Skip interacts with the retinoblastoma tumor suppressor and inhibits its transcriptional repression activity

Abstract

Ski interacting protein (Skip) plays an important role in the transforming activity of both v-Ski and EBNA2 (Epstein-Barr virus encoded latency protein) and is involved in EBNA2 and NotchIC activation of CBF1-repressed promoters. We have previously shown that Skip acts as a transcriptional co-activator on a number of cellular and viral promoters. Here, we report that Skip also interacts with pRb and, in cooperation with Ski, can overcome pRb-induced transcriptional repression. We show a strong and direct interaction between pRb and Skip, and we map the site of interaction to amino acid residues 171-353 of the evolutionarily conserved SNW domain of Skip. Furthermore, the combination of Skip and Ski can successfully overcome the G1 arrest and flat cell phenotype induced by pRb. Taken together, these studies suggest that one potential function of the Skip-Ski complex is to overcome the growth-suppressive activities of pRb.

Figure 1

(A) Skip and c-Ski synergistically overcome pRb-mediated repression of the AdE2 promoter. pRb null Saos-2 cells were transfected with 3 µg of the AdE2 CAT reporter construct together with 0.1 µg of pRb expression plasmid as indicated. Effects of Skip and c-Ski were assessed by transfecting them in increasing amounts either alone or in combination as shown (concentrations are in µg). After 48 h the cells were harvested and CAT assays performed. The upper panel shows the results from a representative assay and the lower panel shows the collated results from at least three independent transfections. Standard deviations are shown. (B) Skip and c-Ski together inhibits transcriptional repression by pRb. A mixture of Gal4 site containing TK CAT reporter, the Gal4-Rb and the plasmid expressing Skip and c-Ski were transfected into U2OS cells as indicated, and after 48 h the cells were harvested and CAT assays performed. The upper panel shows the results from a representative assay and the lower panel shows the collated results from at least three independent transfections. Standard deviations are shown. (C) Analysis of pRb expression in the presence of ectopic Skip and Ski expression. U2OS cells were transfected with the plasmids pCMV-Rb (0.1 µg), pCDNA3-HA-Skip/pCMV-Ski (0.5, 1 and 2 µg of each as shown) and pCMV-βGal (0.1 µg). Cell extracts were analyzed by western blotting for Rb (αRb), Skip (αHA), βGal (α-βGal). The Coomassie protein stain of an SDS–PAGE run in parallel with an aliquot of the extract used for the western blot is shown in the bottom panel and the numbers indicate the position of molecular weight markers.

Figure 1

(A) Skip and c-Ski synergistically overcome pRb-mediated repression of the AdE2 promoter. pRb null Saos-2 cells were transfected with 3 µg of the AdE2 CAT reporter construct together with 0.1 µg of pRb expression plasmid as indicated. Effects of Skip and c-Ski were assessed by transfecting them in increasing amounts either alone or in combination as shown (concentrations are in µg). After 48 h the cells were harvested and CAT assays performed. The upper panel shows the results from a representative assay and the lower panel shows the collated results from at least three independent transfections. Standard deviations are shown. (B) Skip and c-Ski together inhibits transcriptional repression by pRb. A mixture of Gal4 site containing TK CAT reporter, the Gal4-Rb and the plasmid expressing Skip and c-Ski were transfected into U2OS cells as indicated, and after 48 h the cells were harvested and CAT assays performed. The upper panel shows the results from a representative assay and the lower panel shows the collated results from at least three independent transfections. Standard deviations are shown. (C) Analysis of pRb expression in the presence of ectopic Skip and Ski expression. U2OS cells were transfected with the plasmids pCMV-Rb (0.1 µg), pCDNA3-HA-Skip/pCMV-Ski (0.5, 1 and 2 µg of each as shown) and pCMV-βGal (0.1 µg). Cell extracts were analyzed by western blotting for Rb (αRb), Skip (αHA), βGal (α-βGal). The Coomassie protein stain of an SDS–PAGE run in parallel with an aliquot of the extract used for the western blot is shown in the bottom panel and the numbers indicate the position of molecular weight markers.

Figure 1

(A) Skip and c-Ski synergistically overcome pRb-mediated repression of the AdE2 promoter. pRb null Saos-2 cells were transfected with 3 µg of the AdE2 CAT reporter construct together with 0.1 µg of pRb expression plasmid as indicated. Effects of Skip and c-Ski were assessed by transfecting them in increasing amounts either alone or in combination as shown (concentrations are in µg). After 48 h the cells were harvested and CAT assays performed. The upper panel shows the results from a representative assay and the lower panel shows the collated results from at least three independent transfections. Standard deviations are shown. (B) Skip and c-Ski together inhibits transcriptional repression by pRb. A mixture of Gal4 site containing TK CAT reporter, the Gal4-Rb and the plasmid expressing Skip and c-Ski were transfected into U2OS cells as indicated, and after 48 h the cells were harvested and CAT assays performed. The upper panel shows the results from a representative assay and the lower panel shows the collated results from at least three independent transfections. Standard deviations are shown. (C) Analysis of pRb expression in the presence of ectopic Skip and Ski expression. U2OS cells were transfected with the plasmids pCMV-Rb (0.1 µg), pCDNA3-HA-Skip/pCMV-Ski (0.5, 1 and 2 µg of each as shown) and pCMV-βGal (0.1 µg). Cell extracts were analyzed by western blotting for Rb (αRb), Skip (αHA), βGal (α-βGal). The Coomassie protein stain of an SDS–PAGE run in parallel with an aliquot of the extract used for the western blot is shown in the bottom panel and the numbers indicate the position of molecular weight markers.

Figure 2

Skip interacts with pRb in vitro. Purified GST or GST-Rb on glutathione resin was incubated with equal amounts of ³⁵S-labeled wild-type Skip. After extensive washing, bound proteins were resolved by SDS–PAGE and autoradiography. Lower panel shows the Coomassie blue stain of the gel used in the binding assay.

Figure 3

Mapping the pRb-interaction domain on Skip. (A–C) The results from representative GST pull-down assays. Purified GST or GST-Rb on glutathione resin was incubated with the in vitro translated Skip deletion mutants for 1 h at room temperature. After extensive washing, bound proteins were analyzed by SDS–PAGE and autoradiography. (D) Schematic diagram of the Skip deletion mutants used in the analysis. Numbers refer to the amino acid residues and the central red-boxed region corresponds to the highly conserved SNW region where homology with Skip proteins derived from other species is >90%. The relative strength of binding of each protein to Rb is also shown, together with the mean percentage binding from at least three separate assays, where +++ indicates 20–30%, whereas ++ and +/– indicate 10–15% and 2–3% binding, respectively.

Figure 4

The interaction between Skip and pRb is direct. GST-fusion proteins and GST alone were purified on glutathione–agarose columns. These were then incubated with 40 ng of recombinant purified His₆-Skip 1–353 at room temperature for 1 h. After extensive washing, bound Skip was assessed by SDS–PAGE and western blot analysis with an antiserum raised against Skip.

Figure 5

Interaction of Skip with pocket proteins. Purified GST or GST-Skip on glutathione resin was incubated with the in vitro translated pocket proteins: pRb, p130 and p107 for 1 h at room temperature. After extensive washing, bound proteins were analyzed by SDS–PAGE and autoradiography.

Figure 6

Skip and c-Ski synergistically overcome p130-mediated repression of the AdE2 promoter. Saos-2 cells were transfected with 3 µg of the AdE2 CAT reporter construct together with 0.5 µg of p130 expression plasmid as indicated. Effects of Skip and c-Ski were assessed by transfecting them in increasing amounts either alone or in combination as shown (concentrations are in µg). After 48 h, the cells were harvested and CAT assays performed. (A) The results from a representative assay and (B) the collated results from at least three independent transfections. Standard deviations are shown.

Figure 7

Release of pRb-induced effects by Skip and Ski. (A) The effects of Skip and Ski on the G1 block from pRb. pRb null Saos-2 cells were co-transfected with GFP and pRb expression plasmids (1 µg), together with Skip and Ski (5 µg). After 48 h, the cells were assessed by flow cytometry. The results are plotted as the percentage change in the G1 fraction relative to cells transfected with vector alone. The results show collated results from at least three independent transfections. Standard deviations are shown. (B) The effects of Skip and Ski on the flat cell phenotype induced by pRb: Saos-2 cells were transfected with 1 µg of pBabe-Puro expression plasmid and 5 µg each of the indicated plasmids. Transfected cells were subjected to puromycin selection for 7–10 days, and the percentage of flat cells was determined by SA-β-Gal assay. Percent flat cells indicates the total number of SA-β-Gal-positive cells divided by the total number of cells counted, and represents the average from three independent experiments. Standard deviations are shown.

Figure 7

Release of pRb-induced effects by Skip and Ski. (A) The effects of Skip and Ski on the G1 block from pRb. pRb null Saos-2 cells were co-transfected with GFP and pRb expression plasmids (1 µg), together with Skip and Ski (5 µg). After 48 h, the cells were assessed by flow cytometry. The results are plotted as the percentage change in the G1 fraction relative to cells transfected with vector alone. The results show collated results from at least three independent transfections. Standard deviations are shown. (B) The effects of Skip and Ski on the flat cell phenotype induced by pRb: Saos-2 cells were transfected with 1 µg of pBabe-Puro expression plasmid and 5 µg each of the indicated plasmids. Transfected cells were subjected to puromycin selection for 7–10 days, and the percentage of flat cells was determined by SA-β-Gal assay. Percent flat cells indicates the total number of SA-β-Gal-positive cells divided by the total number of cells counted, and represents the average from three independent experiments. Standard deviations are shown.