Association with E2F-1 governs intracellular trafficking and polyubiquitination of DP-1.
The cell cycle-regulated transcription factor E2F is a family of heterodimers 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 the nucleus, the role of DP proteins in E2F-dependent gene expression is not well understood. Using immunolocalization, immunoprecipitation, and cell fractionation experiments, here we show association with E2F subunits governs intracellular trafficking and ubiquitination of DP-1. In transient transfection experiments, DP-1 polypeptides that stably bound E2F-1 entered the nucleus. DP-1 proteins that failed to associate with E2F subunits accumulated in the cell cytoplasm as polyubiquitinated DP-1. A Chinese hamster cell line that conditionally expresses HA-DP-1 was used to examine the effect of DP-1 on cell cycle progression. In serum response experiments, moderate increases in HA-DP-1 led to a threefold increase in E2F DNA binding activity in vitro, a corresponding increase in dhfr gene expression during transition of G1, and higher rates of S phase entry. However, flow cytometry showed cells expressing very high levels of HA-DP-1 failed to enter the S phase. Inhibition 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 degradation of ubiquitinated proteins is required for progression from G0 to S phase even in the presence of activated E2F. Under similar conditions, expression of E2F-1 reduced the levels of ubiquitinated cellular proteins and accelerated cell cycle progression. Our studies indicate association with E2F subunits prevents ubiquitin-dependent degradation of DP-1 in the cytoplasm by promoting nuclear entry of E2F/DP heterodimers.
Magae
J
J
Department of Pathology, University of Vermont College of Medicine, Burlington 05405, USA.
Illenye
S
S
Chang
Y C
YC
Mitsui
Y
Y
Heintz
N H
NH
D000818:Animals; D001692:Biological Transport; D016466:CHO Cells; D002352:Carrier Proteins; D002453:Cell Cycle; D018797:Cell Cycle Proteins; D006224:Cricetinae; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D050704:E2F4 Transcription Factor; D050700:E2F5 Transcription Factor; D015870:Gene Expression; D006801:Humans; D007424:Intracellular Fluid; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D056465:Retinoblastoma-Binding Protein 1; D016196:S Phase; D050680:Transcription Factor DP1; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014452:Ubiquitins / Q000378:metabolism*