Article Figures & Data
Figures
- FIG. 1.
PAI-2 expression in HeLa cells results in posttranscriptional recovery of Rb protein. (A) Western blot analysis of lysates from the parental HeLa line cells (HeLa), HeLa cells transfected with antisense-PAI-2 (A2/7), HeLa cells lines stably expressing PAI-2 (S1a and S1b), HeLa cells stably expressing the PAI-2 C-D interhelical mutant (C-D PAI-2a and C-D PAI-2b), and HeLa cells stably expressing the RSL mutant of PAI-2 (PAI-2 Ala380a and PAI-2 Ala380b). Protein lysates were probed using antibodies specific for PAI-2, Rb, p53, and GAPDH. (B) Western blot analysis of protein lysates from the parental HeLa line cells (HeLa), HeLa cells transfected with antisense-PAI-2 (A2/7), and HeLa cells lines stably expressing PAI-2 (S1a and S1b) by using antibodies specific for the E7 protein from HPV-18. (C) Quantitative real-time RT-PCR expression analysis of E7 (top) and Rb (bottom) mRNA levels. Rb and E7 cDNAs from HeLa cells and A2/7, S1a, and S1b cells were quantified by PCR based on a standard curve established by using dilutions of GAPDH cDNA prepared from the parental HeLa line. Data from three experiments are expressed as the mean fold change ± the standard deviation compared with parental HeLa cells. *, expression levels were undetectable. (D) Treatment of S1a cells with siRNA specific for Rb and a scrambled control (Cont.) siRNA. S1a cells were treated with siRNA, and whole-cell extracts were immunoblotted for Rb, E7, and actin. Control untreated S1a and HeLa cells were run in parallel.
- FIG. 2.
PAI-2 colocalizes with, and binds to, Rb in vivo. (A) Colocalization of Rb and PAI-2 shown by indirect immunofluorescent confocal laser microscopy. S1a, KJD, and phorbol ester-activated U937 cells were dually labeled with antibodies against PAI-2 (rhodamine TRITC, red) and Rb (FITC, green). Overlay of the two images shows yellow staining where colocalization has occurred; arrows indicate colocalization in the nucleus Bar, 50 μm. (B) Immunoprecipitation of PAI-2 from nuclear (I.P. PAI-2 nuclear) and cytoplasmic (I.P. PAI-2 cytoplasmic) fractions from S1a, KJD, and activated U937 cells by using an anti-PAI-2 antibody, followed by electrophoresis and Western blotting of the immunoprecipitates with anti-Rb and anti-PAI-2 antibodies (top and bottom panels in each set, respectively). Control (lane 1) represents Western blotting of nuclear immunoprecipitates where the anti-PAI-2 antibody was omitted. Lysate (lanes 4) shows Western blotting of nuclear lysates.
- FIG. 3.
Involvement of the PAI-2 C-D interhelical region and the C-pocket of Rb in Rb-PAI-2 binding. (A) Summary of data presented in panels B to E. +++, strong binding; +, weak binding; NT, not tested. (B) PAI-2 binds Rb and p130 but not p107. An in vitro GST binding assay used IVT, 35S-radiolabeled PAI-2 and the GST fusion proteins Rb379-928, p107385-1068, and p130414-1135. Input IVT PAI-2 (lane 3) was incubated with glutathione-agarose beads bound to either GST alone (lane 1), GST-Rb379-928 (Rb), GST-p107385-1068 (p107), or GST-p130414-1135 (p130). After washing, bead-bound, 35S labeled PAI-2 was resolved by SDS-PAGE and exposed to X-ray film (lane 2). (C) Binding of PAI-2 and the C-D interhelical deletion mutant of PAI-2 to Rb and Rb LXCXE mutants. Input IVT PAI-2 (lane 5), in the form of either 35S-labeled IVT PAI-2 (top panel) or a 35S-labeled IVT C-D interhelical deletion mutant of PAI-2 (C-D PAI-2) (bottom panel), was incubated with glutathione-agarose beads bound to either GST-Rb379-928 (lane 1), GST-Rb(706F) (Rb379-928 with a mutation in position 706, which disrupts LXCXE binding) (lane 2), or GST-Rb(del exon 21) (Rb379-928 with a deletion in exon 21, which also disrupts the LXCXE binding site) (lane 3), or GST alone (GST) (lane 4). After washing, bead-bound, 35S-labeled PAI-2 or C-D PAI-2 was resolved by SDS PAGE and exposed to X-ray film. (D) The RSL mutant of PAI-2 retains binding to Rb. Input IVT PAI-2 (lane 3, top panel), a C-D interhelical deletion mutant of PAI-2 (C-D PAI-2) (middle panel), or the RSL mutant of PAI-2 (PAI-2Ala380) was incubated with GST-Rb379-928. After washing, the bead-bound, 35S-labeled PAI-2 proteins were resolved by SDS-PAGE and exposed to X-ray film (lane 2). (E) PAI-2 binds to the C-pocket of Rb. Input 35S-labeled IVT PAI-2 (top panel, lane 6) or 35S-labeled C-D PAI-2 (bottom panel, lane 6) was incubated with glutathione-agarose beads bound to either GST (lane 1), GST-Rb379-928 (lane 2), GST-SE (lane 3), GST-SEΔ (lane 4), or GST-13S (lane 5). SE and GST-SEΔ bind c-Abl, but 13S does not. After washing, bead-bound, 35S-labeled PAI-2 or C-D PAI-2 deletion mutant PAI-2 was resolved by SDS-PAGE and exposed to X-ray film.
- FIG.4.
A new Rb-binding motif, the PENF homology region. (A) Alignment of Rb-binding proteins with the C-D interhelical region of PAI-2 reveals a conserved motif (boldfaced). Rb-binding proteins for which Rb binding has been reported to be LXCXE independent include HDAC-1 (16) and -3 (15), yeast RPD3 (28), DNA polymerase delta (DNA pol delta) (30), cyclin D1 (35), c-Abl (50), Ctip, BRG1 (10), BRCA-1 (14), TGMV AL3 (43) and AL1 (29), Epstein-Barr virus nuclear protein 6 (EBNA6) (36), and human cytomegalovirus (HCMV) IE2 86 (18). LXCXE-independent binding has not been reported for the Rb-binding proteins HBRM, cyclin D2 and -3, HDAC-2 (10), Elf-1 (48), and RBP1 and -2 (15). The sequence representing the C-D interhelical region and the sequence deleted in the PAI-2 C-D interhelical mutant are indicated. The peptide sequence that is able to inhibit HDAC-3 binding to Rb379-928 is underlined (PAI-266-95; see panel B). The proposed geminivirus TGMV AL1 Rb-binding site (29) is shown with a dashed underline. The alignments were facilitated by using MENE software, available at the BioNavigator bioinformatics website (www.bionavigator.com.au). (B) Inhibition of HDAC-3 binding to Rb379-928 by using synthetic peptides based on the PAI-2 C-D interhelical region. 35S-labeled IVT HDAC-3 was incubated with the indicated peptides, and the mixture was added to glutathione-agarose beads bound to GST or GST-Rb379-928. After washing, bead-bound, 35S-labeled HDAC-3 was resolved by SDS-PAGE and exposed to X-ray film. This experiment was repeated three times, and scanning densitometry was used to quantify resolved HDAC-3 bands. The mean percent inhibition from the three separate experiments is shown on the right; percent inhibition of binding refers to percent reduction in band intensity compared with that of GST-Rb plus control peptide. The inhibitory PAI-266-95 peptide is underlined, and the TGMV AL1 peptide sequence, AL1132-157, is shown with a dashed underline, in both the sequence and the gel.
- FIG.5.
Increases in Rb protein levels in PAI-2-expressing Jurkat cells. (A) Western blot analysis of nuclear and cytoplasmic proteins extracted from the parental Jurkat cell line (Parent), a vector control Jurkat line (Vector control), Jurkat lines stably expressing wild-type PAI-2 (J.PAI-2a and J.PAI-2b), the C-D interhelical mutant of PAI-2 (J.C-D PAI-2a/b), or the RSL mutant of PAI-2 (J.PAI-2 Ala380a/b). Nuclear and cytoplasmic cell lysates were probed using antibodies specific for PAI-2, Rb, and actin. The low level of the cytoplasmic protein actin in the nuclear extract confirmed the low level of cytoplasmic proteins contaminating these extracts. (B) Quantitative real-time RT-PCR expression analysis of Rb mRNA levels in Jurkat control and PAI-2-expressing lines. Rb cDNA prepared from parental Jurkat cells, Jurkat vector control cells, and PAI-2-expressing Jurkat lines (J.PAI-2a and -b) was performed using GAPDH as a standard. Data from three experiments are expressed as the mean fold change (± standard deviation) compared with parental Jurkat cells. (C) Immunoprecipitation of PAI-2 from nuclear (I.P. PAI-2 nuclear) and cytoplasmic (I.P. PAI-2 cytoplasmic) fractions from PAI-2-expressing Jurkat lines by using an anti-PAI-2 antibody, followed by electrophoresis and Western blotting of the immunoprecipitates with anti-Rb (top panel) and anti-PAI-2 (bottom panel) antibodies. Control and lysate lanes are as for Fig. 2B. (D) Increased Rb protein stability in PAI-2-expressing Jurkat cells. Rb turnover was analyzed by a pulse-chase procedure using the control parental Jurkat cell line (solid squares), the vector control Jurkat cell line (solid triangles), and the PAI-2-expressing Jurkat lines J.PAI-2a (open squares) and J.PAI-2b (open triangles). Cells were metabolically labeled with [35S]methionine-cysteine and washed, and at the indicated times, cell lysates were prepared and immunoprecipitated with an antibody specific for Rb. Immunoprecipitated Rb was resolved by SDS-PAGE and detected by autoradiography. Scanning densitometry was used to quantify the percentage of Rb remaining. (Left) Autoradiogram from one representative experiment. (Right) Graph showing the mean percentage of Rb remaining ± standard deviation as determined from three separate experiments.
- FIG. 6.
Changes in E2F-1-dependent transcription and G1 arrest in PAI-2-expressing cells. (A) Quantitative real-time RT-PCR analysis of E2F-1-dependent gene expression. DHFR and cyclin A cDNAs from parental HeLa cells, antisense PAI-2 (A2/7), PAI-2-expressing HeLa lines (S1a and S1b), parental Jurkat cells, vector control Jurkat cells, and PAI-2-expressing Jurkat lines (J.PAI-2a and -b) were quantified by PCR using GAPDH as a standard. Data from three experiments are expressed as the mean fold change (± standard deviation) compared with parental HeLa and Jurkat cells. P < 0.0001 by the Student's t test comparing DHFR levels in HeLa and A2/7 cells with those in S1a and S1b cells; for cyclin A in the HeLa lines, P < 0.001; for DHFR levels in Jurkat cells and the vector control versus PAI-2a and -b lines, P < 0.008; and for cyclin A in the same Jurkat lines, P < 0.003. (B) Cell cycle analysis of PAI-2-expressing and control cell lines. Cells were analyzed using propidium iodide staining and fluorescence-activated cell sorter analysis to determine the percentage of cells in the G1 phase of the cell cycle. The mean of three experiments ± standard deviation is shown. By Student's t test comparing control and PAI-2-expressing cells, P < 0.0002 for PAI-2-expressing HeLa cells and P < 0.05 for PAI-2-expressing Jurkat lines.
- FIG. 7.
Proposed intracellular Rb protection activity of PAI-2. PAI-2 binds Rb via the PENF homology motif and inhibits the activity of unknown proteases that degrade Rb. Rb turnover is reduced and Rb protein levels rise, resulting in enhancement of Rb-mediated activities. Rb can associate with many transcription factors, although the best characterized is association of Rb with E2F-1, which results in inhibition of E2F-1-dependent gene transcription. Inhibition of E2F-1-dependent transcription is associated with decreased cell cycle progression, reduced proliferation, an increase in tumor suppression activity, promotion of differentiation, inhibition of apoptosis, and transcriptional regulation. PAI-2 expression has been shown to promote similar phenotypes in several systems.
