The PP1–HCF and VP16–HCF complexes in HeLa nuclear extracts. (A) About 10 µg GST-VP16 was used to pull down HCF from 0.4 to 0.5 mg of HeLa nuclear extract. Bound HCF on the beads was resuspended in 50 µl of SDS–PAGE buffer and 15 µl of the HCF pull-down probed with anti-PP1 antibody (bottom panel). The panel at the top shows a western blot of the pull-down probed with anti-HCF antibody. Lane 1 contained nuclear extract. Lanes 2 and 3 had GST and GST-VP16 pull-downs respectively. Lane 4 contained an immunoprecipitation from HeLa nuclear extract with anti-HCF antibody. The arrowheads indicate the major HCF polypeptides and PP1 respectively. Because of the high levels of PP1 in nuclear extract (Fig. 2C), the blot in the lower panel probed with anti-PP1 antibody contained ~15% of the amount of nuclear extract used in the corresponding lane in the top panel western blot probed with anti-HCF antibody. (B) The effect of adding active bacterially expressed PP1 on the VP16 induced complex. No PP1 was added to assays in lanes 1–4. The samples in lanes 5–8 contained ~250 ng of PP1. Varying the quantity of PP1 in the assays to levels below or above 250 ng did not have any significant effect on the formation of the VP16-induced complex. The + signs represent increasing amounts of VP16 while the – signs indicate the absence of VP16. +, 2 ng VP16; ++, 10 ng of VP16; and +++, 50 ng of the protein in the assay. The Oct-1-TAATGARAT complex (Oct 1) and VP16-induced complex (VIC) are indicated.

Linear representation of HCF showing the three overlapping clones that interact with PP1-γ. The diagram is based on the HCF described in (7) with some modifications. The numbers above the rectangles representing clones 1, 15 and 17 show the start and end positions of the amino acid sequence of the HCF fragments expressed by the cDNAs obtained from the library screen.

Recombinant HCF binds PP1. The HCF cDNA fragment in clone 17 was expressed as a β-galactosidase (β-Gal) and GST fusions. (A) Western blot of an SDS–PAGE gel probed with digoxigenin-labelled PP1-γ. The arrowhead indicates recombinant fusion protein. The small arrow indicates a proteolytic product of β-Gal–HCF. Escherichia coli cells were grown to an OD₆₀₀ of 0.9–1. Each lane contained 10 µl of a 25 times concentrated E.coli lysate. Lane 1 contains bacterial lysate of cells expressing β-Gal alone. Lanes 2 and 3 contain bacterial lysates of cells expressing HCF (clone 17) and NIPP1 fusions respectively. (B) The GST–HCF fusion protein was incubated with purified PP1 (~3 nmol of each protein) and glutathione–Sepharose beads. Each GST-fusion pull-down was done in duplicate. After several washes the samples were resolved by SDS–PAGE and immunoblotted with anti-PP1. Lanes 1 and 2 contained the binding assay between the GST–HCF fragment in clone 17 and PP1-γ. Lanes 3 and 4 contained GST and PP1 and lanes 5 and 6 contained a positive control: GST-p53BP2 (p53BP2 is known to bind PP1 avidly) (33). The arrow indicates bands corresponding to the PP1 pulled down. (C) HCF was immunoprecipitated from 0.4–0.5 mg of HeLa nuclear extract using ~5 µg of anti-HCF antibody and the beads resuspended in ~50 µl of SDS–PAGE loading buffer. Fifteen microlitres of the immunoprecipitates of HCF from HeLa nuclear extract were electroblotted as above and probed with anti-HCF antibody (upper panel) and anti-PP1 antibody (lower panel). Each immunoprecipitation experiment contained duplicate samples. Lanes 1 and 2 contained nuclear extract. Lanes 3 and 4 had immunoprecipitates using pre-immune IgG (PI) and lanes 5 and 6 represent immunoprecipitates using anti-HCF antibody. The brace indicates the major HCF polypeptides immunoprecipitated by anti-HCF antibody. The arrow indicates the PP1 co-immunoprecipitated with the HCF polypeptides.

The HCF complex in nuclear extract contains a PP1 activity. Immunoprecipitates of HCF were assayed as described in the methods section for protein phosphatase activity using ³²P-labelled skeletal muscle phosphorylase a as substrate. The results of the assay are represented as shaded bars in the figure. (A) Columns 1 and 2 represent pre-immune IgG (40 µg) immunoprecipitates assayed for protein phosphatase activity with (+) or without (–) trypsinization. Columns 3 and 4 indicate anti-HCF (40 µg) immunoprecipitates with column 4 assayed after trypsinization. Column 5 shows a control containing the enzyme trypsin, soybean trypsin inhibitor and substrate. (B) Protein phosphatase activity was assayed in anti-HCF immunoprecipitates after trypsin treatment using increasing amounts of anti-HCF antibody or pre-immune IgG. The graph with the squares represent protein phosphatase activity in immunoprecipitates using anti-HCF antibody while the graph with the diamond shapes shows the enzyme’s activity in immunoprecipitates using pre-immune IgG. (C) Inhibition of protein phosphatases in trypsin treated anti-HCF immunoprecipitates indicates the presence of a PP1 activity in the HCF complex. Column 1 shows phosphatase activity in pre-immune IgG immunoprecipitate. Column 2 represents the protein phosphatase activity obtained from assaying the anti-HCF immunoprecipitate while column 3 shows phosphatase activity after treatment of the anti-HCF immunoprecipitate with nanomolar amounts okadaic acid. Column 4 indicates the protein phosphatase activity after treatment with micromolar amounts of okadaic acid and column 5 represents the phosphatase activity of the anti-HCF immunoprecipitate after incubation in 0.5 µM inhibitor 2, a specific inhibitor of PP1.

Release of PP1 activity from the HCF complex by a peptide containing the consensus PP1-binding motif. HCF immunoprecipitates were incubated with 10 µM of the peptide containing the putative PP1 binding domain of p53BP2 with sequence: GKRTNLRKTGSERIAMGMRVKFNPLALLLDSC or with a control peptide lacking this domain (38). (A) Columns 1, 2 and 3 represent pre-immune IgG immunoprecipitates incubated with buffer (O), control peptide (C) and p53BP2 peptide (P) respectively. Columns 4, 5 and 6 show the PP1 activities obtained when the anti-HCF immune precipitates were incubated with buffer (O), control peptide (C) and p53BP2 peptide (P) respectively. (B) The immunoprecipitates described in (A) that had been incubated with or without PP1-binding peptides were further treated with trypsin to reveal remaining PP1 activity after incubation with the peptides. Columns marked 1, 2 and 3 were the same samples as in (A) except that the immunoprecipitates were treated with trypsin and then assayed for PP1 activity. The columns labelled 4, 5 and 6 represent the same samples as corresponding columns in (A) but with the anti-HCF immunoprecipitates trypsinised and then assayed for PP1 activity not released by peptides.