a ccelerated cell cycle progression. Our studies indicate association with E2 F subunits prevents ubiquitin-dependent degradation of DP-1 in the cytoplas m by promoting nuclear entry of E2F/DP heterodimers.