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. 1997 May 13;94(10):5095-100.
doi: 10.1073/pnas.94.10.5095.

The subcellular localization of E2F-4 is cell-cycle dependent

G J Lindeman  1 , S Gaubatz, D M Livingston, D Ginsberg
Affiliations

The subcellular localization of E2F-4 is cell-cycle dependent

G J Lindeman et al. Proc Natl Acad Sci U S A. .

Abstract

The E2F family of transcription factors plays a crucial role in cell cycle progression. E2F activity is tightly regulated by a number of mechanisms, which include the timely synthesis and degradation of E2F, interaction with retinoblastoma protein family members ("pocket proteins"), association with DP heterodimeric partner proteins, and phosphorylation of the E2F/DP complex. Here we report that another mechanism, subcellular localization, is important for the regulation of E2F activity. Unlike E2F-1, -2, or -3, which are constitutively nuclear, ectopic E2F-4 and -5 were predominantly cytoplasmic. Cotransfection of expression vectors encoding p107, p130, or DP-2, but not DP-1, resulted in the nuclear localization of E2F-4 and -5. Moreover, the transcriptional activity of E2F-4 was markedly enhanced when it was invariably nuclear. Conversely, it was reduced when the protein was excluded from the nucleus, implying that E2F-4 transcription function depends upon its cytological location. In keeping with this, the nuclear/cytoplasmic ratios of endogenous E2F-4 changed as cells exited G0, with high ratios in G0 and early G1 and a progressive increase in cytoplasmic E2F-4 as cells approached S phase. Thus, the subcellular location of E2F-4 is regulated in a cell cycle-dependent manner, providing another potential mechanism for its functional regulation.

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Figures

Figure 1
Figure 1
Location of endogenous E2F-4 is cell cycle-dependent. (A) Western blot analysis of whole cell (W), nuclear (N) and cytoplasmic (C) protein extracts from asynchronously growing U2OS (Upper) and NIH 3T3 cells (Lower) using mAbs to E2F-4 (GG22) and E2F-1 (KH20) to detect endogenous protein. (B) NIH 3T3 cells were serum-starved for 72 h in 0.5% serum and synchronously released using 10% serum-containing medium. Whole cell, nuclear, and cytoplasmic protein extracts were collected at the times shown above and subject to Western blot analyses. Blots were probed with mAb to E2F-4 or the cytoplasmic protein, β-tubulin, respectively. The cell cycle status of each sample is shown below, together with the relative nuclear:cytoplasmic levels of E2F-4 protein. A total of 100 μg of cellular protein was loaded per lane for U2OS cells, and 30 μg for NIH 3T3 cells.
Figure 2
Figure 2
Subcellular localization of E2F-4 and -5 is affected by coexpression of either the p107 or p130 pocket proteins or DP-2. Asynchronously growing U20S cells were transiently transfected with expression vectors encoding the proteins shown above each panel. After 40 h, overproduced E2F protein was detected in fixed cells by indirect immunofluorescence using the corresponding primary antibody and a FITC- or rhodamine-conjugated secondary antibody. In each case, location of the cotransfected protein was also determined (data not shown). Nuclei were revealed by Hoechst staining (data not shown).
Figure 3
Figure 3
Wild-type and mutant E2F-4 species. E2F-4Δ18 lacks pocket protein binding sequences, E2F-4Δ1–81 lacks the DNA-binding domain. The NLS from SV40 T antigen NLS and the NES from the HIV-1 Rev protein were inserted at the N terminus in E2F-4.NLS and E2F-4.NES, respectively. HA, hemagglutinin epitope; DBD, DNA-binding domain; dimerization domain; TA, pocket protein-binding and transactivation domain.
Figure 4
Figure 4
Effects of p107 and DP-2 on the nuclear localization of E2F-4 mutants. U2OS cells were transiently cotransfected with plasmids encoding either a mutant deficient in the ability to bind pocket proteins (E2F-4Δ18) or to bind DNA (E2F-4Δ1–81) and p107 or DP-2, as indicated above each panel (Top and Middle). E2F-4 was detected by immunofluorescence using mAb GG22 and a FITC- or rhodamine-conjugated secondary antibody. Nuclear location of coexpressed p107 or DP-2 was confirmed in each case (data not shown). (Bottom) The location of E2F-4 in U2OS cells transiently transfected with wt-E2F-4, E2F-4.NLS, or E2F-4.NES is shown.
Figure 5
Figure 5
Nuclear import enhances E2F-4 transcriptional activity. (A) U20S cells were transfected with 2 μg of E2F-luciferase, 2 μg of pCMV-β-Gal, and 150, 500, or 2,000 ng of either wt-E2F-4 or E2F-4.NLS. The resulting cell extracts were analyzed by luciferase reporter assay and values normalized to β-galactosidase units. Fold-activation over wt-E2F-4 is indicated above the E2F-4.NLS bars. (B) Transcriptional activity 150 ng of wt-E2F-4 alone versus wt-E2F-4 cotransfected with 100, 500, or 1,000 ng of either DP-1 or DP-2. A total of 150 ng of the E2F-4.NLS expression vector induced the transactivation noted in the far right bar of this panel. (C) Luciferase reporter assay using 2 μg of either wt-E2F-4, E2F-4.NES or E2F-4.NLS expression vector, alone, or after cotransfection with 1 μg of DP-1 or DP-2.
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