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UV-induced binding of ING1 to PCNA regulates the induction of apoptosis

¹ Departments of Biochemistry and Molecular Biology, Faculty of Medicine, The University of Calgary, 3330 Hospital Drive, NW, Calgary, Alberta T2N 4N1, Canada
² Department of Oncology, Faculty of Medicine, The University of Calgary, 3330 Hospital Drive, NW, Calgary, Alberta T2N 4N1, Canada

View larger version (14K): [in a new window]   Fig. 1. ING1 protein structure. (A) Schematic diagram of three characterized isoforms of ING1 expressed in most tissue culture cells including Hs68 fibroblasts and SNB19 cells and a recently cloned ING1-like (ING1-L) gene. All proteins contain PHD and nuclear localization signal (NLS) domains; p33ING1b contains a PIP domain (P). Darkened regions represent isoform-unique regions and open bars represent a common exon. (B) functional regions of p33ING1b are indicated including the PIP sequence, a partial Bromodomain and the NLS, which consists of two independent nucleolar transport sequences (Scott et al., 2001).

View larger version (67K): [in a new window]   Fig. 2. Glycerol gradient fractionation of protein complexes containing ING1. Extracts prepared from Hs68 fibroblasts and sedimented, as outlined in Materials and Methods, were fractionated from the bottom of tubes. Fraction 15 represents the highest density fraction and fraction 1 represents the least dense fraction that was isolated last from the top of gradients. Samples corresponding to the 15 fractions isolated were electrophoresed through 12.5% gels, transferred to nitrocellulose and probed with (a) -ING1 (Cab1-4) or (b) mouse -PCNA antibodies and visualized by ECL. Numbers below panel b represent the relative migration of denatured protein standards run on control gradients.

View larger version (50K): [in a new window]   Fig. 3. Co-immunoprecipitation of p33ING1b with PCNA. (A) Nondenaturing immunoprecipitations of extracts from 5x106 cells with rabbit polyclonal -ING1 were done in the absence (0) or at the number of hours indicated on the abscissa, after exposure of cells to 25 J/m2 of UV. Immunoprecipitates were electrophoresed and blotted with mouse -PCNA. IgG-H and IgG-L represent crossreactivity with heavy and light chains of immunoprecipitating antibodies, respectively. (B) Cells transfected with parental vector pCIneo (V), or with expression constructs encoding p47ING1a (a) or p33ING1b (b) were harvested 24 hours after electroporation under nondenaturing conditions, immunoprecipitated with a mixture of four mouse -ING1 monoclonals (Cab1-4) and electrophoresed and blotted with rabbit -PCNA. (C) Lysates from the same experiment as shown in B blotted with rabbit -PCNA. (D) Lysates from the same experiment as shown in B and C blotted for ING1 expression with Cab1-4. (E) The PIP motif in p33ING1b where ‘X’ represents any amino acid, ‘h’ represents a hydrophobic residue and ‘a’ represents aromatic residues. ING1b differs from the consensus sequence only by having a valine residue in place of an aromatic amino acid but both valine and phenylalanine have hydrophobic character. (F) Sequence of the PIP box mutations examined in G, showing the relative degree of interaction between PCNA and p33ING1 variants. Values were generated by scanning densitometry, Results shown are the average of two experiments where data for each was generated by three scans at different exposures. (H) Effects of overexpressing the cyclin-dependent kinase inhibitors p21WAF1 and p16MTS1 on the ability of ING1 to bind PCNA. Cells transfected with vector, p21, p16 or p47ING1a were harvested 48 hours later and immunoprecipitated with -ING1 monoclonals (Cab1-4) under non-denaturing conditions. Immunoprecipitates were electrophoresed and probed with rabbit -PCNA. Experiments were done with both Hs68 and SNB19 cells, and gave similar results.

View larger version (103K): [in a new window]   Fig. 4. UV-induced colocalization of ING1 with PCNA but not with p53. Half of a set of Hs68 fibroblasts plated for 24 hours were treated with 25 J/m2 of UV, fixed 2 hours later and stained for DNA, ING1 and PCNA (a-h), or for DNA, ING1 and p53 (panels i-p). Confocal microscopy and calculation of the degree of colocalization was as described in Materials and Methods. Bar, 10 µm.

View larger version (33K): [in a new window]   Fig. 5. p33ING1b PIP mutants that do not bind PCNA do not induce apoptosis. Hs68 fibroblasts electroporated with constructs encoding GFP plus vector (V), wild-type (WT) or mutant (PM1) versions of p33ING1b were allowed to recover for 24 hours and the subset indicated were treated with UV. 48 hours after electroporation, cells were harvested and analyzed by FACS (A), following propidium iodide staining to detect sub-G1 DNA content as a measure of apoptosis. The graph shows the mean ± s.e. from five individual trials. (B) A comparison of results obtained by FACS (examining DNA content) and by TUNEL to confirm FACS results by examining DNA breakage.