pRB binds to and modulates the transrepressing activity of the E1A-regulated transcription factor p120E4F

Abstract

The retinoblastoma protein pRB is involved in the transcriptional control of genes essential for cell cycle progression and differentiation. pRB interacts with different transcription factors and thereby modulates their activity by sequestration, corepression, or activation. We report that pRB, but not p107 and p130, binds to and facilitates repression by p120(E4F), a ubiquitously expressed GLI-Kruppel-related protein identified as a cellular target of E1A. The interaction involves two distinct regions of p120(E4F) and the C-terminal part of pRB. In vivo pRB-p120(E4F) complexes can only be detected in growth-arrested cells, and accordingly contain the hypophosphorylated form of pRB. Repression of an E4F-responsive promoter is strongly increased by combined expression of p120(E4F) and pRB, which correlates with pRB-dependent enhancement of p120(E4F) binding activity. Elevated levels of p120(E4F) have been shown to block growth of mouse fibroblasts in G(1). We find this requires pRB, because RB(-/-) fibroblasts are significantly less sensitive to excess p120(E4F).

Figure 1

p120^E4F associates with pRB. (A) Recombinant p120^E4F associates with cellular pRB. GST pull-down assays were performed with nuclear extracts prepared from confluent cultures of either RB^+/+ (lanes 1, 3, and 5) or RB^−/− NIH 3T3 mouse fibroblasts (lanes 2, 4, and 6). Extracts were incubated with either Sepharose-bound GST–p120^E4F (lanes 3 and 4) or GST–ELK (negative control) (lanes 5 and 6). Bound proteins were released and probed for the presence of pRB by immunoblotting. One-third of the input extract was loaded in lane 1 and 2. (B) Recombinant p120^E4F associates with the hypophosphorylated form of pRB present in arrested cells and does not associate at all with cellular p107 and p130. GST pull-down assays were performed as above by using nuclear extracts from either quiescent (lanes 1 and 3) or exponentionally growing (lanes 2 and 4) NIH 3T3 fibroblasts. Bound proteins (lanes 3 and 4) were probed for the presence of pRB (Top), p107 (Middle), or p130 (Bottom) by immunoblotting. One-third of the input extract was loaded in lane 1 and 2. (C) p120^E4F is easily detectable in U2OS cells. Nuclear extract from U2OS cells is probed by immunoblotting by using an affinity-purified rabbit polyclonal antibody (E4F-88) (lane 3). Specificity of this Ab is tested on nuclear extracts prepared from a Chinese hamster cell line (CCL39-tet/E4F) expressing a tetracycline inducible p120^E4F gene (lanes: 1, + tetracycline; 2, − tetracycline). (D and E) Coimmunoprecipitation of cellular p120^E4F and pRB proteins from U2OS cells extracts. (D) p120^E4F is immunoprecipitated from confluent U2OS nuclear extracts by using the E4F 88.2 Ab, and precipitate is probed for the presence of pRB by immunoblotting (lane 2). The same experiment is performed with the corresponding preimmune serum (lane 3). One-tenth of the input extract was loaded in lane 1. (E) pRB is immunoprecipitated from confluent U2OS nuclear extracts by using the anti-pRB mAb coupled to agarose beads and precipitate is probed for the presence of p120^E4F by immunoblotting (lane 4). The same experiment is performed with control beads coupled to unrelated mAbs (lanes 2 and 3). One-thirtieth of the input extract was loaded in lane 1.

Figure 2

Mapping of the binding domains of pRB and p120^E4F. (A) GST pull-down assays using the indicated GST-pRB constructs (Upper) and ³⁵S-labeled in vitro translated p120^E4F (full-length) (lanes 2–6, Lower). Input of radiolabeled p120^E4F is loaded lane 1 (Lower). As a control, GST pull-down assay is done with GST–ELK protein (lane 7). (B) Schematic representation of the various pRB protein constructs and summary of their capacity to bind p120^E4F. Pocket domains A (aa 379–580), B (aa 639–771), C (aa 771–928) are indicated. The asterisk indicates the C→F mutation of pRB^C706-F. (C) GST pull-down assays using the indicated ³⁵S-labeled in vitro-translated p120^E4F fragments and the GST–pRB construct containing domains A, B, and C of pRB. (D) Schematic representation of the various p120^E4F fragments used in the assay and summary of their capacity to bind pRB. The zinc fingers domains are shown as black boxes. FL, full length (aa 1–784); N/p50^E4F, aa 1–357; C, aa 358–784; S, aa 358–486; F1, aa 184–254; F2, aa 428–521; F3, aa 515–584.

Figure 3

pRB up-regulates the DNA binding and transrepression capabilities of p120^E4F. (A) p120^E4F is a site-dependent transcriptional repressor. NIH 3T3 cells are transiently cotransfected with an E4F-responsive reporter gene E4F-TK-Luc and increasing amounts of the pcDNA–p120^E4F plasmid. TK-Luc (without E4F site) was used as a control. Luciferase activity is shown as fold inhibition of the activity obtained with E4F-TK-Luc or TK-Luc constructs transfected alone. (B) pRB up-regulates the transrepression capabilities of p120^E4F. The maximum amount of p120^E4F plasmid that was unable to inhibit the reporter activity as determined in A is cotransfected with either E4F-TK-Luc or TK-Luc and with (dotted bars) or without (stripped bars) a phosphorylation-defective constitutively active pRB (p34RB). As a control, E4s-TK-Luc and TK-Luc reporters were also transfected alone (filled bars) or with p34RB (open bars). (C) pRB up-regulates the DNA binding activity of p120^E4F in vitro. EMSAs are performed with a probe containing an E4F DNA binding site. Purified GST–p120^E4F is added alone (lane 1) or together with increasing amounts of baculovirus-expressed purified pRB protein (lanes 2–4) or BSA (lane 5).

Figure 4

p120^E4F-mediated growth arrest is pRB-dependent. (A) Rb^+/+ (Upper) or Rb^−/− (Lower) mouse fibroblasts were transfected with the pcDNA–p120^E4F plasmid. Twenty-four hours after transfection with the indicated plasmids, cells were assessed for BrdUrd uptake in their DNA (14 h in BrdUrd) as a marker of cell proliferation. Cells were fixed and labeled with 4′,6′-diamidino-2-phenylindole (DAPI) (blue). p120^E4F-expressing cells were identified by immunofluoresence by using anti-human E4F (E4F 88.2) (red) and monitored for BrdUrd incorporation by using an anti-BrdUrd-specific Ab (green). (B) Diagram showing the average of four independent experiments (200 cells counted per experiment) performed as reported in A. Average of similar experiments performed in p107/p130^−/− cells are also shown as indicated. As a control, Rb^−/− cells are also transfected with p34(RB).